non-confidential report

Transcription

2009AnnualRepor
t
I. Table of Contents
Pages
Cover
1
Table of Contents
2
Overview of the Activities of the Taube-Koret Center for HD Research
3–5
Oversight of the Taube-Koret Center for HD Research
Biographies of our Advisors
Report from Dr. Pagno Paganetti
Report from Dr. Norbert Bischofberger
6
7–10
11–13
Publications and Presentations of the Taube-Koret Center for HD Research
Bibliography of Publications
HD-related Academic Seminars
HD-related Industry Consultations and Seminars
14
15
16
Taube-Koret Center for HD Research and the Community
Press releases
News stories
The Taube-Koret Center for HD Research and
HD Families
17–22
23–33
34
Appendix of Publications
35–113
2
II. Overview of the Activities of the Taube-Koret Center for Huntington’s Disease
Research for 2009
We are pleased to provide this annual report describing the activities of the Taube-Koret Center
for Huntington’s Disease Research during 2009. The Center was established in 2009 with a joint
gift from Taube Philanthropies and the Koret Foundation. It has been a very exciting year. I am
delighted to say that we exceeded all five of the scientific and financial goals we set for the first
year of operation. Our progress in each area is described in detail below.
Goal 1. Establish the Taube-Koret Center for Huntington’s Disease
Our initial goal was to establish a Center focused on developing therapeutics for
Huntington’s disease (HD). We proposed to develop an infrastructure that would be capable
of identifying and validating drug targets for HD and of discovering compounds that modify
HD and have the potential to become drugs. The new Center is housed in rented space within
the Gladstone Center for Translational Research at 1700 Owens Street and in existing space
within the main research building of the J. David Gladstone Institutes at 1650 Owens Street.
The new laboratories have been outfitted with equipment to evaluate potential HD drug
targets and to synthesize potential new therapeutics. Substantial capabilities, including
special robotics, have been added to our existing laboratories to carry out high-throughput
screens to find new therapeutics. One silver lining of the global financial crisis last year was
that it enabled us to purchase equipment and set up these laboratories for less than it would
otherwise have cost.
Goal 2. Integrate industrial experience and capability into the academic framework
In addition to the physical resources necessary to find HD therapeutics, we added critical
human resources. Dr. Stephen Freedman provides assistance in prioritizing drug targets,
designing screens, developing hits into lead programs, and negotiating relationships with
potential industry partners. His decades of drug experience with Merck and Elan have proven
to be extremely helpful. In addition, we recruited experts in medicinal chemistry to help us
develop leads into potential drugs and established relationships with an array of contract
research organizations that can perform critical steps in drug development that are not costeffective to establish in house. We also recruited two external advisors of international
reputation and drug discovery experience to provide a detailed scientific review of our
program. Throughout the year, they have provided advice and oversight. In December, at our
request, they made a site visit to review the program. The review meeting with Dr. Paolo
Paganetti (Novartis) and Dr. Norbert Bischofberger (Gilead) was highly successful and added
considerable input to our future direction. The detailed reports are provided below.
Goal 3. Implement a critical review process and focus on programs most likely to
succeed
Recognizing that our resources are limited, we implemented a hard-nosed strategy to
periodically re-prioritize our programs as results from our experiments become available.
Programs that fail to meet performance criteria are dropped, and resources are redeployed to
more promising leads. Programs that meet performance criteria and progress to the point that
they interest industry are favored. They lead to partnerships that bring in additional resources
from our industry partners, which also allow us to redeploy resources of the Center to other
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promising leads. Industry partners will eventually be needed to carry leads forward into
clinical trials, which require resources that are currently beyond those of the Center. We
began the year with 11 programs, spanning target identification, validation, and lead
development. By year’s end, one program was dropped because it failed to meet performance
milestones. Another program had progressed to the point that it garnered interest by two
competing biotechnology companies, who delivered term sheets to form a partnership. Three
new lead programs have been added.
Goal 4. Use a publication strategy to validate the scientific excellence of the Center,
stimulate scientific discussion and promote scientific awareness in the Huntington’s
disease field
The scientific productivity of the Center during its first year has been exceptional. The
Muchowski and Finkbeiner laboratories published 10 peer-reviewed papers describing results
from their HD research programs. These studies revealed a range of pathogenic mechanisms
in HD and therapeutic strategies. These include ground-breaking work on misfolding and
abnormal clearance of huntingtin, critical neurobiology of cellular mechanisms to rid cells of
protein aggregates, excessive neuroinflammation, new potential drugs to protect neurons
against neurodegeneration induced by polyglutamine stretches, and new methods to use
neurons to find therapeutics. A bibliography and copies of all the original publications from
the Center in 2009 are enclosed.
Publication is the major mechanism for achieving international renown for our HD research
program. Other mechanism are to accept invitations to speak about the work from the Center
all over the world and to participate in service to the National Institutes of Health (NIH) and
on scientific advisory boards (SABs) of drug companies working on HD. Drs. Muchowski
and Finkbeiner both helped to guide NIH HD programs in 2009 and provided SAB service
and consultation to 11 biotechnology and pharmaceutical companies. As a result of these and
other activities, the Center has been featured in the popular press. Some of these news stories
can be found in this annual report.
Goal 5. Leverage additional external funding to support the overall mission of the
center
Another important strategic feature of Center is our commitment to attract additional
resources to leverage the investment by our donors. We were unusually successful in 2009,
raising an additional $7.85M to support our HD therapeutics programs. A $1.7M grant from
the prestigious Keck Foundation will enable us to establish a facility to study electrical
activity in the region of the brain affected by HD in mice while they are awake and behaving.
A $3.7M Grand Opportunity grant from the NIH will enable us to generate inducible
pluripotent stem cells from skin tissue of adults with HD and use them to create human
neurons we will use to search for new therapeutics. Further, the award itself provides
additional recognition for the Center as one of the world’s leading sites for HD research. The
remaining $2.45M came from the NIH in a series of smaller grants. We might never
duplicate the fund raising success we experienced in 2009, but it was an encouraging start for
the new Center nonetheless.
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The Taube-Koret Center for
Huntington’s Disease Research
was established to facilitate the
development of therapeutics for
HD. We proposed a novel
strategy to bridge the gap
between academia and industry
and to create a pipeline for
therapeutics. This year, we
expected to be heavily focused
on building the infrastructure to
develop therapeutics. However,
we are pleased to report the
unexpected news that two of our
lead programs have already
attracted industry interest. The
fact that these programs have
warranted industry interest is an
important validation for the overall strategy of the Taube-Koret Center for Huntington’s Disease
Research.
The need for HD therapeutics is clear. Overall, we are very pleased with the success of the
Taube-Koret Center for HD Research during its first year of operation. We remain as committed
as ever to the primary goal of the Center—to develop therapies that prevent, treat, and eventually
cure HD.
Steven Finkbeiner, M.D., Ph.D.
Director, Taube-Koret Center
for Huntington’s Disease Research
Associate Director, Senior Investigator
Gladstone Institute of Neurological
Disease
Professor, Departments of Neurology
and Physiology
UCSF
Paul Muchowski, Ph.D.
Co-Director, Taube-Koret Center
for Huntington’s Disease Research
Associate Investigator
Gladstone Institute of Neurological
Disease
Associate Professor, Department
of Biochemistry and Biophysics
UCSF
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III. Reports of the External Advisors to the Taube-Koret Center for Huntington’s Disease
Research
We seek to be transparent and accountable in our management of the gifts entrusted to us by the
donors, which enabled us to establish the Taube-Koret Center for Huntington’s Disease
Research. As part of this effort, we recruited expert external advisors to the Center to provide an
outside perspective on our performance. Short biographies of our advisors can be found below.
The advisors provided advice and oversight throughout 2009. On December 15, 2009, we
organized a day-long meeting on-site with our external advisors, who reviewed the structure of
the Center and our major lead programs. Their reports are reproduced verbatim below.
Biographies of the External Advisors to the Taube-Koret Center for Huntington’s Disease
Research
Paolo Paganetti, PhD
Head of Huntington’s Disease Research
Novartis
Dr. Paganetti received his PhD from the University of Zurich, Switzerland in the lab of Prof.
M.E. Schwab, in the Brain Research Institute. His postdoctoral research was done with Prof.
Schwab and Prof. R.H. Scheller, HHMI and Stanford University. He joined Novartis in 1992 as a
lab head and has occupied positions with increasing responsibilities. Within the neuroscience
disease area, Dr. Paganetti was part of the Alzheimer’s disease team responsible for drug
discovery programs for compounds reducing Ab-peptide secretion and inhibiting the aspartic
protease BACE. Currently, he leads the Huntington’s disease team and is involved in several
external research collaborations. He was mentor for six postdoctoral fellows, four PhD students
and seven research assistants and leads a lab with five associates. Dr. Paganetti received the
Novartis Leading Scientist award in 2003 and was appointed senior research investigator II in
2006. He is author of over 60 scientific publications.
Norbert W. Bischofberger, PhD
Executive Vice President, Research and Development and Chief Scientific Officer
Gilead Sciences
Dr. Bischofberger joined Gilead Sciences in 1990 and has served as executive vice president for
research and development since 2000 and chief scientific officer since 2007. He oversees all of
Gilead’s research discovery, preclinical & clinical development, pharmaceutical development
and API manufacturing. Before joining Gilead, Dr. Bischofberger was a senior scientist in
Genentech’s DNA Synthesis Group from 1986 until 1990. He received a PhD in organic
chemistry from Zurich's Eidgenossische Technische Hochschule and performed postdoctoral
research in steroid chemistry at Syntex. He also performed additional research in organic
chemistry and applied enzymology in Professor George Whiteside’s lab at Harvard University.
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Paolo Paganetti, PhD
Senior Research Investigator II
Novartis Institutes for BioMedical Research
Novartis Pharma AG
Basel, Switzerland
External Evaluation
Taube-Koret Center for Huntington’s Disease
Advisory Meeting of December 15, 2009
I had the great pleasure to actively participate at the advisory board meeting of the
Taube-Koret Center for Huntington’s Disease as an external advisor. I was astonished
by the clear and concise presentations of top scientific quality made by Dr. Steve
Finkbeiner and Dr. Paul Muchowski and the other presenters, as well as by the focused
drug discovery activities and the quality of the translational research advancing rapidly
at the Center.
The objective of the Taube-Koret Center is to find a cure for Huntington’s disease (HD)
by 2020. HD is a progressive neurodegenerative genetic disorder that affects muscle
coordination and some cognitive functions. Caused by a dominant mutation in a gene
located on chromosome 4 encoding for the huntingtin protein, HD is inherited with a
50% risk by any child of an affected parent. Mutated huntingtin with a CAG repeat
expansion (for polyglutamine) provokes a gradual damage to the brain by mechanisms
not fully understood. Clinical symptoms usually begin with subtle changes in physical
skills, personality, or cognition in middle age. Lethal complications such as pneumonia
or heart disease result in a life expectancy of ~20 years after onset of clinical symptoms.
HD is an orphan disease with no cure available, but with treatments improving some
symptoms. Approved in 2008, Tetrabenazine has specific use for reducing the severity
of chorea in HD.
There is a lot of confidence that a pharmacological intervention reducing the amount of
mutant huntingtin in the brain would lead to an effective cure for HD. On the other hand,
the length of the CAG repeat accounts for only 50% of the variation in age of onset and
rate of disease progression, implying that other “modifying” genes or to environmental
factors influence the disease mechanism and explain the remaining variation. The drug
discovery activities progressing at the Taube-Koret Center are targeting both
intervention nodes making the aim to find a cure for HD within the proposed timeline an
achievable mission.
Fulfilling this goal requires a deep understanding of the pathogenic mechanisms of HD
and the application of this knowledge to develop more effective methods of early
detection and treatment. This is crucially dependent on advances in genomics, cell
biology, chemistry and computational science. The most modern tools and techniques
in these areas have been developed by the scientists of the Taube-Koret Center or are
accessible through affiliated Institutes (Gladstone and UCSF to only mention the two
most important) or well established scientific and technical collaborations. This is an
excellent basis for propelling basic science and drug discovery, in particular because
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the Taube-Koret Center will bridge these two disciplines and fill an historic gap in the
discovery of new therapies.
The Taube-Koret Center has been created this year and is directed by is by Dr. Steven
Finkbeiner and Dr. Paul Muchowski, two world-wide recognized scientists who have
made critical contributions to advancing basic knowledge by dissecting
pathomechanisms underlying the development and progression of Huntington’s
disease. This is not only evident by an impressive number of recent peered reviewed
publications in top-ranked scientific journals, but also by a well running network of
collaborations that is among the most impressive existing in the field. Clear recognition
for this achievement is demonstrated by the fact that their work has attracted financial
support through a handful of grants for a yearly funding that surpasses by more than
fivefold the initial investment made by the donors who made the creation of the TaubeKoret Center possible.
In this report, I would like to give a feedback on different projects that attracted my
attention during the meeting and include some recommendations.
Drug Target Identification
Identification of new drug targets for a cure of HD at the Taube-Koret Center is based
on well-established unbiased screening capabilities in cultured cells. Dr. Muchowski has
long-standing expertise in successfully applying yeast to identify genetic modifiers of the
toxic properties of mutant huntingtin. Dr. Finkbeiner has developed over the last 10
years a powerful automated microscopy screening model with mammalian neurons in
cultures that not only has proven its use as a screening assay but represent a worldwide unique test paradigm for drug target validation in vitro. In addition to other target
screening and validation techniques, already these two models (yeast and primary
neurons) led the researchers at the Center promising starting point for drug discovery.
Such candidate drug target genes are currently validated not only with the mentioned in
vitro test assays but through a battery of in vivo mouse lines. These models are
recognized by the scientific community as golden standard for HD-relevant pathological
and clinical measures and thus of robust translational medicine potential. In this contest,
at The Gladstone Institute there are excellent facilities for neurobehavioral and
neuropathological studies to which as good access.
Medicinal Chemistry
Medicinal chemistry with best pharmaceutical practice and decades of know-how is
present at the Center including computational chemistry and other modern techniques.
Although small, these capacities have already delivered series of proprietary small
molecular weight compounds with proven in vitro and in vivo activities. It is suggested to
make appropriate use of these assets in the different programs and seek external
partners with the adequate resources to accelerate the most advanced programs.
Partnering will also allow access capabilities not yet available at the Center and
leverage the investments made to date by the donors as pointedly recognized by the
presenters.
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Animal Models
Use of animal experiments needs careful evaluation. Their importance as a powerful
translational medicine tool is obvious. On the other hand, in the field of
neurodegenerative disorders efficacy studies in mouse models often acquire proportion
similar to those of clinical trials with long study length, substantial costs and often
requiring a large number of animals because only few measurable endpoints are
available. The scientists at the Center are well aware of these issues and beside
pharmacokinetic studies of compound distribution, gave high priority to demonstrate
target engagement, as well as adequate safety margins by the experimental drugs. For
programs directly aiming at reducing the load of toxic huntingtin in the brain, the link
with mechanism of action and efficacy is well accepted. In contrast, for putative toxicity
modifiers the link between brain pathology, animal behavioral endpoints and clinical
efficacy is weaker and may require significant tailoring for each program. The search for
powerful biomarkers of disease onset and progression is one of the priority activities in
the HD field and the Center has established privileged relationship with the most
important HD center in the US and Europe.
KMO Program
Dr. Muchowski has demonstrated a relation between this target and HD in several
cellular and animal models by tenaciously championing this program to steady
progress. This year, the Center has unequivocally validated the target in vivo making
KMO world-wide one of maybe two-three preclinically validated targets. This
contribution is outstanding and of excellent quality. The animal data indicate that KMO
inhibition will affect disease progression, prolonging survival and rescuing some of the
pathological measurements. Further morphological analysis of brain atrophy and striatal
markers, such as DARP32, may represent an additional asset of the program, as well
as attempts to better understand the mechanism of action possibly also in peripheral
tissues. Dose chronically one or more of the KMO metabolites may also contribute in
elucidating the mechanism. Overall, there was good agreement on the path forward,
such as integrating the key enzymatic tests within the Center, convincing enzymatic
studies, the need for an efficient measure for short-term mouse compound screen and a
mechanistic readout in corticospinal fluid. In the near future, the established IP position
needs an aggressive protection strategy as the design of adequate partnering plans.
The preliminary positive outcome in animal models of other neurodegenerative
disorders, such as Alzheimer’s disease, is remarkable and of wonderful potential.
Autophagy Program
Macroautophagy is a cellular defense mechanism for degradation of defective
organelles and toxic protein aggregates that has attracted recently a lot of attention by
the scientific community and drug discovery researchers. Also neurons make us of
autophagy but the regulatory mechanisms in these cells are poorly understood as the
classical inducing treatment paradigms are ineffective. Dr. Finkbeiner has made perfect
use of his automated microscopy technique by screening a large number of marketed
drugs and identifying a small molecular weight drug which efficiently induce mutant
huntingtin degradation by autophagy in neurons. This discovery is of upmost importance
and combined with the identification of a marketed drug with proven safe clinical use,
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this program pushes the Center in unique competitive advantage. The path forward was
endorsed by all participants: a concise medicinal chemistry program with the aim of
obtaining a small increase in potency to allow validation of the hypothesis in vivo, proof
of concept could also be envisaged in peripheral tissues and thus limit the program
should not be limited to CNS active compounds, demonstration of a specific mechanism
and not related to the known biology of the current leads. The well progressed
partnering negotiation for licensing biology and chemistry to one of the two companies
Proteostatis or LINK is fully supported.
Additional Programs
IDO/TDO represents a very attractive back-up program to KMO. It is expected that the
identified modulatory compounds, as well as use of the knock-out mice, are adequate to
rapidly validate this program in vivo. Additional exploratory activities to assess the
possible role of inflammatory cytokines in the brain with the potential to produce a
biomarker strategy as well as therapeutic approach are well founded.
Mgmt, a DNA repair enzyme identified in the yeast screen, if validated, has a lot of
potential since compounds in advance clinical trials exist for oncology indications. Also
here, compound treatment and knock-out mice are adequate to rapidly validate this
program in vivo.
CB2 and Nrf2 are in an early exploratory phase, and their potential as drug targets
difficult to assess at the present date.
Huntingtin modifying strategies have an excellent rationale, and the programs on
polyglutamine conformation and phosphorylation have great potential. It is unfortunate
that the compound leads identified in the screen can not be pursued with the necessary
determination for lack of resources. If a reprioritization would endanger more advanced
program, then partnering seems the best solution.
General comment
When testing strategies reducing toxic huntingtin, it is advised to analyze additional
neurodegeneration-linked proteins, such as alpha synuclein or tau, in the HD models.
Integration of human models in the current screen would further increase the value of
the screening models developed at the Center.
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MEMORANDUM
TO:
Steven Finkbeiner, M.D., Ph.D.
Professor, Departments of Neurology and Physiology
University of California, San Francisco
Senior Investigator and Associate Director, Gladstone Institute of
Neurological Disease
Director, Taube-Koret Center for Huntington’s Disease Research
1650 Owens St., Office 308
San Francisco, CA 94158
CC:
Paul Muchowski, Ph.D.
Stephen Freedman, Ph.D.
FROM:
Norbert Bischofberger, Ph.D.
Executive Vice President, R&D and Chief Scientific Officer
Gilead Sciences, Inc.
333 Lakeside Drive
Foster City, CA 94404
DATE:
January 29, 2010
Re:
Report of the December 15, 2009 External Advisory Meeting of the
Taube-Koret Center for HD Research
The following constitutes my report following The External Advisory meeting of
the Taube-Koret Center for HD Research which took place December 15th 2009 at
the Gladstone Institute in San Francisco. I was one of two external advisors
attending the meeting. My expertise is mainly in drug discovery and drug
development including regulatory issues and translational medicine.
Overall, I was very impressed with the progress that is being made with the
work by Paul Muchowski and Steve Finkbeiner. I sensed a high awareness and
desire to advance basic scientific findings into therapeutics which in my
experiences is not at all common in academic settings. Both Paul and Stephen
are very much aware of the issues that have to be addressed and the hurdles that
have to be overcome in early lead optimization, preclinical development and in
human clinical studies. The progress made so far is impressive particularly
1
more extensive and expensive phase III studies. Also, the design and nature of
the POC studies can shape discovery and preclinical development strategies.
In summary, I was impressed by the efforts of the group. With Paul and
Stephen, The Taube-Koret Center has two world-class biologists and experts in
CNS biology. The choice of targets is judicious and there is a goal oriented
approach to research. Near term, some of the advanced projects have to be
pushed further to answer basic questions, earlier projects need to be focused and
the promise has to be further defined.
I am looking forward to reviewing the progress at our next meeting.
Sincerely,
Norbert Bischofberger, Ph.D.
Executive Vice President, Research & Development
Chief Scientific Officer
Gilead Sciences, Inc.
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IV. Publications and Presentations of the Taube-Koret Center for Huntington’s Disease
Research
A. Bibliography of Publications
Daub A, Sharma P, Finkbeiner S. High content screening in primary neurons, Curr. Opin.
Neurobiol. 2009, 19, 1–7. (Advanced online publication doi:10.1016).
Gu X, Greiner ER, Mishra R, Kodali R, Osmand A, Finkbeiner S, Steffan JS, Thompson LM,
Wetzel R, and Yang XW. Ser13 and Ser16 are critical determinants of full-length human mutant
huntingtin induced disease pathogenesis in HD mice. Neuron. 2009, 64:828–840.
Legleither J, Lotz GP, Miller J, Ko J, Ng C, Williams GL, Finkbeiner S, Patterson PH,
Muchowski PJ. Monoclonal antibodies recognize distinct conformational epitopes formed by
polyglutamine in a mutant huntingtin fragment. J. Biol. Chem. 2009, 284: 21647–21658.
Miller J, Rutenber E, Muchowski P. Polyglutamine dances the conformational cha-cha-cha.
Structure. 2009, 17: 1151–1153.
Mitra S, Tsvetkov A, Finkbeiner S. Single-neuron ubiquitin-proteasome dynamics
accompanying inclusion body formation in Huntington’s disease. J. Biol. Chem. 2009, 284:
4398–4403.
Mitra S, Tsvetkov, AS, Finkbeiner S. Protein turnover and inclusion body formation. Autophagy
2009, 5: 1037–1038.
Montie HL, Cho MS, Holder L, Liu Y, Tsvetkov AS, Finkbeiner S, Merry DE. Cytoplasmic
retention of polyglutamine-expanded androgen receptor ameliorates disease via autophagy in a
mouse model of spinal and bulbar muscular atrophy. Hum. Mol. Gen. 2009, 18: 1937–1950.
Thompson LM, Aiken CT, Agrawal N, Kaltenbach LS, Illes K, Khoshnan A, Martinez-Vincente
M, Arrasate M, O’Rourke JG, Lukacsovich T, Zhu Y-Z, Lau AL, Massey A, Hayden MR,
Zeitlin SO, Finkbeiner S, Huang L, Lo DC, Patterson PH, Cuervo AM, Marsh JL, and Steffan
JS. The IKK complex phosphorylates huntingtin and targets it for degradation by the proteasome
and lysosome, J. Cell Bio. 2009, 187:1083–1099.
Tsvetkov A, Wong J, Rao V, Finkbeiner S. Differential regulation of autophagy in neuronal and
non-neuronal cells. Autophagy 2009, PMID: 19411824.
Wacker JL, Huang SY, Steele AD, Aron R, Lotz GP, Nguyen Q, Giorgini F, Roberson ED,
Lindquist S, Masliah E, Muchowski PJ.Loss of Hsp70 exacerbates pathogenesis but not levels of
fibrillar aggregates in a mouse model of Huntington's disease.J Neurosci. 2009 Jul
15;29(28):9104-14.
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B. Huntington’s Disease-Related Academic Seminars
Discussion leader, Gordon Research Conference on CAG Triplet Repeat Disorders, Science
Session: Inflammation in CAG Triplet Repeat Disorders, Waterville Valley, Vermont
(Muchowski).
Keynote speaker, "The pathomechanisms of brain diseases: new technologies and approaches"
(sponsored by RIKEN), Sapporo, Japan (Muchowski).
Moderator, World Congress of Huntington’s Disease (HD), Science Session: Inflammatory and
Metabolic Changes in HD, Vancouver, Canada (Muchowski).
Invited speaker, The Fourth International Congress on Stress Responses in Biology and
Medicine, Sapporo, Japan (Muchowski).
Keynote speaker, Protein Misfolding and Neurological Disorders Conference, Port Douglas,
Australia (Muchowski).
Invited speaker, Adler Symposium on Proteotoxicity in Neurodegeneration; Salk Institute,
Torrey Pines, California 2009 (Muchowski).
Invited speaker, Huntington’s Disease Society of America, Coalition for the Cure; Vancouver,
Canada (Finkbeiner).
Invited speaker, Towards Treatment of Spinocerebellar Ataxia (EuroSCA) Conference;
Tübingen, Germany (Finkbeiner).
Symposium chair, Society for Neuroscience; Nanomedicine Symposium, Chicago (Finkbeiner).
Invited speaker, Washington University School of Medicine, Department of Neurobiology; St.
Louis (Finkbeiner).
Invited speaker, Cornell Medical Center, New York Presbyterian Hospital, Department of
Neurology and Neuroscience; New York (Finkbeiner).
Invited speaker, High Impact Science Seminar, Burnham Institute; La Jolla (Finkbeiner).
Invited speaker, Institute for Systems Biology; Seattle (Finkbeiner).
Invited talk, Cytometry Development Workshop; Asilomar (Finkbeiner).
Invited talk, University of California Irvine, Departments of Neurobiology and Behavior, Irvine
(Finkbeiner).
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C. Huntington’s Disease-related Industry Consultations and Seminars
FivePrime Therapeutics; San Francisco (Finkbeiner).
Vertex Pharmaceuticals, Inc.; San Diego (Finkbeiner).
LINK Medicine Corporation; Cambridge (Finkbeiner).
iPierian; San Francisco (Finkbeiner).
Pescadero Technologies; San Francisco (Finkbeiner).
Valla Technologies; San Diego (Finkbeiner).
Amgen; Thousand Oaks (Muchowski).
Genentech; South San Francisco (Muchowski).
Lundbeck; San Francisco (Muchowski).
Merck; San Francisco (Muchowski).
Proteostasis; Cambridge (Muchowski and Finkbeiner).
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V. Taube-Koret Center for Huntington’s Disease Research and the Community
A. Press releases in 2009
Contact:
Valerie Tucker
415-734-2019
[email protected]
For Immediate Release
GLADSTONE INSTITUTES ESTABLISHES TAUBE-KORET CENTER FOR
HUNTINGTON’S DISEASE RESEARCH
Targeted program to cure Huntington’s by 2020
SAN FRANCISCO, CA – March 25, 2009 – The J. David Gladstone Institutes has joined forces
with Taube Philanthropies and the Koret Foundation to initiate a groundbreaking research
program aimed at preventing, treating, or curing Huntington’s disease (HD) by the year 2020.
The new Taube-Koret Center for Huntington’s Disease Research has been established at the
Gladstone Center for Translational Research at Mission Bay, with $3.6 million in funding from
the two organizations.
HD, also called ‘Huntington’s chorea’ and ‘Woody Guthrie’s disease,’ is a devastating inherited,
degenerative brain disorder. More than 100,000 Americans and more than 10 times that number
worldwide have HD or are at risk of inheriting the disease from a parent.
Investigators Steven Finkbeiner, MD, PhD, and Paul Muchowski, PhD, of the Gladstone Institute
of Neurological Disease (GIND) will build on their leading-edge research, which has led to the
development of powerful assays for the identification of potential drug targets and a pipeline of
several molecular targets that may modulate HD progression. Taube Philanthropies has
supported the work of Drs. Finkbeiner and Muchowski, as well as other researchers for several
years. This new research program is called “HD Cure 2020.”
“We believe that the focus and evolving new technologies of the HD Cure 2020 program
provide a real chance to close in on a cure,” said Tad Taube, chairman of Taube Philanthropies
and president of the Koret Foundation. “It is our hope that Gladstone’s depth of understanding
about how Huntington’s progresses, combined with a well-defined and integrated therapeutic
screening strategy, will enable real progress to be made toward treating or curing this devastating
disease.”
-more-
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Taube-Koret Center
Page 2
“While so much is known about Huntington’s disease, it remains an unsolved mystery,” said
GIND Associate Director Steven Finkbeiner. “Over the last few years, we have been able to find
new points of entry into how the disease progresses and where we might possibly intervene.”
Dr. Finkbeiner has pioneered new technologies that have added important new understanding to
HD etiology and pathology. Dr. Muchowski has focused his work on identifying key
intracellular pathways that modify progression of the disease. Both investigators have developed
innovative technological and biological approaches for finding and screening small molecules
that may work to modulate the disease.
“While Gladstone brings a unique and impressive foundation of Huntington’s research to this
program, we are extremely grateful for the visionary leadership of the Koret Foundation and the
Taube Philanthropies for their creation of this center and their support of our approach,” said
Andrew S. Garb, Trustee of The J. David Gladstone Institutes.
The Taube-Koret Center is located in Gladstone’s Center for Translational Research where
Gladstone is collaborating with several pharmaceutical companies on potential therapies for
Alzheimer’s disease (Merck), HIV (Gilead Sciences and JT Pharma), and for applying induced
pluripotent stem (iPs) cell technology to cardiovascular disease (iZumi Bio).
About Taube Philanthropies
Guided by a long-term commitment to both secular and Jewish life, Taube Philanthropies
provide direct and indirect support to projects and institutions that advance the philosophies and
vision of founder Tad Taube. Central to these are the concepts and principles of a free,
democratic society, including open economic enterprise, self-reliance, academic freedom of
inquiry and limited government, and programs that support Jewish heritage, survival and cultural
celebration.
About the Koret Foundation
An entrepreneurial spirit guides Koret in addressing societal challenges and strengthening Bay
Area life. In the San Francisco Bay Area, Koret adds to the region’s vitality by promoting
educational opportunity, contributing to a diverse cultural landscape, and bolstering
organizations that are innovative in their approaches to meeting community needs. With roots in
the Jewish community, Koret embraces the community of Israel, especially through Koret Israel
Economic Development Funds, believing that economic stability and free market expansion offer
the best hope for a prosperous future
-more-
18
Taube-Koret Center
Page 3
About the Gladstone Institutes
Established in 1979, The J. David Gladstone Institutes is an independent, nonprofit
biomedical research organization that operates in close affiliation with the University of
California, San Francisco (UCSF). Gladstone is dedicated to the health and welfare of
humankind through research into the causes and prevention of some of the world’s most
devastating diseases. Gladstone is comprised of the Gladstone Institute of Cardiovascular
Disease, the Gladstone Institute of Virology and Immunology, the Gladstone Institute of
Neurological Disease, and the Gladstone Center for Translational Research. More
information can be found at: www.gladstone.ucsf.edu
About Huntington’s disease
Huntington’s disease (HD), also called Woody Guthrie’s disease, is a devastating degenerative
brain disorder that is inherited from a parent with the disease. Over a period of 10 to 25 years,
HD slowly but steadily reduces a person’s ability to walk, think, talk, and reason. Ultimately, HD
renders its victims totally dependent upon others for their care. Patients with HD ultimately die
from complications, such as choking, infection, or heart failure. Men and women of all racial and
ethnic groups are equally susceptible to contracting HD. A child of a parent with HD is 50%
likely to inherit the fatal “huntingtin” gene. Tragically, every person who carries the HD gene
ultimately develops the disease.
The typical patient with HD is aged 30 to 50, although manifestations of the disease may arise in
children as young as 2 years of age. Young people who are afflicted with the juvenile form of
HD rarely live to adulthood. Today, more than 250,000 Americans—and more than 10 times that
number worldwide—have HD or are at risk of inheriting the disease from a parent with HD. The
disease affects as many people as hemophilia, cystic fibrosis, and muscular dystrophy.
The HD gene was successfully isolated in 1993. Subsequently, a genetic blood test was
developed to determine precisely whether a person has inherited the HD gene. However, no test
can predict when HD symptoms will begin. As with other diseases that are inherited, many of
those who have a parent with HD elect not to take the HD gene test.
Over the years, biomedical research involving HD has yielded a wealth of knowledge about the
disease and its basic mechanisms. However, no effective method exists for preventing, treating,
or curing HD. In fact, no validated drug targets for HD, besides the huntingtin gene itself, have
been discovered. Although HD is one of the most cruel and devastating diseases, those afflicted
are too few in number to interest most major pharmaceutical companies in developing relevant
HD-targeted drug discovery programs.
###
19
Valerie Tucker
(415) 734-2019
E-mail: [email protected]
Web:www.gladstone.ucsf.edu
FOR IMMEDIATE RELEASE
GLADSTONE AND PARTNERS RECEIVE $3.7 MILLION TO USE
STEM CELL TECHNOLOGY FOR HUNTINGTON’S DISEASE RESEARCH
NIH Funds Effort to Develop Disease Models for Pathogenesis and Drug Discovery
SAN FRANCISCO, CA – October 13, 2009 – The National Institutes of Health (NIH) has
awarded a “Grand Opportunity” grant of $3.7 million to a consortium formed with the Gladstone
Institute of Neurological Disease (GIND) and the Taube-Koret Center for Huntington’s Disease
Research to use stem cell technology to better understand Huntington’s disease (HD) and to
develop potential therapies. The consortium comprises a partnership of five leading Huntington’s
research laboratories at the University of Wisconsin, Massachusetts General Hospital, the
University of California at Irvine, Johns Hopkins and the Gladstone Institutes. The consortium will
use induced pluripotent stem (iPS) cell technology pioneered by Gladstone and Kyoto University’s
Shinya Yamanaka, MD, PhD, to develop human neurons with Huntington’s disease characteristics.
iPS technology enables stem cells to be generated from skin samples from adults and avoids the
ethical issues surrounding the use of fetal stem cells.
“One of the challenges of Huntington’s (and many other neurological diseases) is that many of the
potential therapies that show promise in animal models are ineffective in people. We think that
molecular differences between mice and humans may be an important cause for this failure,” said
Steven Finkbeiner MD, PhD, consortium co-leader and Director of the Taube-Koret Center for
Huntington’s Disease Research and Associate Director of GIND.
-more-
20
Huntington’s Consortium
2-2-2
“One of the promises of iPS technology is to be able to develop models from Huntington’s disease
patients that can give us more detailed information about the disease and better predict how
therapies could work in humans,” he said.
HD, which is also called “Huntington’s chorea” and “Woody Guthrie’s disease,” is a devastating
inherited, degenerative brain disorder. More than 100,000 Americans and more than 10 times that
number worldwide have HD or are at risk of inheriting the disease from a parent.
iPS cells are generated by reprogramming adult cells from skin or other tissues. They are almost
identical to human embryonic stem cells with the ability to self-renew for long periods and to
differentiate into all cell lineages. More importantly, iPS cells can be generated from adult patients
with genetically inherited and sporadic diseases making it possible to study some diseases, such as
Alzheimer’s and Parkinson’s disease, for which the causes remain largely unknown.
“HD is caused by a single mutation, which provides an ideal paradigm to generate a panel of
patient-specific lines,” Finkbeiner explained. “This offers hope that these models can teach us why
some patients experience certain symptoms and why some family members develop symptoms
later rather than sooner, which then can potentially be used to develop treatments that can act
before symptoms appear.”
Finkbeiner added, “the convergence of a dedicated, collaborative group of committed investigators
targeting HD, the need for new treatments based on the root causes of the disease, and the
emergence of powerful new technologies herald a truly grand opportunity to make a real difference
for those afflicted with Huntington’s.”
Dr. Finkbeiner’s primary affiliation is with the Gladstone Institute of Neurological Disease where
his laboratory is located and all of his research is conducted. He is also associate professor of
neurology and physiology at the University of California, San Francisco.
-more-
21
Huntington’s Disease Consortium
3-3-3
Established in 1979, The J. David Gladstone Institutes is an independent, nonprofit
biomedical research organization that operates in close affiliation with the University of
California, San Francisco (UCSF). Gladstone is dedicated to the health and welfare of
humankind through research into the causes and prevention of some of the world’s most
devastating diseases. Gladstone is comprised of the Gladstone Institute of Cardiovascular
Disease, the Gladstone Institute of Virology and Immunology, the Gladstone Institute of
Neurological Disease, and the Gladstone Center for Translational Research. More
information can be found at: www.gladstone.ucsf.edu
About the Taube-Koret Center for Huntington’s Disease Research.
The Center was established in 2009 with gifts from Taube Philanthropies and the Koret
Foundation for the sole purpose of identifying strategies and developing therapeutics to
treat people with Huntington’s disease and related neurodegenerative diseases.
###
22
B. The Taube-Koret Center for Huntington’s Disease Research in the News
23
New HD Research Center Tasked with Preventing, Treating, or Curing Disease by 2020
10/17/09 11:58 PM
Mar 26 2009, 11:30 AM EST
New HD Research Center Tasked with Preventing, Treating,
or Curing Disease by 2020
GEN News Highlights
The J. David Gladstone Institutes, Taube Philanthropies, and the Koret Foundation joined forces to initiate a research
program aimed at preventing, treating, or curing Huntington's disease (HD) by 2020.
The new Taube-Koret Center for Huntington's Disease Research has been established at the Gladstone Center for
Translational Research at Mission Bay, CA, with $3.6 million in funding from the two organizations. The program is
called HD Cure 2020.
The center will build on research from investigators Steven Finkbeiner, M.D., Ph.D., and Paul Muchowski, Ph.D., of the
Gladstone Institute of Neurological Disease (GIND) related to assay development and molecular targets that may
modulate HD progression.
Dr. Finkbeiner’s technologies reportedly aid in the understanding of HD etiology and pathology. Dr. Muchowski’s
studies have identified intracellular pathways that modify progression of the disease. Together they have also
developed methods to find and screen small molecules that may work to modulate the disease.
“While so much is known about Huntington's disease, it remains an unsolved mystery,” notes Dr. Finkbeiner. “Over the
last few years, we have been able to find new points of entry into how the disease progresses and where we might
possibly intervene.”
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Medical News Today News Article - Printer Friendly
10/30/09 8:47 PM
Gladstone Institutes Establishes Taube-Koret Center For Huntington's Disease Research,
Aims To Cure Huntington's By 2020
30 Mar 2009
Click to Print
The J. David Gladstone Institutes has joined forces with Taube Philanthropies and the Koret
Foundation to initiate a groundbreaking research program aimed at preventing, treating, or curing
Huntington's disease (HD) by the year 2020. The new Taube-Koret Center for Huntington's
Disease Research has been established at the Gladstone Center for Translational Research at
Mission Bay, with $3.6 million in funding from the two organizations.
HD, also called 'Huntington's chorea' and 'Woody Guthrie's disease,' is a devastating inherited,
degenerative brain disorder. More than 100,000 Americans and more than 10 times that number
worldwide have HD or are at risk of inheriting the disease from a parent.
Investigators Steven Finkbeiner, MD, PhD, and Paul Muchowski, PhD, of the Gladstone Institute of
Neurological Disease (GIND) will build on their leading-edge research, which has led to the
development of powerful assays for the identification of potential drug targets and a pipeline of
several molecular targets that may modulate HD progression. Taube Philanthropies has supported
the work of Drs. Finkbeiner and Muchowski, as well as other researchers for several years. This
new research program is called "HD Cure 2020."
"We believe that the focus and evolving new technologies of the HD Cure 2020 program provide a
real chance to close in on a cure," said Tad Taube, chairman of Taube Philanthropies and
president of the Koret Foundation. "It is our hope that Gladstone's depth of understanding about
how Huntington's progresses, combined with a well-defined and integrated therapeutic screening
strategy, will enable real progress to be made toward treating or curing this devastating disease."
"While so much is known about Huntington's disease, it remains an unsolved mystery," said GIND
Associate Director Steven Finkbeiner. "Over the last few years, we have been able to find new
points of entry into how the disease progresses and where we might possibly intervene."
Dr. Finkbeiner has pioneered new technologies that have added important new understanding to
HD etiology and pathology. Dr. Muchowski has focused his work on identifying key intracellular
pathways that modify progression of the disease. Both investigators have developed innovative
technological and biological approaches for finding and screening small molecules that may work
to modulate the disease.
"While Gladstone brings a unique and impressive foundation of Huntington's research to this
program, we are extremely grateful for the visionary leadership of the Koret Foundation and the
Taube Philanthropies for their creation of this center and their support of our approach," said
Andrew S. Garb, Trustee of The J. David Gladstone Institutes.
The Taube-Koret Center is located in Gladstone's Center for Translational Research where
Gladstone is collaborating with several pharmaceutical companies on potential therapies for
Alzheimer's disease (Merck), HIV (Gilead Sciences and JT Pharma), and for applying induced
pluripotent stem (iPs) cell technology to cardiovascular disease (iZumi Bio).
About Taube Philanthropies
Guided by a long-term commitment to both secular and Jewish life, Taube Philanthropies provide
http://www.medicalnewstoday.com/printerfriendlynews.php?newsid=144184
Page 1 of 3
10/30/09 8:47 PM
direct and indirect support to projects and institutions that advance the philosophies and vision of
founder Tad Taube. Central to these are the concepts and principles of a free, democratic society,
including open economic enterprise, self-reliance, academic freedom of inquiry and limited
government, and programs that support Jewish heritage, survival and cultural celebration.
About the Koret Foundation
An entrepreneurial spirit guides Koret in addressing societal challenges and strengthening Bay
Area life. In the San Francisco Bay Area, Koret adds to the region's vitality by promoting
educational opportunity, contributing to a diverse cultural landscape, and bolstering organizations
that are innovative in their approaches to meeting community needs. With roots in the Jewish
community, Koret embraces the community of Israel, especially through Koret Israel Economic
Development Funds, believing that economic stability and free market expansion offer the best
hope for a prosperous future
Established in 1979, The J. David Gladstone Institutes is an independent, nonprofit biomedical
research organization that operates in close affiliation with the University of California, San
Francisco (UCSF). Gladstone is dedicated to the health and welfare of humankind through
research into the causes and prevention of some of the world's most devastating diseases.
Gladstone is comprised of the Gladstone Institute of Cardiovascular Disease, the Gladstone
Institute of Virology and Immunology, the Gladstone Institute of Neurological Disease, and the
Gladstone Center for Translational Research.
About Huntington's disease
Huntington's disease (HD), also called Woody Guthrie's disease, is a devastating degenerative
brain disorder that is inherited from a parent with the disease. Over a period of 10 to 25 years, HD
slowly but steadily reduces a person's ability to walk, think, talk, and reason. Ultimately, HD
renders its victims totally dependent upon others for their care. Patients with HD ultimately die from
complications, such as choking, infection, or heart failure. Men and women of all racial and ethnic
groups are equally susceptible to contracting HD. A child of a parent with HD is 50% likely to
inherit the fatal "huntingtin" gene. Tragically, every person who carries the HD gene ultimately
develops the disease.
The typical patient with HD is aged 30 to 50, although manifestations of the disease may arise in
children as young as 2 years of age. Young people who are afflicted with the juvenile form of HD
rarely live to adulthood. Today, more than 250,000 Americans-and more than 10 times that
number worldwide-have HD or are at risk of inheriting the disease from a parent with HD. The
disease affects as many people as hemophilia, cystic fibrosis, and muscular dystrophy.
The HD gene was successfully isolated in 1993. Subsequently, a genetic blood test was developed
to determine precisely whether a person has inherited the HD gene. However, no test can predict
when HD symptoms will begin. As with other diseases that are inherited, many of those who have
a parent with HD elect not to take the HD gene test.
Over the years, biomedical research involving HD has yielded a wealth of knowledge about the
disease and its basic mechanisms. However, no effective method exists for preventing, treating, or
curing HD. In fact, no validated drug targets for HD, besides the huntingtin gene itself, have been
discovered. Although HD is one of the most cruel and devastating diseases, those afflicted are too
few in number to interest most major pharmaceutical companies in developing relevant HDtargeted drug discovery programs.
Source
Gladstone Institutes
Page 2 of 3
10/30/09 8:47 PM
Article URL: http://www.medicalnewstoday.com/articles/144184.php
Main News Category: Huntingtons Disease
Also Appears In: Neurology / Neuroscience,
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medical advice and you should not take any action before consulting with a health care
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Page 3 of 3
PND - News - Koret Foundation, Taube Philanthropies Award $...
http://foundationcenter.org/pnd/news/story_print.jhtml?id=248...
Print • Close Window
Posted on April 4, 2009
Koret Foundation, Taube Philanthropies Award $3.6 Million for Huntington's Disease Research
The Koret Foundation has announced a joint $3.6 million grant with Taube Philanthropies to establish a center for Huntington's
disease (HD) research at the Gladstone Center for Translational Research in Mission Bay, California.
The new Taube-Koret Center for Huntington's Disease Research will house a program designed to help prevent, treat, and cure
HD by 2020. The program will build on previous research by the center's investigators that has led to the development of
powerful assays for the identification of potential drug targets and a pipeline of molecular targets that could modulate HD
progression.
Also called Huntington's chorea and Woody Guthrie's disease, HD is an inherited, degenerative brain disorder. More than
100,000 Americans — and more than one million worldwide — have HD or are at risk of inheriting the disease from a parent.
"We believe that the focus and evolving new technologies of the HD Cure 2020 program provide a real chance to close in on a
cure," said Tad Taube, chairman of Taube Philanthropies and president of the Koret Foundation. "It is our hope that Gladstone's
depth of understanding about how Huntington's progresses, combined with a well-defined and integrated therapeutic screening
strategy, will enable real progress to be made toward treating or curing this devastating disease."
“Gladstone Institutes Establishes Taube-Koret Center for Huntington's Disease Research.” Koret Foundation Press Release
4/25/09.
Primary Subject: Health
Secondary Subject(s): Medical Research
Location(s): California
FC013233
©2009 Foundation Center
All rights reserved.
1 of 1
4/6/09 9:59 AM
Gladstone, Stanford share $3.9M to study Huntington’s - San Francisco Business Times:
10/30/09 8:47 PM
Members: Log in | Not Registered? Register for free extra services.
San Francisco Business Times - March 30, 2009
/sanfrancisco/stories/2009/03/30/story18.html
Friday, March 27, 2009
Gladstone, Stanford share $3.9M to study
Huntington’s
San Francisco Business Times - by Ron Leuty
Taube Philanthropies and the Koret Foundation have donated a total $3.9 million to the J. David
Gladstone Institutes and Stanford University to find a treatment or cure for Huntington’s disease.
The bulk of that money — $3.6 million from both Taube and Koret — is earmarked over three years
to the Gladstone Institutes in San Francisco.
The money will create the Taube-Koret Center for Huntington’s Disease Research at the Gladstone
Center for Translational Research at Mission Bay.
Dr. Steven Finkbeiner and Paul Muchowski will hire at least five new staffers to help translate their
basic research into promising drug candidates and — perhaps as soon as the next 12 months — ink a
partnership with a biopharmaceutical company like Merck & Co., Novartis or Elan.
That makes the gifts critical for crossing the so-called “valley of death” between basic research
funded largely by the National Institutes of Health and the point where a biotech or pharmaceutical
company would be interested in pursuing a drug.
“There’s a critical gap,” Finkbeiner said.
Huntington’s, a genetic disorder that strikes seven in every 100,000 people globally, is marked by
progressively uncoordinated, jerky body movements of the hands, feet, face and trunk and the loss of
some mental abilities. There is no cure.
At least one Bay Area company, Medivation Inc., has undertaken a Phase II trial of its drug,
Dimebon, as a potential Huntington’s treatment.
The other $300,000 — from Taube Philanthropies alone — will be used over two years by Dr. Frank
Longo, who leads Stanford’s department of neurology and neurological sciences. He is undertaking a
massive trial-and-error process testing thousands of potential drugs on mice.
The Taube-Koret Center is looking at small molecules that Finkbeiner and Muchowski hope will stop
or even roll back Huntington’s damage, Finkbeiner said.
Hladstone and Stanford are working toward a Huntington’s cure by 2020.
“That’s our collective light,” said Tad Taube, chairman of Taube Philanthropies in Belmont and
president of the Koret Foundation in San Francisco. “They hope and are optimistic that by 2020
there should be some results that lead to a positive drug therapy or a cure.”
[email protected] / (415) 288-4939
All contents of this site © American City Business Journals Inc. All rights reserved.
http://sanfrancisco.bizjournals.com/sanfrancisco/stories/2009/03/30/story18.html?t=printable
Page 1 of 1
Gladstone and partners receive $3.7 million for Huntington's disease research
10/30/09 8:52 PM
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Find more articles on taube-koret-center
Gladstone and partners receive $3.7 million for Huntington's
disease research
October 13th, 2009
The National Institutes of Health (NIH) has awarded a "Grand Opportunity" grant of $3.7 million to a
consortium formed with the Gladstone Institute of Neurological Disease (GIND) and the Taube-Koret
Center for Huntington's Disease Research to use stem cell technology to better understand Huntington's
disease (HD) and to develop potential therapies. The consortium comprises a partnership of five leading
Huntington's research laboratories at the University of Wisconsin, Massachusetts General Hospital, the
University of California at Irvine, Johns Hopkins and the Gladstone Institutes. The consortium will use
induced pluripotent stem (iPS) cell technology pioneered by Gladstone and Kyoto University's Shinya
Yamanaka, MD, PhD, to develop human neurons with Huntington's disease characteristics. iPS technology
enables stem cells to be generated from skin samples from adults and avoids the ethical issues surrounding
the use of fetal stem cells.
"One of the challenges of Huntington's (and many other neurological diseases) is that many of the potential
therapies that show promise in animal models are ineffective in people. We think that molecular differences
between mice and humans may be an important cause for this failure," said Steven Finkbeiner MD, PhD,
consortium co-leader and Director of the Taube-Koret Center for Huntington's Disease Research and
http://www.physorg.com/wire-news/16901103/gladstone-and-partners-receive-37-million-for-huntingtons-diseas.html
Page 1 of 8
10/30/09 8:52 PM
Associate Director of GIND.
"One of the promises of iPS technology is to be able to develop models from Huntington's disease patients
that can give us more detailed information about the disease and better predict how therapies could work in
humans," he said.
HD, which is also called "Huntington's chorea" and "Woody Guthrie's disease," is a devastating inherited,
degenerative brain disorder. More than 100,000 Americans and more than 10 times that number worldwide
have HD or are at risk of inheriting the disease from a parent.
iPS cells are generated by reprogramming adult cells from skin or other tissues. They are almost identical to
human embryonic stem cells with the ability to self-renew for long periods and to differentiate into all cell
lineages. More importantly, iPS cells can be generated from adult patients with genetically inherited and
sporadic diseases making it possible to study some diseases, such as Alzheimer's and Parkinson's disease,
for which the causes remain largely unknown.
"HD is caused by a single mutation, which provides an ideal paradigm to generate a panel of patientspecific lines," Finkbeiner explained. "This offers hope that these models can teach us why some patients
experience certain symptoms and why some family members develop symptoms later rather than sooner,
which then can potentially be used to develop treatments that can act before symptoms appear."
Finkbeiner added, "the convergence of a dedicated, collaborative group of committed investigators targeting
HD, the need for new treatments based on the root causes of the disease, and the emergence of powerful
new technologies herald a truly grand opportunity to make a real difference for those afflicted with
Huntington's."
Source: Gladstone Institutes
Ads by Google
New Stem Cell Treatment - High standard German clinic treats degenerative diseases. Request info www.xcell-center.com/StemCells
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http://www.physorg.com/wire-news/16901103/gladstone-and-partners-receive-37-million-for-huntingtons-diseas.html
Page 2 of 8
10/30/09 8:45 PM
Public release date: 13-Oct-2009
[ Print | E-mail |
Share ] [ Close Window ]
Contact: Valerie Tucker
[email protected]
415-734-2019
Gladstone Institutes
Gladstone and partners receive $3.7 million for Huntington's
disease research
NIH funds effort to develop disease models for pathogenesis and drug discovery
The National Institutes of Health (NIH) has awarded a "Grand Opportunity" grant of $3.7 million to a
consortium formed with the Gladstone Institute of Neurological Disease (GIND) and the Taube-Koret
Center for Huntington's Disease Research to use stem cell technology to better understand
Huntington's disease (HD) and to develop potential therapies. The consortium comprises a
partnership of five leading Huntington's research laboratories at the University of Wisconsin,
Massachusetts General Hospital, the University of California at Irvine, Johns Hopkins and the
Gladstone Institutes. The consortium will use induced pluripotent stem (iPS) cell technology
pioneered by Gladstone and Kyoto University's Shinya Yamanaka, MD, PhD, to develop human
neurons with Huntington's disease characteristics. iPS technology enables stem cells to be generated
from skin samples from adults and avoids the ethical issues surrounding the use of fetal stem cells.
"One of the challenges of Huntington's (and many other neurological diseases) is that many of the
potential therapies that show promise in animal models are ineffective in people. We think that
molecular differences between mice and humans may be an important cause for this failure," said
Steven Finkbeiner MD, PhD, consortium co-leader and Director of the Taube-Koret Center for
Huntington's Disease Research and Associate Director of GIND.
"One of the promises of iPS technology is to be able to develop models from Huntington's disease
patients that can give us more detailed information about the disease and better predict how
therapies could work in humans," he said.
HD, which is also called "Huntington's chorea" and "Woody Guthrie's disease," is a devastating
inherited, degenerative brain disorder. More than 100,000 Americans and more than 10 times that
number worldwide have HD or are at risk of inheriting the disease from a parent.
iPS cells are generated by reprogramming adult cells from skin or other tissues. They are almost
identical to human embryonic stem cells with the ability to self-renew for long periods and to
differentiate into all cell lineages. More importantly, iPS cells can be generated from adult patients
with genetically inherited and sporadic diseases making it possible to study some diseases, such as
Alzheimer's and Parkinson's disease, for which the causes remain largely unknown.
"HD is caused by a single mutation, which provides an ideal paradigm to generate a panel of patientspecific lines," Finkbeiner explained. "This offers hope that these models can teach us why some
patients experience certain symptoms and why some family members develop symptoms later rather
than sooner, which then can potentially be used to develop treatments that can act before
symptoms appear."
Finkbeiner added, "the convergence of a dedicated, collaborative group of committed investigators
targeting HD, the need for new treatments based on the root causes of the disease, and the
emergence of powerful new technologies herald a truly grand opportunity to make a real difference
for those afflicted with Huntington's."
###
http://www.eurekalert.org/pub_releases/2009-10/gi-gap100909.php
Page 1 of 2
10/30/09 8:45 PM
Dr. Finkbeiner's primary affiliation is with the Gladstone Institute of Neurological Disease where his
laboratory is located and all of his research is conducted. He is also associate professor of neurology
and physiology at the University of California, San Francisco.
Established in 1979, The J. David Gladstone Institutes is an independent, nonprofit biomedical
research organization that operates in close affiliation with the University of California, San Francisco
(UCSF). Gladstone is dedicated to the health and welfare of humankind through research into the
causes and prevention of some of the world's most devastating diseases. Gladstone is comprised of
the Gladstone Institute of Cardiovascular Disease, the Gladstone Institute of Virology and
Immunology, the Gladstone Institute of Neurological Disease, and the Gladstone Center for
Translational Research. More information can be found at: www.gladstone.ucsf.edu
About the Taube-Koret Center for Huntington's Disease Research.
The Center was established in 2009 with gifts from Taube Philanthropies and the Koret Foundation
for the sole purpose of identifying strategies and developing therapeutics to treat people with
Huntington's disease and related neurodegenerative diseases.
[ Print | E-mail |
Share ] [ Close Window ]
http://www.eurekalert.org/pub_releases/2009-10/gi-gap100909.php
Page 2 of 2
C. The Taube-Koret Center for Huntington’s Disease Research and HD Families
1. Huntington’s Disease Education. People who are newly diagnosed with HD often have
many questions. Nowadays, the internet is a common place to look for information, and the first
result from a Google search for Huntington’s disease is an entry from Wikipedia.
To help improve the quality and access to information about HD, we collaborated with Lee vanJackson, an author at Wikipedia, to develop and improve their entry. The result was an article
that got promoted to featured article. Less than 0.1% of articles in Wikipedia receive that
distinction, which is given by their editors based on the quality and accuracy of the article.
Wikipedia gets 65 million visitors a month, so we think this is a worthwhile investment of our
effort. The Taube-Koret Center is acknowledged as the source of the image that first appears as
the Wikipedia web page on HD opens.
2. Supporting Families with Huntington’s Disease. This year, members of the Center
participated in the annual “Walk for Hope” sponsored by the Huntington’s Disease Society of
America. The event brings HD families from all over Northern California to San Francisco, and
it gave us an opportunity to answer questions about the Center. The members of the Taube-Koret
Center are committed to showing our support for HD families and we raised funds from our
friends and family. Overall we raised nearly $6,000.
One of the most moving experiences of establishing the Taube-Koret Center has been the
outpouring of gratitude from the HD community for the hope that it offers patients and their
families. We have included an example of the sort of encouragement we receive from HD
families.
Michelle from Denver wrote:
Hello,
I just found out about the grant establishing the Taube-Koret Center for
Huntington's Disease Research and your involvement in this project. Your
research into the cause, treatment and dare I say, cure of this disease is the most
fabulous kernel of hope that I have come across on this subject. My family has
been affected for generations by HD and I just want to thank you for your efforts.
I am in no way able to put into words how much this means to me.
Thank you.
Shellie
34
VI. Appendix of Publications
35
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Author's personal copy
Available online at www.sciencedirect.com
High-content screening of primary neurons: ready for prime time
Aaron Daub1,2,3,*, Punita Sharma1,3,* and Steven Finkbeiner1,3,4,5
High-content screening (HCS), historically limited to drugdevelopment companies, is now a powerful and affordable
technology for academic researchers. Through automated
routines, this technology acquires large datasets of
fluorescence images depicting the functional states of
thousands to millions of cells. Information on shapes, textures,
intensities, and localizations is then used to create unique
representations, or ‘phenotypic signatures,’ of each cell.
These signatures quantify physiologic or diseased states, for
example, dendritic arborization, drug response, or cell coping
strategies. Live-cell imaging in HCS adds the ability to
correlate cellular events at different points in time, thereby
allowing sensitivities and observations not possible with fixed
endpoint analysis. HCS with live-cell imaging therefore
provides an unprecedented capability to detect
spatiotemporal changes in cells and is particularly suited for
time-dependent, stochastic processes such as
neurodegenerative disorders.
Addresses
1
Gladstone Institute of Neurological Disease, San Francisco, CA 94158,
United States
2
Medical Scientist Training Program and Program in Bioengineering,
University of California, San Francisco, 94143, United States
3
Taube-Koret Center for Huntington’s Disease Research and the
Consortium for Frontotemporal Dementia Research, San Francisco, CA
94158, United States
4
Program in Biomedical Sciences, Neuroscience Graduate Program,
Biomedical Sciences Program, University of California, San Francisco,
94143, United States
5
Departments of Neurology and Physiology, San Francisco, CA 94143,
United States
*
These authors contributed equally to this work.
Corresponding author: Finkbeiner, Steven
([email protected])
Current Opinion in Neurobiology 2009, 19:537–543
This review comes from a themed issue on
New technologies
Edited by Ehud Isacoff and Stephen Smith
Available online 4th November 2009
0959-4388/$ – see front matter
# 2009 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.conb.2009.10.002
Introduction
Biological research is entering a new era. Molecular
biology will be combined with novel engineering technologies and increased computational power to examine
living systems in exciting new ways.
www.sciencedirect.com
We are only beginning to understand the benefits — in
fact, the necessity — of studying biological systems with
large-scale unbiased screens [1]. Here we focus on highcontent screening (HCS) and considerations needed to
use this method effectively to study normal and disease
physiology in primary cells, currently the most biologically relevant models.
Why high-content screening?
HCS is a multiplexed, functional screening method based
on extracting multiparametric fluorescence data from
multiple targets in intact cells [2,3]. By temporally and
spatially resolving fluorescent readouts within individual
cells, HCS yields an almost unlimited number of kinetic
and morphometric outputs. HCS was developed to facilitate drug-target validation and lead optimization before
costly animal testing [4]. Today it is broadly used to
catalog cellular, subcellular, and intercellular responses
to multiple systematic perturbations and is applicable to
basic science, translational research, and drug development [5–8]. We distinguish HCS from high-content
analysis (HCA). HCA refers to extracting information
from image data. HCS is the automated, high-throughput
application of HCA.
HCS can fill a gap in academic research. Our growing
awareness of biological complexity underscores the need
to examine more than one variable at a fixed point in time.
Traditional low-throughput methods have severe limitations. In complex systems with many interacting genes,
measuring any single perturbation is not very informative.
In gain-of-function diseases, especially those with late
onset, a toxic protein effect may not be related to the
protein’s normal function. Unbiased screens therefore
identify potential pathogenic mechanisms faster and
more comprehensively, and the large datasets are less
prone to sampling error when analyzing stochastic events.
HCS assays capture cell-system dynamics and exploit
typically confounding cell-to-cell variability. For
example, a recent study used simultaneous tracking of
1000 proteins in lung carcinoma cells after drug treatment to detect time-dependent proteomic changes that
predicted individual cell fate [9]. Hypotheses in HCS
are used to design tracked variables and outputs that
maximize the likelihood of meaningful results. We
labeled mutant huntingtin and measured cell survival
to determine the role of inclusion bodies in Huntington’s
disease (HD) [10], a question unanswered by 10 years of
time-invariant, low-throughput approaches. HCS provides large datasets that unveil multiple, often nonintuitive, correlations that seed subsequent lines of thought.
538 New technologies
Table 1
Neuronal cell models for HCS
Property
Immortalized cells
Primary neurons
Embryonic stem cells
Current use in HCS
Ready for HCS
Source
Ubiquitous
Yes
Specific to cell line
Limited
Yes
Animal tissue
Specific brain
regions
Differentiation screens
No
Established or new cell line
From human or animal
embryos
Differentiation screens
No
Established or new cell line
From human or animal
fibroblasts (most common)
Freeze/Thaw
Proliferative capacity
Yes
Very High
Once
Post-mitotic
Yes
High
Murine better than human
Yes
High
Murine better than human
Differentiation required
Population type
In some cases
Clonal or
Heterogeneous
Durable
High
Low
Low
No
Heterogeneous
Yes
Clonal ! Heterogeneous
Yes
Clonal ! Heterogeneous
Sensitive
Limited
High
High
Sensitive
Medium to high
Medium
Medium to high
Sensitive
Medium to high
Medium
Medium to high
Limited human
source
Labor intensive
Limited human source
Dedifferentiation
Differentiation
Quality control
Differentiation
Quality control
Quantity
Quantity
Diversity of cell types
Diversity of cell types
Patient-specific screening
Handling
Ability to be engineered
Cost
Physiologic relevance
Major challenge for HCS
Major benefits for HCS
Physiologic
relevance
Quantity
Physiologic
relevance
Engineering
Induced pluripotent stem cells
The advantages and disadvantages of different cell types are summarized for their use in HCS. Adapted from Eglen et al. [10].
Thus, HCS accelerates the iterative process of classical
hypothesis-driven research [11].
Primary cells or cell lines?
Choosing the best cell type for a particular HCS assay is
challenging. Each option comes with inherent benefits
and drawbacks (Table 1). Primary cells provide highquality models for several reasons. They are more physiologically relevant than immortalized cell lines [12]. They
form synapses, thus incorporating significant neuromodulatory and trophic inputs. Neuronal physiology
and disease are also notoriously cell-type specific, and
neurons differentiated in vivo best recapitulate actual
neuronal subpopulations. One study found that hepatoma cell lines differ profoundly from primary hepatocytes, consistent with a shift from oxidative to anaerobic
metabolism, upregulation of mitotic proteins, and downregulation of typical hepatocyte functions [13]. High
attrition rates for candidate neuropharmacologics
(Figure 1) suggest even more striking differences in
neurons.
Most screenings have involved cell lines, but future
screenings will use primary and stem cells [14,15].
Embryonic stem (ES) cells can be differentiated into
motor neurons in large numbers [16]. Mouse and
human induced pluripotent stem (iPS) cells [17,18]
may better predict in vivo drug side effects and are
particularly attractive for disease-focused HCS [15–21].
For example, iPS cells from patients with spinal muscular atrophy differentiated into motor neurons retained
pathological deficits and drug responses consistent with
the disease. More work is needed to characterize iPS cell
lines, and better dedifferentiation protocols will avoid
viral vectors and oncogenes [21–24]. Ultimately, HCS
will place additional demands on dedifferentiation and
Figure 1
Success rates and millions of dollars spent from first-in-man clinical
trials to registration by therapeutic area. The overall clinical success rate
is 11% with 900 million dollars spent. However, when the analysis is
carried out by therapeutic area, big differences emerge, with central
nervous system (CNS) and oncology trailing far behind cardiovascular
diseases in the % success rate versus the dollars spent [54,55].
Screening of primary neurons Daub, Sharma and Finkbeiner 539
Table 2
Recommended fluorescent proteins
Fluorescent
protein a
Spectral
class
Excitation
peak (nm)
Emission
peak (nm)
Brightness b
Photostability c
pKa c
Association
state c
Filter set d
EBFP2
mCerulean
mEGFP
mEmerald
EYFP
mCitrine
mOrange2
TagRFP-T
mCherry
mKate2
Blue
Cyan
Green
Green
Yellow
Yellow
Orange
Orange
Red
Far-red
383
433/445
488
487
514
516
549
555
587
588
448
475/503
507
509
527
529
565
584
610
633
18
27/24
34
39
51
59
35
33
17 f
25
55
36
174
101 e
60
49
228
337
96
118
5.3
4.7
6.0
6.0
6.9
5.7
6.5
4.6
<4.5
5.4
Weak dimer
Monomer
Monomer
Monomer
Weak dimer
Monomer
Monomer
Monomer
Monomer
Monomer
DAPI/BFP
CFP
FITC/GFP
FITC/GFP
FITC/YFP
FITC/YFP
TRITC/DsRed
TRITC/DsRed
TxRed
TxRed
Physical properties for fluorescent proteins (FPs) in each spectral class.
Common abbreviation.
b
Product of the molar extinction coefficient and the quantum yield (mM cm)1.
c
Literature values except as noted.
d
Specialized applications may require choosing filter combinations that closely match the spectral profiles [56].
e
Measured in live cells with mEGFP (t1/2 = 150 s) as a control.
f
Averages of literature values. Adapted from Shaner et al. [27,30].
a
redifferentiation, including high efficiency and reproducibility. High throughput screens are already helping to
address these needs [25,26].
Despite technical challenges in isolating, culturing, and
transfecting primary neurons, their use decreases false
negatives and saves time and money wasted on pursuing
false positives. Until protocols are improved for differentiating ES and iPS cells into many neuronal cell types,
primary cells will remain the most physiologically
relevant model for large-scale screens.
HCS planning for live-cell imaging
Assay development encompasses selecting fluorophores
and proteins to label, choosing a transfection method,
migrating to 96-well or 384-well formats, upgrading automation, and completing preliminary experiments to
determine the robustness of readouts. None of these
steps are trivial. Migrating to a new format alone requires
re-optimizing labware, intra-well and inter-well cell
distributions, and transfection and image-acquisition protocols. During this time, a lab data management system
must also be integrated.
Fluorophores. Excellent reviews describe fluorophores for
HCA [27,28]. Notably, mKate [29] (now mKate2), mOrange2 and TagRFP-T [30], and EBFP2 [31] provide
improved brightness and photostability. After balancing
these features, the best options for live-cell imaging are
listed in Table 2. HCS allows up to four fluorophores with
sufficient spectral separation to avoid crosstalk. In the
future, more channels will be simultaneously acquired
with spectral imaging [32].
Transfection. Lipid-based methods, Ca2+-phosphate coprecipitation, viral infection, electroporation, and nucleowww.sciencedirect.com
fection all have benefits and drawbacks [33]. Primary
neurons pose additional challenges: they are susceptible
to transfection toxicities and plagued by low transfection
efficiency [34]. We found Lipofectamine 2000 (Invitrogen) best for efficiency, cell viability, and automation in
assays that require transfection after cell plating. With this
reagent, most transfection variability results from cellplating density, total mass of DNA, and ratio of transfection reagent to DNA. These factors must be optimized for
specific cells and DNAs. Reverse transfection with this
reagent now makes arrayed libraries of transfection-ready
DNA and siRNA a reality for HCS [35,36]. Although
biochemical assays utilizing large numbers of pooled cells
rely on high transfection efficiencies, this actually complicates microscopy-based screening of individual cells.
Identifying the same cell over time can be confounded by
cell movement. The researcher must strike a balance
between maximizing transfected cell number per field
and verifying the ability of image-analysis algorithms to
accurately track the cells.
Automation. Automation can be applied to each step of
HCS, including sample preparation, image acquisition
and analysis, quality-control measures, and data reporting.
Highly capable liquid-handling robots are increasingly
affordable for individual labs. They provide scalable
options for liquid aspiration and dispensing of large
and small volumes. Multiple high-content microscopy
systems are now available [37]. The most popular use
confocal or wide-field microscopes, and all offer hardware
autofocus, options for environmental control, and data
management and image-analysis software. They provide
out-of-the-box access to HCS for many scientific applications. Downsides to these solutions include expense,
proprietary image formats and algorithms, and the inability
to write ground-level scripts for true user customization.
Lab automation upgrades should be integrated early into
low-throughput assay development so quality measures are
determined from datasets reflecting the automation.
Robustness. Minimizing assay variability is essential for
HCS. The Z0 -factor is a useful way to estimate assay
quality and is calculated as a signal detection window
between positive and negative controls scaled by the
dynamic range [38]. It is an excellent measure of
single-output assays. Since HCS allows powerful multiparametric analyses with potentially hundreds of quantified parameters, a Z0 -factor can be calculated individually
for each parameter [39]. Alternatively, multivariate
criteria without informational losses due to averaging
can be instituted from the beginning [40]. In either
case, large datasets from positive and negative controls
should be used to determine assay quality before screening is initiated.
Data Management. HCS datasets are large. Live-cell imaging of a single 96-well plate with three channels and nine
images per well yields 30 GB of raw image data. A
reliable informatics infrastructure is needed. Data should
flow seamlessly from acquisition to storage on a server
where it can be accessed for offline image analysis.
Initially, hierarchical file structures can be used, but
optimal management should include a central database
for storing images and metadata that can be accessed by
both acquisition and image-analysis software [41].
Image analysis: the new bottleneck
Automation advancements have been valuable for HCS,
but extracting meaningful data from complex image sets
poses major challenges. These challenges arise from a
combination of microscopy and image-processing limitations and the need for new statistical tools. Neuroscience
poses particular difficulties due to complexities in
neuronal morphology and subcellular trafficking. Most
laboratories use image-analysis algorithms and manual
labor to analyze images, but the throughput is too low
for HCS. More robust and accurate image-analysis algorithms that can be applied to entire datasets with minimal
user intervention are necessary [42]. Zhang et al. published a neurite extraction algorithm [43] for HCS, and
multiple commercial packages quantify neuronal bodies
and neurites. To understand HCS informatics problems
more fully, we refer you to excellent reviews [44–46].
HCA uniquely provides multiplexed quantification of
individual cell features with temporal and spatial resolution. Image analysis comprises image segmentation and
cell tracking, extraction of individual cell features, and
data modeling and classification [46]. Image-analysis programs routinely measure size, shape, intensity, texture,
moments, and subcellular localization that, when combined, yield hundreds of parameters that characterize a
specific cellular phenotype [47]. For example, Loo et al.
used 300 unbiased parameters and a multivariate clustering algorithm to determine separation between drugtreated HeLa cells and controls [40]. The redundancy of
this parameter set was reduced, resulting in a minimal
phenotypic signature of the treated cells at various drug
dosages. With the signatures, a drug class could be predicted, and therapeutic windows could also be deduced.
The close relationship of neuronal morphology and functional state [48] holds promise for similar phenotypic
signatures to emerge from HCS focused on neuronal
development, physiology, and disease. For instance, an
HCS study of cultured rat primary cortical neurons identified Ab1–42 induced reduction in neurite outgrowth with
no apparent effect on neuron number, pointing to more
subtle morphological changes that can precede cell death.
These studies used fixed-cell imaging; however, the full
potential of HCS will be realized by imaging live cells
over time [49,50].
HCS and live-cell imaging of primary neurons:
putting it all together
HCS with live-cell imaging in relevant neuronal models
promises to elucidate physiologic and pathophysiologic
processes with unprecedented sensitivity and correlative
power. Live-cell imaging captures changes in cellular
phenotypes. Thus, previously static features are transformed into dynamic features where timed occurrences
and rates of change generate more informative phenotypic signatures. Imaging in live cells also permits causeand-effect relationships to be determined. We use this
novel approach to investigate pathogenic mechanisms of
neurodegenerative disorders, including HD, Parkinson’s
disease, amyotrophic lateral sclerosis, and frontotemporal
dementia. Our system (Figure 2) allows us to correlate
events in thousands of neurons to individual cell fates —
enabling us to determine if the events are adaptive,
pathogenic, or incidental to disease progression [51].
For instance, we used live-cell imaging in a primary
neuron model of HD to establish a mitigating role for
inclusion bodies [6] and reveal the interplay between
ubiquitin-proteasome system function and inclusion body
formation [52]. Such studies necessitate large sample
sizes and the ability to follow individual neurons over
time. They highlight the power of HCS, when coupled
with live-cell imaging, to reveal causal relationships in
biological processes.
Repeated measures of individual cells by automated
microscopy allow use of powerful statistical techniques,
such as Cox proportional hazards (CPH) analysis [53].
CPH integrates a user-defined number of parameters to
determine whether they explain time-to-event outcomes,
for instance cell survival. Much as in a prospective cohort
study, we allow cells, through stochastic diversification, to
‘take on’ certain traits and then retrospectively determine
how significant these traits are in predicting outcomes.
Our goal is to find robust, disease-specific phenotypic
Figure 2
Workflow of our second-generation high-content screening system for live-cell imaging.
Our system uses primary neurons from embryonic mice. A Microlab STARlet (Hamilton, Reno, CA) automated pipetting workstation prepares and
transfects cells in 96-well plates, which are then transferred to the plate stacker of a KiNEDx 4-axis robot (Peak Robotics, Colorado Springs, CO). The
plates are loaded onto an MS-2000 stage (Applied Scientific Instruments, Eugene, OR) fixed to a Nikon TE-2000 (Nikon, Melville, NY) microscope. The
robot and microscope are enclosed in an environmental chamber (InVivo Scientific, St Louis, MO) to enable around-the-clock imaging for six to seven
days. Wide-field images are acquired according to in-house scripts. At each time point, montage images are generated for each well and fluorophore
channel. Image analysis algorithms then extract cell-based information. Metadata generated from image acquisition and analysis flows into a central
database for data modeling, mining and classification.
signatures for screening small-molecule pharmacological
agents and genome-wide siRNA libraries. CPH takes
advantage of inherent cell-to-cell heterogeneity, and
the increased sensitivity resulting from temporal analysis
permits fewer cells to be analyzed. We therefore avoid
two main drawbacks of screening in primary cells —
decreased transfection efficiency and lack of cell homogeneity.
Conclusion
HCS is a technology with vast potential for academic
researchers and particularly neuroscientists. Large-scale
screens are strategically essential in understanding complex biological systems and gain-of-function diseases.
HCS can be applied to diverse assay types, depending
on the experimental conditions and labeled proteins.
Challenges still remain in image analysis and data
interpretation, and new statistical tools will be necessary
to analyze time-dependent processes of millions of cells
across thousands of conditions. Advances in HCS will
result from new microscopy techniques, such as spectral
imaging, better fluorescence proteins, and the maturation
of stem cell technology. Greater knowledge of which
proteins to probe for particular physiologic and disease
processes will increase HCS sensitivity. HCS with livecell imaging in primary neurons is practical and will help
answer some of the most elusive questions in neurobiology and related disease.
Acknowledgements
We thank the members of the Finkbeiner Lab for their generous support
and advice. We thank G. Howard and S. Ordway for editorial assistance and
K. Nelson for administrative assistance. This work was supported by the
Consortium for Frontotemporal Dementia Research, the Taube-Koret
Center for Huntington’s Disease Research, National Institutes of Health
(NIH) grants 2R01 NS039074 and 2R01045491 from the National Institutes
of Neurological Disorders and Stroke and 2P01 AG022074 from the
National Institutes of Aging and by the J. David Gladstone Institutes (to
S.F.). Support was also provided by the NIH-NIGMS UCSF Medical
Scientist Training Program (to A.C.D.) and the California Institute of
Regenerative Medicine (P.S.).
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Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2009/06/02/M109.016923.DC1.html
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 32, pp. 21647–21658, August 7, 2009
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Monoclonal Antibodies Recognize Distinct Conformational
Epitopes Formed by Polyglutamine in a Mutant
Huntingtin Fragment*□
S
Received for publication, March 24, 2009, and in revised form, May 4, 2009 Published, JBC Papers in Press, June 2, 2009, DOI 10.1074/jbc.M109.016923
Justin Legleiter‡§1,2, Gregor P. Lotz‡§, Jason Miller‡¶储3, Jan Ko**, Cheping Ng‡, Geneva L. Williams‡,
Steve Finkbeiner‡§储‡‡¶¶, Paul H. Patterson**, and Paul J. Muchowski‡§ §§¶¶4
From the ‡Gladstone Institute of Neurological Disease, Departments of §Neurology and ¶Chemistry and the Chemical Biology
Graduate Program, 储Medical Scientist Training Program, and Departments of ‡‡Physiology and §§Biochemistry and Biophysics,
University of California, San Francisco, California 94158 and the ¶¶Taube-Koret Center for Huntington’s Disease Research and
**Biology Division, California Institute of Technology, Pasadena, California 91125
Huntington disease (HD)5 is a fatal neurodegenerative disorder that is caused by an expansion of a polyglutamine (polyQ)
domain in the protein huntingtin (htt), which leads to its aggregation into fibrils (1). HD is part of a growing group of diseases
* This work was supported, in whole or in part, by National Institutes of Health
Grants R01NS047237 and R01NS054753 (to P. J. M.), P01AG022074 (to
S. F.), R01NS039074 (to S. F.), and R01NS045091 and R01NS055298 (to
P. H. P.). This work was also supported by the Hereditary Disease Foundation and the Cure Huntington’s Disease Initiative.
□
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Fig. 1 and Movies S1 and S2.
1
Supported by a postdoctoral fellowship from the Hereditary Disease
Foundation.
2
Current address: The C. Eugene Bennett Dept. of Chemistry, Wes Virginia
University, Morgantown, WV 26505.
3
Supported by the National Institutes of Health-NIGMS UCSF Medical Scientist Training Program and a fellowship from the University of California at
San Francisco Hillblom Center for the Biology of Aging.
4
To whom correspondence should be addressed: Gladstone Institute of Neurological Disease,1650 Owens St., San Francisco, CA 94158. Tel.: 415-7342515; Fax: 415-355-0824; E-mail: [email protected].
5
The abbreviations used are: HD, Huntington disease; polyQ, polyglutamine;
htt, huntington; PD, Parkinson disease; polyP, polyproline; AFM, atomic
force microscopy; GST, glutathione S-transferase; GFP, green fluorescent
protein.
AUGUST 7, 2009 • VOLUME 284 • NUMBER 32
that are classified as “conformational diseases,” which include
Alzheimer disease (AD), Parkinson disease (PD), the prion
encephalopathies, and many more (2– 4). The length of polyQ
expansion in HD is tightly correlated with disease onset, and a
critical threshold of 35– 40 glutamine residues is required for
disease manifestation (5). Biochemical and electron microscopic studies with htt fragments demonstrated that expanded
polyQ repeats (⬎39) form detergent-insoluble aggregates that
share characteristics with amyloid fibrils (6 – 8), and the formation of amyloid-like fibrils by polyQ was confirmed by studies
with synthetic polyQ peptides (9). Collectively, these studies
demonstrated a correlation between polyQ length and the
kinetics of aggregation. This phenomenon has been recapitulated in cell-culture models that express htt fragments (10 –12).
Although it is clear that proteins with expanded polyQ repeats
assemble into fibrils in vitro, recent studies have reported that
htt fragments can also assemble into spherical and annular oligomeric structures (13–16) similar to those formed by A␤ and
␣-synuclein, which are implicated in AD and PD, respectively.
While the major hallmark of HD is the formation of intranuclear and cytoplasmic inclusion bodies of aggregated htt (17),
the role of these structures in the etiology of HD remains controversial. For instance, the onset of symptoms in a transgenic
mouse model of HD follows the appearance of inclusion bodies
(18), while other studies indicate that inclusion body formation
may protect against toxicity by sequestering diffuse, soluble
forms of htt (10, 19, 20). Based on the direct correlation
between polyQ length, htt aggregation propensity, and toxicity
(6), it has been hypothesized that the aggregation of htt may
mediate neurodegeneration in HD. However, there is no clear
consensus on the aggregate form(s) that underlie toxicity, and
there likely exist bioactive, oligomeric aggregates undetectable
by traditional biochemical and electron microscopic approaches whose formation precedes disease symptoms.
Although identification of the one or more toxic species of htt
that trigger neurodegeneration in HD remains elusive, such
species might exist in a diffuse, mobile fraction rather than in
inclusion bodies (19). A thioredoxin-polyQ fusion protein was
recently reported to exhibit toxicity in a meta-stable, ␤-sheet-rich,
monomeric conformation (21), suggesting that polyQ can adopt
multiple monomeric conformations, only some of which may be
toxic. Consistent with such a scenario, molecular dynamic simuJOURNAL OF BIOLOGICAL CHEMISTRY
21647
Downloaded from www.jbc.org at UCSF Library & CKM, on April 8, 2010
Huntington disease (HD) is a neurodegenerative disorder
caused by an expansion of a polyglutamine (polyQ) domain in
the N-terminal region of huntingtin (htt). PolyQ expansion
above 35– 40 results in disease associated with htt aggregation
into inclusion bodies. It has been hypothesized that expanded
polyQ domains adopt multiple potentially toxic conformations
that belong to different aggregation pathways. Here, we used
atomic force microscopy to analyze the effect of a panel of antihtt antibodies (MW1–MW5, MW7, MW8, and 3B5H10) on
aggregate formation and the stability of a mutant htt-exon1
fragment. Two antibodies, MW7 (polyproline-specific) and
3B5H10 (polyQ-specific), completely inhibited fibril formation
and disaggregated preformed fibrils, whereas other polyQ-specific antibodies had widely varying effects on aggregation. These
results suggest that expanded polyQ domains adopt multiple
conformations in solution that can be readily distinguished by
monoclonal antibodies, which has important implications for
understanding the structural basis for polyQ toxicity and the
development of intrabody-based therapeutics for HD.
Antibodies Recognize Distinct Conformers of Huntingtin
6
C. Peters-Libeu, E. Rutenber, J. Miller, Y. Newhouse, P. Krishnan, K. Cheung, E.
Brooks, K. Widjaja, T. Tran, D. Hatters, S. Mitra, M. Arrasate, L. Mosquera, D.
Taylor, K. Weisgraber, and S. Finkbeiner, submitted for publication.
21648 JOURNAL OF BIOLOGICAL CHEMISTRY
while another (mEM48) ameliorates neurological symptoms in
a mouse model of HD (48).
Three of the antibodies examined in this study (MW1, MW2,
and MW7) modulate htt-induced cell death when co-transfected as single-chain variable region fragment antibodies
(scFvs) in 293 cells with htt exon 1 containing an expanded
polyQ domain (46). In these studies MW1 and MW2, which
bind to the polyQ repeat in htt, increased htt-induced toxicity
and aggregation (46). Conversely, MW7, which binds to the
polyproline (polyP) regions adjacent to the polyQ repeat in htt,
decreased its aggregation and toxicity (46). Interestingly, MW7
has also been shown to increase the turnover of mutant htt in
cultured cells and reduce its toxicity in corticostriatal brain
slice explants (49).
Given the difficulty in understanding which specie(s) of htt
exist and mediate pathogenesis in the putative toxic diffuse
fraction of neurons, we sought to rigorously characterize the
conformational specificity of a panel of anti-htt antibodies, the
best in situ probes currently available for distinguishing specie(s) of htt. We reasoned that if htt can adopt multiple conformations that mediate different aggregation pathways, then
anti-htt antibodies should differentially alter htt aggregation
pathways by stabilizing or sequestering the specific conformers
or aggregates they recognize. We therefore examined the
effects of various antibodies on mutant htt fragment fibril formation and stability by atomic force microscopy (AFM). Our
results are consistent with the hypothesis that monoclonal antibodies recognize distinct conformational epitopes formed by
polyQ in a mutant htt fragment.
EXPERIMENTAL PROCEDURES
Protein Purification—GST-HD53Q fusion proteins were
purified as described (52). Cleavage of the GST moiety by PreScission Protease (Amersham Biosciences) initiates aggregation. Fresh, unfrozen GST-HD53Q was used for each experiment. GST-HD53Q was centrifuged at 20,000 ⫻ g for 30 min at
4 °C to remove any preexisting aggregates before the addition of
the PreScission protease. MW series of antibodies were
obtained as described previously (39). 3B5H10 was purified as
described before (53).
Western Blot Analysis—For Western blotting analysis, purified GST-HD53Q proteins were incubated at 37 °C with shaking at 1400 rpm. Solutions were sampled at 0, 5, and 20 h after
the addition of PreScission Protease. Proteins and aggregates
were separated by SDS-PAGE and then transferred onto Protran BA85 nitrocellulose membranes (pore-size ⫽ 0.45 ␮m,
Whatman) by standard Western transfer techniques. The
membranes were incubated for 1 h at 37 °C with MW1, MW2,
MW3, MW4, MW5, MW7, MW8, or 3B5H10 at a dilution of
1:1000. The membranes were then incubated with horseradish
peroxidase-conjugated rabbit anti-mouse IgG or IgM (Jackson
ImmunoResearch) at a 1:5000 dilution for 1 h at room temperature. The horseradish peroxidase was detected using an ECL
Advance Western blotting Detection System (Amersham Biosciences), and the membranes were exposed to x-ray films.
Neuronal Culture, Transfection, and Immunocytochemistry—
Primary cultures of rat striatal neurons were prepared from
embryos (embryonic days 16 –18) and transfected with plasVOLUME 284 • NUMBER 32 • AUGUST 7, 2009
lations and fluorescence correlation spectroscopy experiments
with synthetic polyQ peptides indicate that polyQ domains can
adopt a heterogeneous collection of collapsed conformations that
are in equilibrium before aggregation (22–25).
Although biochemical, biophysical, and computational
approaches have yielded insight into the structures formed by
polyQ in vitro, whether such structures form in vivo remains
largely unknown. Indeed, determining the conformational state
of any misfolded/aggregated protein in situ and/or in vivo
remains a major technical challenge.
Toward this goal, antibodies have been explored as a potentially powerful tool for detecting specific conformations or multimeric states of aggregated proteins in situ. Antibodies specific
for amyloid fibrils often do not react with natively folded globular proteins from which they are derived, suggesting that such
antibodies recognize a conformational epitope (26, 27). Several
antibodies display conformation-dependent interactions with
amyloids, aggregation intermediates, or natively folded precursor proteins. For example, there are antibodies specific for
paired helical filaments of Tau (28 –31), of aggregated forms of
A␤ ranging from dimers to fibrils (32–34), and of native (35) or
disease-related (36) forms of the prion protein. Antibodies have
also been developed that are specific for common structural
motifs associated with amyloid diseases, such as oligomers (37)
and fibrils (38), independent of the peptide sequence of the
amyloid forming protein from which they are derived, suggesting the potential for a common mechanism of aggregation and
toxicity for these diseases.
With regard to htt, several antibodies (MW1, MW2, MW3,
MW4, MW5, IC2, and IF8), which are specific for polyQ
repeats, stain Western blots of htt with expanded polyQ repeats
much more strongly than htt with normal polyQ length (39, 40),
suggesting that these antibodies may recognize abnormal
polyQ conformations. Furthermore, these polyQ-specific antibodies have distinct staining patterns in immunohistochemical
studies of brain tissue sections (39). In one study, the affinity
and stoichiometry of MW1 binding to htt increased with polyQ
length, suggesting a “linear lattice” model for polyQ (41). This
model is supported by a crystal structure of polyQ bound to
MW1, which showed that polyQ can adopt an extended, coillike structure (42). However, an independent structural study
showed that the anti-polyQ antibody 3B5H10 binds to a compact ␤-sheet-like structure of polyQ in a monomeric htt fragment.6 These results clearly indicate that polyQ domains can
fold into at least two unique, stable, monomeric conformations
and suggest that the “linear lattice” model is not generally applicable to all polyQ structures.
Not only are antibodies useful for understanding what polyQ
structures exist in situ, especially in the diffuse htt fraction of
neurons, but antibodies and/or intrabodies may also have
potential as therapeutic agents. For example, several studies
showed that intrabodies reduce htt toxicity in cellular models
(44 – 49). Moreover, one intrabody (C4) slows htt aggregation
and prolongs lifespan in a Drosophila model of HD (50, 51),
fibrils and were compared using a t test. Aggregate populations
based on height were compared using a Spearman’s rank correlation performed with GraphPad Prism.
RESULTS
Anti-htt Antibodies Recognize a Variety of SDS-stable Oligomeric Species of HD53Q—All experiments in this study, with
the exception of the immunocytochemistry studies described
later, were performed with a mutant htt fragment that
expresses exon 1 with 53Q (HD53Q). HD53Q was purified
from Escherichia coli as a soluble fusion with glutathione
S-transferase (GST) (Fig. 1) (52). After purification, GSTHD53Q appeared non-aggregated as determined by AFM analysis and size-exclusion chromatography (data not shown). The
HD53Q fragment contains epitopes specifically recognized by
the panel of eight independent monoclonal anti-htt antibodies
(Fig. 1A) used in this study. MW1, MW2, MW3, MW4, MW5,
and 3B5H10 are specific for the polyQ domain. MW7 is specific
for the polyP domains. MW8 is specific for the last seven residues of the C terminus of htt exon 1.
Cleavage of a unique peptide sequence between the GST
moiety and HD53Q with a site-specific protease (PreScission
protease) released the HD53Q fragment, initiating aggregation
in a time-dependent manner as reported (7, 15). Western blots
of HD53Q were used to monitor cleavage 0, 5, and 20 h after the
addition of the protease (Fig. 1B). Before proteolytic cleavage
(t ⫽ 0 h), most antibodies specific for the polyQ domain
detected a prominent band of intact htt-GST fusion protein
that migrated at an apparent molecular mass of ⬃53 kDa, and a
less intense band that migrated at an apparent molecular mass
of a dimer of the fusion protein (⬃106 kDa). At later time
points, MW1 and MW3 recognized the intact fusion protein
and a band that migrated at a lower apparent molecular mass
that may represent monomeric HD53Q (⬃40 kDa). MW2 did
not recognize this ⬃40-kDa species after proteolytic cleavage
but did react with a larger, potentially dimeric species (⬃80
kDa) at later time points. MW4, MW5, and 3B5H10 recognized
a ⬃40-kDa species and a variety of SDS-stable bands of HD53Q,
some of which may be fragments of HD53Q. Only antibodies
that were not specific for the polyQ domain (MW7 and MW8)
recognized large aggregated forms of HD53Q that remained in
the wells of the gel, indicating that the polyQ epitopes recognized by these anti-polyQ antibodies are not accessible or
absent in large aggregates. Of the two antibodies that bound the
large aggregated form, only MW7 stained the ⬃40-kDa species
of HD53Q. These results indicate quite remarkably that six
independent anti-polyQ antibodies (MW1–5 and 3B5H10)
detect a variety of stable polyQ epitopes formed by HD53Q,
even after apparent htt denaturation in SDS. Two antibodies
against regions outside the polyQ stretch of htt exon1 (MW7
and -8) appear to expand the repertoire of recognizable htt
species further.
Anti-htt Antibodies Recognize a Variety of htt Species in Neurons in Situ—To determine if these anti-htt antibodies could
distinguish different htt epitopes in neurons, we applied immunocytochemistry to an established neuronal model (19) in
which primary striatal neurons are transiently transfected with
a mutant htt exon1 fragment fused to enhanced green fluoresJOURNAL OF BIOLOGICAL CHEMISTRY
21649
mids (6 –7 days in vitro) as described (10). Neurons were cotransfected with pGW1-Httex1-Q46 or 97-GFP in a 1:1 molar
ratio, using a total of 1– 4 ␮g of DNA in each well of a 24-well
plate. After transfection, neurons were maintained in serumfree medium. All immunocytochemistry was performed as
described (54). Cy3-conjugated secondary antibodies targeted
to the appropriate primary antibody were acquired from Jackson Immunolabs.
Atomic Force Microscopy—For experiments on monomeric
preparations, GST-HD53Q was incubated at 20 ␮M alone or
with anti-htt antibodies (MW1, MW2, MW3, MW4, MW5,
MW7, MW8, or 3B5H10) in a 1:1 ratio of protein to antigen
binding sites in buffer A (50 mM Tris-HCl, pH 7, 150 mM NaCl,
1 mM dithiothreitol). PreScission protease (4 units/100 ␮g of
fusion protein) was added at time zero to initiate GST cleavage
and aggregation. Samples were incubated at 37 °C with shaking
at 1400 rpm for the duration of the experiment. At time 1, 5, 8,
and 24 h after cleavage of the GST, a sample (5 ␮l) of each
incubation solution was deposited onto freshly cleaved mica
(Ted Pella Inc., Redding, CA) and allowed to sit for 1 min. The
substrate was washed with 200 ␮l of ultrapure water and dried
under a gentle steam of air. For experiments on preformed
fibrils, 40 ␮M solutions of HD53Q were incubated alone for 5– 6
h after the removal of the GST tag to allow the formation of
fibrils. Buffer or anti-htt antibodies (MW1, MW2, MW3,
MW4, MW5, MW7, MW8, or 3B5H10) were added so that the
final concentration of HD53Q was 20 ␮M, and the ratio of
HD53Q to anti-htt antigen binding sites was 1:1. These solutions were sampled immediately and 3 h after the addition of
the buffer or anti-htt antibody. Dose dependence studies of
fibril disaggregation by MW7 and 3B5H10 were performed
similarly, except that the ratio of HD53Q to antibody binding
site varied (10:1, 5:1, and 1:1) and solutions were sampled at 0, 1,
and 3 h after the addition of the antibodies.
Each sample was imaged ex situ using an MFP3D scanning
probe microscope (Asylum Research, Santa Barbara, CA).
Images were taken with silicon cantilevers with nominal spring
constants of 40 newtons (N)/m and resonance frequency of
⬃300 kHz. Typical imaging parameters were: drive amplitude
150 –500 kHz with set points of 0.7– 0.8 V, scan frequencies of
2– 4 Hz, image resolution 512 by 512 points, and scan size of 5
␮m. All experiments were performed in triplicate.
For in situ AFM experiments tracking individual fibrils, solutions containing preformed fibrils of HD53Q were allowed to
rest on mica until several fibrils were present on the surface.
Then, the substrate was washed with buffer A to remove proteins remaining in solution. The deposited fibrils were either
imaged in clean buffer as a control or in the presence of anti-htt
antibodies (2.5 ␮M final concentration). Images were taken with
V-shaped oxide-sharpened silicon nitride cantilevers with a
nominal spring constants of 0.5 N/m. Scan rates were set at 1–2
Hz with cantilever drive frequencies ranging from ⬃8 –12 kHz.
Statistics—All error bars in quantification of ex situ AFM
experiments (number of fibrils or oligomers per ␮m2) represent
the standard error of at least three independent experiments
and were compared using a t test. All error bars in quantification of in situ AFM experiments (change in fibril length) represent the standard error measured from at least eight individual
VOLUME 284 • NUMBER 32 • AUGUST 7, 2009
fibril formation, AFM images from
all incubations were analyzed by
counting the number of fibrils per
␮m2 (Fig. 3A). For this analysis, the
number of fibrils in the AFM images
for a given sample was divided by
the total area covered by the AFM
images. Fibrils were defined as
objects with a height larger than 5
nm and a length-to-width (aspect)
ratio ⬎3.
The AFM images of HD53Q
incubated alone displayed fibril
growth and an increase in fibril
abundance per unit area over the
24-h time course of the experiment
(Figs. 2 and 3A). At 1 h after removal
of GST, only a small number of
fibrils were present, and these
increased in number and grew from
several hundred nanometers to ⬃1
␮m in length at later time points.
The fibrils were ⬃6 – 8 nm tall and
12 nm wide (measured at half
height). Fibril formation in solutions of HD53Q co-incubated with
FIGURE 1. Anti-htt antibodies recognize a variety species of HD53Q in vitro and in situ. A, a schematic MW1, MW2, or MW4 altered
representation of the GST-htt exon 1 fusion protein with 53Q (HD53Q) shows a PreScission protease site aggregation similarly (Figs. 2 and
located between GST and the htt fragment (not drawn to scale) and the locations of epitopes for the antibodies
that were used in this study. B, Western blots of HD53Q after incubation with protease for varying times, 3A). After 1 h of incubation, the
probed with antibodies as labeled. The location of bands representing intact GST-HD53Q fusion protein at ⬃53 number of fibrils/␮m2 significantly
kDa is indicated by a green arrow. A band that migrated at an apparent molecular mass of a dimer of the fusion increased in the presence of these
protein (⬃106 kDa) is indicated by a red arrow. A blue arrow indicated the location of the wells of the gel where
larger HD53Q aggregates are observed. C, primary cultures of rat striatal neurons expressing a GFP-labeled antibodies. Despite this early
mutant htt-exon1 fragment with 97Q were analyzed by immunocytochemistry with antibodies as labeled.
increase in the number of fibrils,
MW1, MW2, and MW4 all had sigcent protein (GFP) (Fig. 1C). We compared the GFP signal, nificantly fewer fibrils than the controls at later time points.
which exhibited fluorescence in a diffuse cytoplasmic localiza- Co-incubation of HD53Q with MW8 also resulted in an initial
tion and in inclusion bodies, to that detected by specific anti- increase in the number of fibrils formed, with a significant
bodies. Consistent with the results with Western blots, only reduction compared with controls at later time points. HowMW7 and MW8 labeled large htt inclusion bodies based on ever, MW8 appeared to be the least effective antibody in reducco-localization with the GFP signal from htt. MW7 also stained ing fibril formation after 24 h. At early time points, the number
diffuse htt. PolyQ-specific antibodies did not stain inclusion of fibrils formed in the presence of MW3 and MW5 did not
bodies; rather, they recognized a diffuse population of htt pro- significantly differ from controls (Figs. 2 and 3A). By 24 h of
teins. All of these results were consistent with Western blots co-incubation, however, both MW3 and MW5 had signififrom Fig. 1B. This diffuse population might contain a heteroge- cantly inhibited HD53Q fibril formation. These results suggest
neous mix of monomeric conformers and soluble, oligomeric that MW1–5 may recognize one or more conformers of mutant
aggregates. The Western blot and immunocytochemistry stud- htt that are required for efficient fibril formation.
Unlike all other antibodies tested, MW7 and 3B5H10 comies suggest that these antibodies recognized different conformpletely prevented fibril formation of HD53Q over the entire
ers or oligomeric forms of HD53Q.
Anti-htt Antibodies Modulate htt Aggregation Differentially— time course of the experiment (Figs. 2 and 3A). Instead of fibrils,
We next used AFM to analyze the effects of anti-htt antibodies compact globular structures were observed in co-incubations
on HD53Q aggregation. Co-incubation experiments were per- of HD53Q with MW7 or 3B5H10. The height of individual
formed with monomeric preparations of HD53Q and each anti- globular structures was analyzed at all time points for HD53Q
body. Representative AFM images of aliquots removed from with or without MW7 or 3B5H10 (Fig. 3, B–D). Height was
solutions of HD53Q in the presence and absence of anti-htt chosen for analysis because it is the most accurately measured
antibodies after 1, 5, 8, and 24 h of incubation are shown in Fig. dimension in AFM, and it does not contain artifacts due to the
2. The concentration of HD53Q in all solutions was 20 ␮M, and finite shape and size of the probe tip. Fibrillar structures were
the ratio of antigen binding sites on the antibody to HD53Q was not included in the analysis with HD53Q alone. In incubations
1:1. In an effort to quantify the effect the anti-htt antibodies on of HD53Q alone, globular oligomers gradually increased in
JOURNAL OF BIOLOGICAL CHEMISTRY
21651
distributions under each condition
did not change over time based on
Spearman’s rank correlation coefficient (p ⬍ 0.001). That is, the size of
globular species formed upon co-incubation of HD53Q with MW7 was
the same at all time points, as was
true for co-incubations of HD53Q
with 3B5H10. This indicated that
globular species observed in these
co-incubations were different from
those formed in incubations of
HD53Q alone. Overall, the quantitative AFM analyses demonstrate that
antibodies specific for the polyQ
domain modulate HD53Q aggregation differentially and that antibodies
with specificity for other domains of
htt can also alter this process.
We next performed biochemical experiments to confirm the
AFM results, in which antibodies
were added to monomeric preparations of GST-HD53Q before initiating aggregation with protease.
20 ␮M HD53Q solutions were
sampled after 8 h for Western blot
analysis of aggregate formation by
staining with MW8 (supplemental
Fig. S1). Before addition of protease (t ⫽ 0 h), no aggregated HD53Q
was detected. Aggregated HD53Q
was detected in the wells for
HD53Q alone after 8 h of incubation; however, there appeared to be
fewer aggregates detected for
HD53Q incubated with MW1–
FIGURE 2. Anti-htt antibodies modulate htt aggregation differentially. Representative 2 ␮m ⫻ 2 ␮m AFM
images of 20 ␮M HD53Q incubated in the absence or presence of antibodies as labeled for 1, 5, 8, and 24 h after MW5 and MW8. For co-incubacleavage of the GST moiety. The ratio of antigen binding sites to HD53Q was 1:1. For HD53Q alone and with tions of HD53Q with MW7 and
MW1-MW5 or MW8, fibrillar structures (black arrows) appeared after 1–5 h of incubation. The number of fibrils 3B5H10, no aggregates were deincreased at 8 and 24 h. However, it appeared that there were more fibrils for HD53Q alone. For incubations of
HD53Q with MW7 or 3B5H10, no fibrillar structures appeared throughout the 24-h experiment. In incubations tected in the well, confirming the
with MW7, globular aggregates (blue arrows) around ⬃3.5 nm tall were the dominant species observed at all complete inhibition of aggregate
time points. For incubation with 3B5H10, smaller globular species (green arrows) ⬃2.5 nm tall were present at
all time points. Shown are representative AFM images. Quantification of the number of fibrils per ␮m2 in these formation by these antibodies.
Anti-htt Antibodies MW7 and
experiments is shown in Fig. 3.
3B5H10 Disassemble htt Aggregates—
height as a function of time (Figs. 2 and 3B). The oligomers To test the effects of different antibodies on pre-aggregated
observed at 1 and 24 h represented distinct populations of HD53Q, GST was first removed from HD53Q by proteolytic
HD53Q aggregates, because the height distributions were no cleavage, and then HD53Q was incubated for 6 – 8 h prior to
longer similar based on a Spearman’s rank correlation coeffi- addition of anti-htt antibodies. The preincubation resulted in a
cient (p ⫽ 0.37). MW7 and 3B5H10 appeared to stabilize dis- large population of HD53Q fibrils (time point 0 h in Fig. 4).
tinct globular structures, which likely are complexes of anti- After the initial incubation time, aliquots were deposited on
body and HD53Q, with globular structures observed for mica, dried, and imaged. Approximately 10 –20 fibrils were
co-incubations of HD53Q with MW7 being slightly larger than observed per 5 ␮m2 by ex situ AFM. These pre-aggregated
those observed with 3B5H10 (compare Fig. 3C with 3D). HD53Q solutions were divided into several aliquots to which
Whereas the mean height of HD53Q oligomers observed in buffer (for control) or antibodies were added to a final antigen
controls at 24 h was 5.3 ⫾ 1.65 nm, globular species observed binding site to HD53Q ratio 1:1, with a final HD53Q concenfrom co-incubations of HD53Q with MW7 and 3B5H10 were tration of 20 ␮M. Immediately after buffer or antibody were
4.4 ⫾ 1.76 nm and 2.6 ⫾ 0.74 nm tall, respectively. The height added, the HD53Q solutions were re-sampled and imaged to
verify that fibrils were still present
to obtain a time point 0-h measurement (Fig. 4). The solutions were
then incubated for an additional 3 h,
sampled, and imaged (Fig. 4). Preformed fibrils that were treated with
buffer, MW1, MW2, MW3, MW4,
MW5, or MW8 appeared to be stable, as the number of fibrils per ␮m2
was unchanged between 0 and 3 h
(Figs. 4 and 5A). Importantly, the
two anti-htt antibodies that prevented fibril formation (MW7 and
3B5H10) also significantly reduced
the number of preformed fibrils. At
the 1:1 ratio of antigen binding sites
to HD53Q, MW7 and 3B5H10 completely disaggregated preformed
FIGURE 3. Quantification over time of HD53Q aggregates in the absence and presence of anti-htt fibrils.
antibodies. A, the number of fibrils/␮m2 was calculated from AFM images of HD53Q incubated in the
We next evaluated the dose
absence and presence of anti-htt antibodies analyzed at 1, 5, 8, and 24 h of incubation. Compared with
dependence
of HD53Q fibril disagcontrol experiments of HD53Q alone, all of the antibodies significantly reduced the number of fibrils
formed at later time points. However, there was a significant increase in the number of fibrils formed after gregation by MW7 and 3B5H10
1 h for incubations with MW1, MW2, MW4, and MW8. MW7 and 3B5H10 completely inhibited the forma- (Fig. 5, B and C). Preformed fibrils of
tion of fibrils over the time course of the experiments. #, a significant increase (p ⬍ 0.05) in the number of
fibrils/␮m2 in comparison to HD53Q alone at the same time point (Student’s t test). * and denote HD53Q were treated with MW7 or
significant decreases (* ⫽ p ⬍ 0.01, ⫽ p ⬍ 0.05) in the number of fibrils/␮m2 in comparison to HD53Q 3B5H10 at an antigen binding site to
alone at the same time point (Student’s t test). 䉬 indicates that no fibrils were observed. The experiment HD53Q ratio of 1:10, 1:5, and 1:1.
was replicated six times, and the error bars represent standard error. B–D, height histograms for globular
structures observed in HD53Q alone (B) and with MW7 (C) or 3B5H10 (D) as a function of time. Whereas the Controls consisting of preformed
height of HD53Q oligomers gradually increased over time, both MW7 and 3B5H10 stabilized distinct HD53Q fibrils treated with buffer
globular structures that likely represent complexes of HD53Q and antibody. The legend applies to all
were also prepared. The final conpanels in the figures.
centration of HD53Q was 20 ␮M in
all experiments. These solutions were sampled at 0, 1, and 3 h
after the addition of buffer, MW7, or 3B5H10 and imaged with
AFM. Preformed fibrils present on mica were significantly
reduced at all ratios of antibody:htt, with a clear antibody dose
dependence for the disaggregation.
Tracking the Fates of Individual HD53Q Fibrils Exposed to
Anti-htt Antibodies in Situ—To further explore the stability of
preformed fibrils of HD53Q, we took advantage of the ability of
AFM in solution to track morphological changes of individual
fibrils as a function of time (Fig. 6 and supplemental movies S1
and S2). Preformed HD53Q fibrils were deposited on mica and
imaged continuously. Buffer (control) or anti-htt antibodies
were injected directly into the fluid cell of the AFM. This
allowed for the tracking of the fate of individual fibrils exposed
to different anti-htt antibodies. Fibrils that were treated with
buffer remained stable with no apparent change in length for
FIGURE 4. Ex situ AFM analysis indicates that the anti-htt antibodies
MW7 and 3B5H10 disassemble htt aggregates. Samples of HD53Q were over 300 min, verifying that the continual scanning of the AFM
incubated for 6 – 8 h after removal of the GST moiety to form a large pop- probe tip was not sufficient to invoke mechanical disruption of
ulation of fibrils. Then, buffer (as control), MW1-MW5, MW7, MW8, or
3B5H10 was added. The ratio of antigen binding sites to HD53Q was 1:1. fibril integrity (supplemental movie S1). Similarly, the majority
The solutions were sampled directly after the addition of buffer/antibod- of fibrils treated with MW1, MW2, MW3, MW4, MW5, or
ies (t ⫽ 0 h) and deposited on mica for AFM imaging. Fibrils (black arrows) MW8 did not exhibit large morphological changes for up to 300
were present in all samples at this time. The solutions were incubated for
3 h after the addition of buffer or antibodies and re-sampled. Fibrils (black min during continuous imaging (data not shown). Consistent
arrows) were still present in samples that had been treated with buffer, with the co-incubation experiments described above, fibrils
MW1-MW5 or MW8. However, fibrils were no longer detected in samples
exposed to MW7 and 3B5H10 gradually shortened in length
treated with MW7 or 3B5H10. Treatment with MW7 resulted in a large
population of globular species (blue arrows) that varied greatly in size with (supplemental movie S2). In the case of MW7, some fibrils
the majority of species ranging in height from 4 to 8 nm. Treatment with completely disappeared from the surface. We then quantified
3B5H10 resulted in globular species (green arrows) that were only ⬃2.5 nm
tall. Shown are representative 2 ␮m ⫻ 2 ␮m AFM images. Quantification of the change in length of individual fibrils as a function of time
(Fig. 7, A–I) by subtracting the length at time 0 from the length
the number of fibrils per ␮m2 in these experiments is shown in Fig. 5.
FIGURE 5. Quantification of the number of fibrils/␮m2 for pre-aggregated HD53Q treated with buffer or anti-htt antibodies. A, the number
of fibrils/␮m2 was calculated from AFM images of incubations of fibrillar
preparations of HD53Q taken immediately after (t ⫽ 0 h) and 3 h after the
addition of buffer, MW1-MW5, MW7, MW8, or 3B5H10. The ratio of antigen
binding sites to HD53Q was 1:1. For comparison, all bars are normalized to
the number of fibrils/␮m2 at t ⫽ 0 h for that sample. With the addition of
buffer (control), MW1-MW5, or MW8, there was no change in the number
of fibrils present after 3 h. With MW7 and 3B5H10, the number of fibrils was
significantly reduced, indicating that these antibodies were able to disassemble preformed fibrils. *, p ⬍ 0.001 (Student’s t test). Error bars represent standard error. B and C, the dose dependence of fibril disaggregation
was studied by quantitative analysis of AFM images of fibrillar preps of
HD53Q taken immediately after (t ⫽ 0 h), 1 h, and 3 h after the addition of
B, MW7 or C, 3B5H10. The ratio of antigen binding sites to HD53Q was 10:1,
5:1, and 1:1. For comparison, all bars are normalized to the number of
fibrils/␮m2 at t ⫽ 0 h for that sample. The disaggregation of fibrils by MW7
(B) and 3B5H10 (C) appeared to be dose-dependent. *, p ⬍ 0.01; **, p ⬍
0.001 (Student’s t test).
of the fibril at any given time. While the length of fibrils did not
vary as a function of time for HD53Q treated with buffer or
MW1-MW5 or MW8 (Fig. 7, A–F and H), all fibrils treated with
MW7 or 3B5H10 displayed a negative change in length. The
average rate of change in fibril length was calculated based on
measurements on individual fibrils under all conditions (Fig.
7J). Fibrils exposed to MW7 or 3B5H10 exhibited significant
rates of decreasing contour length compared with control
fibrils, with MW7 disaggregating fibrils at a faster rate than
3B5H10. The other antibodies did not differ significantly from
the buffer control. These results indicate that some, but not all,
anti-htt antibodies can disassemble fibrils in solution.
MW7 and 3B5H10 Disassemble Fibrils by Forming Different
Complexes with htt—Because MW7 and 3B5H10 both prevented fibril formation and destabilized preformed fibrils, we
next compared the height of the globular complexes formed by
htt with the antibodies when the antibodies were added to
monomeric or fibrillar HD53Q (Fig. 8). Globular species
formed after incubation of HD53Q in the absence of antibodies
were predominately 4 –5 nm tall with a large number of oligomers taller than 6 nm (Fig. 8A). In contrast, globular species
observed from co-incubations of MW7 or 3B5H10 with monoJOURNAL OF BIOLOGICAL CHEMISTRY
21653
FIGURE 6. Monitoring disassembly of single htt aggregates incubated
with MW7 or 3B5H10 by in situ AFM. Samples of HD53Q were incubated for
6 – 8 h after removal of the GST moiety to form a large population of fibrils.
These fibrils were deposited on mica and imaged using in situ AFM, which
allows for the tracking of the fate of individual fibrils as a function of time.
These fibrils were imaged in the absence or presence of anti-htt antibodies.
Fibrils appeared to be stable after treatment with buffer, MW1-MW5, or MW8
(location of stable fibrils indicated by black arrows). However, treatment with
MW7 or 3B5H10 caused fibrils to disaggregate and/or shorten in length (location of disaggregating fibrils indicated by green arrows). Scale bar represents
500 nm and is applicable to all images. See also supplemental movies S1
and S2.
DISCUSSION
Expanded polyQ repeats in htt have been postulated to adopt
multiple conformations, but it is unclear which conformations
may exist in neurons and are pathogenic. To study the existence
and effects of different htt conformations in neurons, appropriate conformational probes must be first be established and
characterized. The ability of antibodies to be used in situ makes
them attractive tools to measure htt conformations in neurons
and to ultimately determine their functional significance in HD
pathogenesis. We therefore set out to characterize the range of
htt conformations that can be detected by a panel of anti-htt
antibodies, including many that are specific for expanded
polyQ repeats. Because various htt conformations have been
linked to different aggregation pathways in vitro (15), we reasoned that different anti-htt antibodies may have disparate
effects on aggregation if the antibodies are recognizing different
htt conformational epitopes.
In this study we showed that a panel of antibodies (MW1–
MW5 and 3B5H10) that are all specific for polyQ sequences
detected different aggregated species of HD53Q in Western
blots and in cultured neurons. These antibodies also had widely
FIGURE 7. Quantification of change in length and rate of change of fibrils
treated with anti-htt antibodies. A–I, the change in length (⌬length) of individual fibrils imaged in the absence and presence of anti-htt antibodies was
tracked as a function of time as measured by in situ AFM. Fibril length
appeared stable with the addition of buffer (A), MW1-MW5 (B–F), or MW8 (H).
The length of individual fibrils steadily decreased after treatment with MW7
(G) or 3B5H10 (I). J, the average rate of change of fibril length for fibrils treated
with buffer (as control), MW1-MW5, MW7, MW8, or 3B5H10 was calculated,
showing that only MW7 and 3B5H10 caused a significant change in fibril
length (*, p ⬍ 0.01 with a Student’s t test).
meric HD53Q were only 3– 4 and 2–3 nm tall, respectively (Fig.
8, B and C). Interestingly, when MW7 was added to preformed
fibrillar HD53Q and allowed to completely disaggregate the
fibrils (3 h after addition MW7), the resulting oligomeric species were much larger than those observed following incubation
of this antibody with monomeric HD53Q (Fig. 8B). These globular structures were predominately 5– 6 nm tall with a large
number of globular structures taller than 6 nm. Based on a
Spearman’s rank correlation coefficient, this difference in size
was statistically significant, demonstrating that the final size of
the complex formed between MW7 and HD53Q can vary,
depending upon the initial aggregation state of HD53Q. This
result may indicate that MW7 can recognize both monomeric
and aggregated forms of htt, consistent with the immunocytochemical experiments and Western blot analysis (Fig. 1). Surprisingly, the globular structures observed from the complete
disaggregation (3 h after the addition of antibody) of preformed
HD53Q fibrils by 3B5H10 were precisely the same size as those
formed when 3B5H10 was added to monomeric HD53Q, based
on Spearman’s rank correlation coefficient. This indicates that,
in contrast to MW7, 3B5H10, which has been previously shown
to bind a monomer of htt,6 forms the same complex with
HD53Q regardless of its initial aggregation state (Fig. 8C). This
suggests that 3B5H10 is incapable of recognizing oligomeric
species of htt. Because MW7 apparently recognizes both aggregated and diffuse forms of htt, MW7 may be physically disrupting fibril structure by stabilizing a population of oligomeric
structures. However, as 3B5H10 only recognizes soluble, nonaggregated forms of htt, it may be tightly binding and sequestering a monomeric conformation of htt that is in direct equilibrium with fibril ends.
21655
FIGURE 8. Size analysis of aggregate observed with MW7 or 3B5H10
added to monomeric or fibrillar HD53Q. A, HD53Q oligomers (HD53Q
incubated alone) after 5 h of incubation were predominantly 4 –5 nm in
height with several as tall as 6 – 8 nm. B, when MW7 was incubated (added
at t ⫽ 0 h) with monomeric HD53Q (black diamonds), the height of globular aggregates formed after 5 h of co-incubation were predominantly
3– 4 nm tall, although there was a large portion of taller globular aggregates (shoulder on the right of the histogram). In contrast, when MW7 was
incubated with pre-aggregated fibrillar HD53Q (gray circles), globular
aggregates (conditions where fibrils disaggregated) observed when
imaged 3 h after addition of MW7 were much taller (4 –5 nm) in comparison to those formed by adding MW7 to monomeric HD53Q, with a larger
portion of aggregates being 5–10 nm tall. C, when 3B5H10 was incubated
(added at t ⫽ 0 h) with monomeric HD53Q (black diamonds), the majority
of globular aggregates observed after 5 h co-incubation were 2–3 nm in
height. Similarly, when 3B5H10 was incubated with pre-aggregated fibrillar HD53Q (gray circles), globular species (conditions where fibrils disaggregated) observed 3 h after the addition of 3B5H10 again were predominantly 2–3 nm tall.
varying effects on HD53Q aggregation, and some even disassembled preformed htt fibrils. MW1, MW2, and MW4 initially
increased fibril formation before suppressing it at later time
points. MW3, MW5, and MW8 slowed fibril formation. MW7
(polyP-specific) and 3B5H10 (polyQ-specific) completely prevented the formation of fibrillar structures. These two antibodies also destabilized preformed fibrils despite being specific for
different regions of htt. These results are consistent with the
hypothesis that expanded polyQ repeats can adopt multiple
conformation-specific epitopes that can be easily discriminated
by the immune system.
While compared with controls at later time points, all of the
polyQ-specific antibodies at least partially inhibited the formation of fibrils. MW1, MW2, and MW4 appeared to initially
boost fibril formation. This initial increase in aggregation is
consistent with previous reports that MW1 and MW2
enhanced aggregation, which was associated with increased
htt-induced toxicity, when they were expressed as scFvs in a
cellular model of HD (46). Among the polyQ-specific antibodies we tested, 3B5H10 appears to recognize a unique polyQ
conformation, because it was the only polyQ-specific antibody
to completely prevent fibril formation and destabilize preformed fibrils. Recent structural studies lend further support to
the notion that polyQ repeats can exist in stable conformations
of different structure. For example, a crystal structure of a
polyQ peptide bound to MW1 showed that polyQ can adopt an
extended, coil-like structure (42). However, an independent
structural study showed that 3B5H10 binds to a compact
␤-sheet-like structure of polyQ.6 We speculate that MW1 binding to a range of conformations on single-stranded polyQ may
initially catalyze the collapse of polyQ into aggregation-prone
structures, accounting for the early increase in fibril formation
for HD53Q incubated with MW1 compared with HD53Q incubated in buffer. However, as aggregation starts, the accumulation of MW1 antibody on each HD53Q molecule may eventually sterically hinder further aggregation, accounting for the late
attenuation in fibril formation for HD53Q incubated with
MW1 compared with HD53Q incubated in buffer. In contrast,
3B5H10’s binding to a compact, double-stranded structure of
polyQ may fully bury the edges of the polyQ conformation that
seeds aggregation, accounting for 3B5H10’s ability to completely block aggregation. Therefore, our results indicate
unequivocally that polyQ domains can sample at least two
unique monomeric conformations, but the polyQ domains are
likely to adopt other stable or meta-stable structures as well. For
example, fluorescence correlation spectroscopy experiments
and molecular dynamics simulations (23) indicate that polyQ
peptides can form a heterogeneous population of collapsed
structures in aqueous solution. In the absence of antibodies, htt
appears to be able to sample multiple conformations; however,
a collapsed conformation appears to be the dominant species as
detected by small-angle x-ray scattering.6
The antibodies MW7 (anti-polyP) and 3B5H10 (anti-polyQ)
both destabilized polyQ fibrils. However, the mechanisms
appear to be different, based on size analysis of the aggregate/
complex after disaggregation. Although MW7 and 3B5H10 are
specific for different regions of htt, there are other notable differences between the two antibodies. MW7 is an IgM while
7
M. Arrasate, J. Miller, E. Brooks, C. Peters-Libeu, J. Legleiter, D. Hatters, J.
Curtis, K. Cheung, P. Krishnana, S. Mitra, K. Widjaja, B. Shaby, Y. Newhouse,
G. Lotz, V. Thulasiramin, F. Sandou, P. J. Muchowski, M. Segal, K. Weisgraber,
and S. Finkbeiner, submitted for publication.
sequence to the C terminus of a polyQ peptide altered both
aggregation kinetics and conformational properties of the
polyQ tract (56). Flanking polyP sequences can also inhibit the
formation of ␤-sheet structure in polyQ peptides by inducing a
PPII-like helix structure, extending the length of the polyQ
domain necessary to induce fibril formation (57). Flanking
sequences in htt exon1 of various polyQ domain lengths modulate toxicity in yeast models, not only in cis, but also in trans
during aggregation (58, 59). Interestingly, the proline-rich
regions of htt exon1 reduced polyQ-related toxicity in these
studies (58, 59).
Protein interactions with the polyP sequence in htt may have
a major influence on the conformation of the adjacent polyQ
domain. Other studies have demonstrated that the polyP
domain of htt interacts with vesicle trafficking proteins (i.e.
HIP1, SH3GL3, and dynamin), which may lead to sequestration
of these proteins in inclusion bodies (61). By analogy, MW7
binding to the polyP domains of HD53Q may stabilize a conformation of the polyP domains that can, in turn, prevent the
necessary conformational changes in the polyQ domain that
lead to fibril formation. Such findings underscore the critical
importance of protein context in polyQ aggregation and aggregate stability. There are currently nine diseases related to polyQ
expansions in proteins that are broadly expressed, and the
nature of the proteins that contain the polyQ domain and their
associated pathologies differ substantially. That is, each mutant
polyQ protein causes a distinct neurodegenerative disease that
is associated with a different population of affected neurons. It
is likely that the protein context of the expanded polyQ
domains associated with each disease, and concomitant protein
interactions that vary due to protein context, could help
explain, at least in part, the striking cell specificity that is
observed in each disease.
Because MW7 and MW8 displayed similar behavior in recognizing aggregated forms of htt by Western blot analysis and
immunocytochemical studies of a HD neuronal model, it is
interesting that MW7 was much more effective in preventing
htt aggregation from monomers. This provides further evidence that the polyP region plays an important role in htt aggregation compared with the specific motif recognized my MW8.
Further, it appears that binding of an antibody to aggregated
forms of htt is not sufficient to disrupt aggregate stability as
MW8, which recognized aggregated forms of htt, was not able
to disaggregate preformed fibrils.
The AFM studies presented here are consistent with previous reports that MW7 suppresses aggregation and toxicity
when it is expressed as a scFv in cellular (46, 49) and Drosophila
(60) models of HD. Co-transfection of MW7 with mutant htt
exon 1 in corticostriatal rat brain slices increased the number of
healthy medium spinal neurons (49). Interestingly, expression
of the MW7 scFV promotes turnover of htt in cellular models of
HD (49). These results indicate that the ability of MW7 to prevent htt aggregation and destabilize htt fibrils, observed in this
study, may play a pivotal role in the ability of MW7 to reduce
cellular toxicity in a variety of HD models.
An important finding in the present study is that htt aggregation can be reversed by antibodies. There is a great deal of
interest in the use of antibodies and intrabodies as potential
3B5H10 is an IgG. MW7 recognizes aggregated and diffused
forms of htt by Western blot and immunocytochemistry,
whereas 3B5H10 does not recognize aggregates of htt. MW7
can block fibril formation from monomeric HD53Q by binding
to a specific conformer, resulting in a stable complex with a
narrow size distribution. However, MW7 can also bind aggregates and may physically bind to fibrils, disrupting their stability, and resulting in a different population of oligomeric complexes with a broader size distribution. Although we did not
observe any direct binding of MW7 to htt fibrils by in situ AFM,
this possibility cannot be ruled out because the ⬃8-min interval
between images may not be fast enough to capture such an
event. Although 3B5H10 also formed a stable complex with
monomeric HD53Q, it did not appear to bind to large htt aggregates observed by Western blot, biochemical, and immunocytochemical methods, suggesting that 3B5H10 disaggregates
fibrils by sequestering monomeric HD53Q and shifting the
equilibrium toward soluble forms of HD53Q.7 This notion is
supported by the finding that 3B5H10 forms stable complexes
of the same size regardless of whether it was added to monomeric or fibrillar HD53Q. Our AFM data also suggest that
3B5H10 is unable to bind oligomeric species of htt, consistent
with 3B5H10’s demonstrated conformational specificity for a
compact, double-stranded conformation of monomeric htt.6
Of the polyQ-specific antibodies used in this study, only
MW1 and 3B5H10 are IgG-type antibodies; the rest are IgM.
We attempted to control for this difference by calculating the
ratio of HD53Q to antibody in all experiments based on antigen
binding sites on the respective antibody. Antibody type did not
appear to have a clear impact on htt aggregation. For instance,
co-incubation of MW1 (IgG) or MW2 (IgM) with monomeric
HD53Q resulted in very similar aggregation profiles; whereas,
3B5H10 (IgG) prevented fibril formation. In regards to fibril
destabilization, antibody type did not appear to play a role,
because 3B5H10 reduced the number of fibrils even at a ratio of
five peptides per antigen binding site, which is analogous to the
dilution factor used to control for IgM type antibodies. 3B5H10
was still effective in destabilizing fibrils even at a dilution of
10:1, yet none of the polyQ-specific IgM type antibodies destabilized fibrils. The other IgG-type polyQ-specific antibody,
MW1, did not disaggregate fibrils even at a ratio of 1:1. Therefore, the effects of these antibodies on htt aggregation and
aggregate stability cannot be simply correlated to their antibody type. This notion is further supported by the observations
that MW7 (polyP-specific), which is an IgM type antibody, was
able to completely prevent fibril formation and destabilize preformed fibrils.
The ability of MW7 to prevent fibril formation and destabilize preformed HD53Q fibrils provides additional support for
the importance of the polyP domains in htt aggregation. More
broadly, it also indicates the critical importance of flanking
sequences on polyQ structure and aggregation. Studies on synthetic peptides revealed that the addition of a 10-residue polyP
Acknowledgments—We acknowledge Carl Johnson for insightful discussions and Gary Howard for editorial assistance.
Addendum—Consistent with the data we present here, a recent
study showed that a mutant htt fragment can misfold into distinct
amyloid conformations, and, depending on whether or not the polyQ
domain was exposed or buried in a ␤-sheet, the amyloids can be
either toxic or nontoxic, respectively (Nekooki-Machida et al. (63)).
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therapeutic agents to treat HD and other polyQ disorders (44 –
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aggregation and also destabilizing pre-existing aggregated
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Structure
Previews
required by the fact they are regulated by
small molecule binding. The somewhat
larger displacement proposed for TorS
(Moore and Hendrickson, 2009) is not
unreasonable since it binds a regulatory
protein, TorT, and the resulting proteinprotein interaction could perhaps generate
enough binding free energy to drive larger
changes in side chain and ridges-grooves
interactions. Second, transmembrane
signals in bacterial receptors must span
distances of 150 Å or more from the periplasmic ligand binding site to the cytoplasmic domain, and thus must be transmitted over a remarkably long distance.
To a first approximation, the H-bonding
framework of an a helix is incompressible
along the helix axis, ensuring that a piston
force pushing on one end of a helix will be
faithfully transmitted throughout the entire
helix length. By contrast, helix bends, rotations, or tilts can be more easily damped by
long-range helix flexibility over these
distances. Third, a small 1-2 Å displacement is large enough to directly regulate
the on-off switching of a kinase active
site, or trigger a larger structural rearrangement in a signal conversion module such as
the HAMP domain. Thus, it appears likely
that chemoreceptors and His kinase
receptors have retained the same piston
mechanism of transmembrane signaling
for good biophysical reasons.
Falke, J.J., Bass, R.B., Butler, S.L., Chervitz, S.A.,
and Danielson, M.A. (1997). Annu. Rev. Cell Dev.
Biol. 13, 457–512.
ACKNOWLEDGMENTS
Marina, A., Waldburger, C.D., and Hendrickson,
W.A. (2005). EMBO J. 24, 4247–4259.
Falke, J.J., and Hazelbauer, G.L. (2001). Trends
Biochem. Sci. 26, 257–265.
Hazelbauer, G.L., Falke, J.J., and Parkinson, J.S.
(2008). Trends Biochem. Sci. 33, 9–19.
Hughson, A.G., and Hazelbauer, G.L. (1996). Proc.
Natl. Acad. Sci. USA 93, 11546–11551.
Support provided by NIH R01 GM-040731.
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Milburn, M.V., Prive, G.G., Milligan, D.L., Scott,
W.G., Yeh, J., Jancarik, J., Koshland, D.E., Jr.,
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Polyglutamine Dances
the Conformational Cha-Cha-Cha
Jason Miller,1,2,3 Earl Rutenber,1 and Paul J. Muchowski1,4,*
1Gladstone
Institute of Neurological Disease
and Chemical Biology Graduate Program
3Medical Scientist Training Program
4Departments of Biochemistry and Biophysics, and Neurology
University of California, San Francisco, CA 94158, USA
*Correspondence: [email protected]
DOI 10.1016/j.str.2009.08.004
2Chemistry
While polyglutamine repeats appear in dozens of human proteins, high-resolution structural analysis of these
repeats in their native context has eluded researchers. Kim et al. now describe multiple crystal structures and
demonstrate that polyglutamine in huntingtin dances through multiple conformations.
There are 66 human proteins with a homopolymeric stretch of five glutamines or
more. The overrepresentation of polyglutamine (polyQ)-containing proteins in transcription-related processes suggests
a critical function for these repeats (Butland et al., 2007). At least 9 of these 66
proteins have a polyQ stretch that, when
expanded beyond a critical threshold,
misfold, aggregate, and cause neurode-
generative diseases. Although the structural basis that underlies the toxicity of
proteins with expanded polyQ repeats is
not clear, numerous laboratories have
hypothesized that a variety of misfolded
conformers, including monomers, oligomers, and fibrils, are the toxic culprits.
Into this debate enters the heroic crystallography feat of Kim et al. (2009). The
authors solved seven independent crystal
structures of a Q17-containing exon1 fragment of wild-type huntingtin (Httex1), a
multifunctional protein that, when mutated
in the polyQ stretch (>Q36), causes
a devastating neurodegenerative disorder
called Huntington’s chorea (chorea,
derived from Greek, describes the involuntary dance-like movements of Huntington’s patients). Reminiscent of the dancelike contortions of affected patients, the
Structure 17, September 9, 2009 ª2009 Elsevier Ltd All rights reserved 1151
Structure
Previews
wild-type polyQ stretch in
proteins in transcriptionHttex1 was surprisingly crysrelated processes suggest
tallized in multiple conforconformational flexibility is
mational contortions, most
especially important for these
convincingly forming a helices
processes? Another interesting
that varied from 1–15 polyQ
question raised by this study is
residues in length (Figure 1A).
whether the polyQ stretch
Although the structure of the
jumps between defined conforpolyQ sequences C-terminal
mations (Nagai et al., 2007;
to these helices was not
Tuinstra et al., 2008) or fluidly
always well resolved in the
flows through conformational
crystal structures, the authors
space. Because Kim et al.
suggest that these sequences
(2009) observed a wide range
likely adopted a random coil or
of conformations for the polyQ
extended-loop conformation.
stretch, one may assume that
The sequences surrounding
fluid conformational sampling
the polyQ stretch, the strucmay predominate. On the other
tures of which have also been
hand, it is hard to imagine how
contested, generally demonHttex1 crystallized if there was
strated less conformational
not at least a limited set of
flexibility. The 17 amino acids
conformations that the polyQ
N-terminal to the polyQ
stretch samples.
sequence in Httex1 (N17) were
From the perspective of
invariably a-helical in every
neurodegenerative diseases,
structure that was solved,
it is interesting to speculate
consistent with structure prewhether the conformational
Figure 1. Conformational Cha-cha-cha: X-Ray Crystallography
Reveals That PolyQ and Polyproline Adopt Multiple Conformations
diction programs and circular
sampling of space by the polyQ
in Htt Exon1
dichroism (CD) spectroscopy
region increases, decreases,
17
(A) Four a helices are shown. Each extends from the N-terminal residue of the N
(Atwal et al., 2007). C-terminal
or stays the same when the
region (Met 371-Phe 387) of Htt Exon1 (blue) and continues as a helix for a varying
to the polyQ region is a polypolyQ stretch expands into
number of glutamine residues (cyan = 5, yellow = 9, magenta = 12, and salmon =
15). Glutamines C-terminal to the a-helical structured residues may adopt other
proline stretch, which formed
the mutant (>Q36) range. For
conformations, including random coil, extended loop, or b strand.
a classic proline helix, also as
example, while the structure
(B) Five of the seven observed polyproline regions of Htt Exon1 are shown
suggested by CD experiments
of fully aggregated fibrillar
superimposed on their five C-terminal residues. Note that all demonstrate
a proline-helix conformation, but some are kinked while others are extended.
(Darnell et al., 2007). InterestpolyQ in many proteins is
This figure was generated using PyMol (www.pymol.org).
ingly, the polyproline stretch
composed predominantly of
was either straight or kinked
b sheet, Kim et al. (2009) did
(Figure 1B), suggesting that
not observe this conformation
this sequence in huntingtin may itself by recognizing that the structures of the in the crystal structures of wild-type Httex1.
N17 and polyproline regions are relatively Does this conformation exist among the
exhibit some conformational flexibility.
Before interpreting and digesting this constant, while the polyQ region varied.
portions of polyQ in Httex1, whose electron
The conformational flexibility of the density was unresolved by Kim et al.
wealth of structural information, it is worth
reflecting upon this astounding technical polyQ region in Httex1 raises several inter- (2009)? Alternatively, does this b strand/
feat. Since the huntingtin gene was esting questions about the functional role sheet conformation emerge only in monocloned more than sixteen years ago, of these stretches. For example, of the 66 mers of mutant Httex1 (>Q36) or only upon
numerous laboratories have attempted human proteins with R Q5 stretch, aggregation? Notably, there is evidence
and failed to determine the structure of approximately half (including all proteins that polyQ in monomeric mutant Httex1
various huntingtin fragments. Indeed, associated with polyQ-expansion disease) can adopt a collapsed b sheet conformathis is the first crystal structure of any demonstrate significant length polymor- tion (Nagai et al., 2007). Further, while
polyQ-containing (>Q10) protein in its phisms in the polyQ stretch in the normal a wide range of aggregate morphologies
native protein context. The fact that the human population. Are polyQ stretches for mutant Httex1 species exists (Wacker
polyQ stretch in the Httex1 fragment only conformationally flexible in the et al., 2004), it is unknown whether a single
adopts different conformations within the proteins with length polymorphism? A conformation of polyQ in monomeric
asymmetric unit of each crystal that the protein that must be functional within mutant Httex1 leads to a single type of
authors solved, combined with the fact a wide range of polyQ lengths may have aggregated species or, alternatively,
that Kim et al. (2009) analyzed diffraction to consequently demonstrate significant whether a single monomeric conformation
from 30 crystals and obtained structures conformational flexibility in this region. can produce all observed aggregate
for seven crystal forms, speaks to the How does this conformational flexibility species. While a recent study with monodaunting nature of the entire effort. The assist in cellular functions? For example, clonal antibodies strongly implicated the
authors demonstrated significant insight does the overrepresentation of polyQ existence of multiple monomeric polyQ
1152 Structure 17, September 9, 2009 ª2009 Elsevier Ltd All rights reserved
Structure
Previews
conformations in mutant Httex1 (Legleiter
et al., 2009), Kim et al. (2009) provide direct
structural evidence of this, suggesting that,
at least in principle, each conformation
may seed a unique type of aggregate.
Even if we fully understood how different
monomeric conformations of polyQ in
Httex1 lead to various aggregated species,
the questions of which species contribute
to neurotoxicity and how they do it are still
open questions. Kim et al. (2009) propose
two general mechanisms for polyQ-mediated toxicity. By one mechanism, the
expanded polyQ stretch adopts a de novo
conformation that mediates toxicity or is
the precursor to a toxic species. By the
second mechanism, the expanded polyQ
stretch is largely unstructured but presents
a very large linear binding surface for
proteins with a polyQ affinity. The structures
from Kim et al. (2009) leave open the possibility that either mechanism may be correct.
The study by Kim et al. (2009) also
provides interesting insight into the relationship between the polyQ stretch and the
surrounding sequences in Httex1. The N17
sequence, which is important for the
subcellular localization of Httex1 and is highly
conserved (100% similarity) in all vertebrate
species (Atwal et al., 2007), was invariably
a-helical in all solved structures. Interest-
ingly, the N17 a-helix appears to ‘‘bleed’’
into the C-terminal adjacent polyQ region,
causing 1–15 glutamines to participate in
the extended a helix (Figure 1A). The structural data from Kim et al. (2009) also hint
that the polyQ repeat in Httex1 may be influenced by the C-terminal polyproline region.
Because Httex1 may be more aggregation
prone (and possibly more toxic) when the
polyQ region is more compact, it is interesting to speculate whether the polyproline
region may serve both its known function as
a protein-interaction domain and a lessappreciated function as a protector against
polyQ conformational collapse. Indeed, this
structural explanation may account for why
Httex1 with the polyproline stretch is less
toxic and aggregation prone than Httex1
without this sequence (Bhattacharyya
et al., 2006; Darnell et al., 2007; Duennwald
et al., 2006). Thus, N17 and polyproline
dance partners may keep the Cha-chacha-prone polyQ stretch of huntingtin in step, and thereby prevent a toxic
conformational stumble.
Bhattacharyya, A., Thakur, A.K., Chellgren, V.M.,
Thiagarajan, G., Williams, A.D., Chellgren, B.W.,
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Darnell, G., Orgel, J.P., Pahl, R., and Meredith, S.C.
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Duennwald, M.L., Jagadish, S., Muchowski, P.J.,
and Lindquist, S. (2006). Proc. Natl. Acad. Sci.
USA 103, 11045–11050.
Kim, M.W., Chelliah, Y., Kim, S.W., Otwinowski, Z.,
and Bezprozvanny, I. (2009). Structure 17, this
issue, 1205–1212.
Legleiter, J., Lotz, G.P., Miller, J., Ko, J., Ng, C.,
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Keeping an Eye on Membrane Transport by TR-WAXS
Jeff Abramson1,* and Vincent Chaptal1
1Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles 90095, CA
*Correspondence: [email protected]
DOI 10.1016/j.str.2009.08.003
In this issue of Structure, Andersson et al. apply time-resolved wide angle X-ray scattering (TR-WAXS) to
follow light-induced conformational changes for both bacteriorhodopsin and proteorhodopsin and probe
real-time dynamics at atomic resolution.
Membrane transport proteins perform
a multitude of cellular reactions, including
energy and signal transduction, regulation
of ion concentrations, and transport of
metabolites into the cell and noxious substances out. Altered membrane protein
function underlies many human diseases,
and thus, a deeper understanding of membrane protein structure and dynamics
remains a critical objective for basic and
medical research. It is well established
that membrane transport proteins require
distinct temporally regulated structural
rearrangements to carry out their biological functions. However, structural details
of these dynamic macromolecules have
only been studied as snapshots of individual static (and, in most cases, stable)
conformations. What is lacking is the ability to capture the transition between these
conformations and to probe the role of
specific domains and ligands in the process as they proceed through the membrane.
In recent years, our knowledge of
membrane protein structure has dramatically increased, providing unforeseen
Structure 17, September 9, 2009 ª2009 Elsevier Ltd All rights reserved 1153
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 7, pp. 4398 –4403, February 13, 2009
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Single Neuron Ubiquitin-Proteasome Dynamics
Accompanying Inclusion Body Formation in
Huntington Disease*□
S
Received for publication, August 14, 2008, and in revised form, December 9, 2008 Published, JBC Papers in Press, December 10, 2008, DOI 10.1074/jbc.M806269200
Siddhartha Mitra‡§1, Andrey S. Tsvetkov‡¶2, and Steven Finkbeiner‡§¶3
From the ‡Gladstone Institute of Neurological Disease, San Francisco, California 94158 and the §Biomedical Sciences Program,
Medical Scientist Training Program, and ¶Neuroscience Program, Departments of Neurology and Physiology, University of
California, San Francisco, California 94143
The accumulation of mutant protein in intracellular aggregates is a common feature of neurodegenerative disease. In
Huntington disease, mutant huntingtin leads to inclusion body
(IB) formation and neuronal toxicity. Impairment of the ubiquitin-proteasome system (UPS) has been implicated in IB formation and Huntington disease pathogenesis. However, IBs
form asynchronously in only a subset of cells with mutant huntingtin, and the relationship between IB formation and UPS
function has been difficult to elucidate. Here, we applied singlecell longitudinal acquisition and analysis to monitor mutant
huntingtin IB formation, UPS function, and neuronal toxicity.
We found that proteasome inhibition is toxic to striatal neurons
in a dose-dependent fashion. Before IB formation, the UPS is
more impaired in neurons that go on to form IBs than in those
that do not. After forming IBs, impairment is lower in neurons with IBs than in those without. These findings suggest
IBs are a protective cellular response to mutant protein mediated in part by improving intracellular protein degradation.
Huntington disease (HD)4 is a progressive incurable neurodegenerative disorder caused by the expansion of a polyglutamine (polyQ) stretch in the N-terminal end of the huntingtin
(htt) protein above a threshold length of ⬃36 (1). The deposition of polyQ-expanded aggregated mutant htt in inclusion
bodies (IBs) is a hallmark of HD, and IBs are found in human
post-mortem samples, transgenic mouse brain, and cell-culture
models (2). The accumulation of ubiquitinated proteins in IBs
* This work was supported, in whole or in part, by National Institutes of Health
Grants R01 2NS039074 and R01 NS045191 from the NINDS (to S. F.) and
Grant P01 AG022074 from the NIA. This work was also supported by the
Taube Family Foundation Program in Huntington Disease, and the Gladstone Institutes (to S. F.). The costs of publication of this article were
defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
□
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Fig. S1.
1
Supported by NIH-NIGHMS UCSF Medical Scientist Training Program and a
fellowship from the UC-wide adaptive biotechnology (GREAT) program.
2
Supported by the Milton Wexler fellowship from the Hereditary Disease
Foundation.
3
To whom correspondence should be addressed: Gladstone Institute of Neurological Disease, 1650 Owens St., San Francisco, CA 94158. Tel.: 415-7342508; Fax: 415-355-0824; E-mail: [email protected].
4
The abbreviations used are: HD, Huntington Disease; UPS, ubiquitin-proteasome system; IB, inclusion body.
has implicated the ubiquitin-proteasome system (UPS) in the
pathogenesis of HD, amyotrophic lateral sclerosis, Parkinson
disease, and polyQ-mediated disorders (3).
The UPS is a major pathway of intracellular protein degradation. After a series of three reactions, each catalyzed by a different set of enzymes, ubiquitin, a 76-amino acid polypeptide,
forms an isopeptide bond with the amino group of lysine residues on substrate proteins. Several lysine residues within ubiquitin are sites for more ubiquitin additions. Once a protein
accumulates four or more ubiquitins, it is efficiently targeted to
the proteasome for degradation. The proteasome binds polyubiquitinated substrates and hydrolyzes ubiquitin isopeptide
bonds, releasing ubiquitin moieties before degrading substrate
proteins through chymotrypsin-like, trypsin-like, and post-glutamyl peptidase activities (3).
Increased polyubiquitin levels and changes in ubiquitin linkages accompany the accumulation of UPS substrates in the
brains of HD patients and transgenic mice and in cellular HD
models (4). UPS substrates accumulate throughout the cell in
polyQ models, even before IB formation (5, 6). This has added
to the confusion over whether polyQ expansion leads to toxicity
through direct impairment of proteasomal degradation. Proteasomes have been reported to cleave polyQ stretches efficiently (7), inefficiently (8), or essentially not at all (9). In vivo,
polyQ-dependent degeneration occurs with no detectable proteasome inhibition (10, 11) or is tightly linked to it (12, 13). The
inability of some studies to detect UPS impairment in HD models may be due to the limited sensitivity of conventional
approaches to identify cell-to-cell variations in UPS function.
The relationship between IB formation and UPS function has
been difficult to determine. Protein turnover in cells with IBs is
evidently reduced and accompanied by the accumulation of
cellular proteins (14 –16); HEK293 cells containing mutant htt
IBs have a greater degree of UPS impairment than those without IBs (5). Proteasome subunits and heat shock proteins colocalize with IBs, but it is unclear if this colocalization facilitates
protein delivery or unfolding at the mouth of active proteasomes, or if it harms proteasome function by sequestering
essential cellular machinery (18). Some IBs are relatively static
(8, 25), but the proteins in others are dynamically exchanged
with cytoplasmic and nuclear pools (19, 20).
UPS function is critical to cellular homeostasis. Deletion of
one of the two inducible polyubiquitin genes in mice leads to
lower intracellular ubiquitin levels in germ cells and hypothaVOLUME 284 • NUMBER 7 • FEBRUARY 13, 2009
Ubiquitin-Proteasome Dynamics in Huntington Disease
lamic neurons. These same populations undergo cell-cycle
arrest and hypothalamic neurodegeneration, respectively (22,
23). Cell lines expressing mutant huntingtin accumulate ubiquitinated proteins and undergo cell-cycle arrest in G2/M (5). In
neurons, UPS impairment may lead to cell death through an
accumulation of signals for apoptosis, a decrease in NF-␬B signaling, sensitization to other toxic stimuli, remodeling of synapses, retraction of neurites, or other unidentified mechanisms
(24). The effect of UPS impairment depends on cell type and
cell cycle, and the relationship between UPS impairment and
striatal neuronal survival is largely unknown.
Diffuse species of mutant htt induce IB formation and neuronal death in a protein concentration-dependent manner (2).
IB formation delays neuronal death, suggesting that IB formation helps neurons cope with toxic diffuse mutant htt. Whether
the effect of IB formation on survival is mediated through UPS
function has been difficult to determine. IB formation and neuronal death occur asynchronously in overlapping but distinct
subsets of neurons that express mutant htt. The observation
that IB formation is not required for UPS impairment also complicates population analysis (6, 26).
To explore this problem, we applied single-cell analysis. We
tracked single neurons over their entire lifetimes, gaining spatial and temporal resolution while simultaneously monitoring
IB formation, UPS inhibition, and neuronal toxicity.
EXPERIMENTAL PROCEDURES
Plasmids—mRFP (27), pCS2-Venus (28), and pEGFPCL1(5), pGW1-GFP, pGW1-httQ72-eGFP, pGW1-mRFP (2)
have been described. pGW1-httQ72-CFP was generated from
pGW1-httQ72-eGFP. pGW1-mRFPu (mRFPu) was generated
by subcloning mRFP1 from pcDNA3.1(⫹) into pEGFP-CL;
mRFP1-CL1 was then subcloned into pGW1. pGW1-GFPu was
constructed by excising EGFP-CL1 from pEGFP-CL1 and
inserting it into pGW1. pGW1-Venus-CL1 (Venusu) was generated by subcloning Venus from pCS2-Venus into
pcDNA3.1(⫹). The stop codon from Venus was removed and
replaced by the sequence AGATCTCG. The CL1 sequence (5)
was introduced at the 3⬘-end of Venus. Venus-CL1 was then
subcloned into pGW1. pCS2-UbG76V-Venus (UbG76V-Venus)
was generated by PCR of UbG76V from UbG76V-GFP (29).
Cell Culture—Striata from rat embryos (E17–18) were dissected, dissociated, and plated on 24-well tissue-culture plates
(5.8 ⫻ 105/well) coated with poly-D-lysine and laminin (BD Biosciences, San Jose, CA) as described (2, 30). The cells were
grown in 1 ml of modified neuronal culture medium (NCM).
Cells were fed every 5–7 days by replacement with equal measures of conditioned and fresh neuronal culture medium.
Transfection, Pharmacology, and Colocalization—Primary cultures were transfected 5–7 days in vitro with combinations of
pGW1-GFPu and pGW1-mRFP, pGW1-mRFPu, and pGW1GFP, pGW1-Venusu, or pCS2-UbG76V-Venus, and pGW1-CFP,
and pGW1-httQ72-eGFP, pGW1-mRFPu, and pGW1-BFP, or
pGW1-httQ72-CFP and pYFP-LMP2 in a 1:1 or 1:1:1 molar ratio
with 2– 4 ␮g of total plasmid DNA per well. Our transfection
protocol was described (2). MG132 (Sigma-Aldrich), epoxomicin (Boston Biochem, Cambridge, MA), and Bafilomycin A1
FEBRUARY 13, 2009 • VOLUME 284 • NUMBER 7
(Sigma-Aldrich) were added in 1 ml of conditioned NCM per
well 12– 60 h after transfection.
Colocalization of fluorescence was calculated using Metamorph. Briefly, images of fluorescence from CFP-htt, ubiquitin
staining, or LMP-YFP were analyzed, and pixels were classified
as “positive” if their intensity was 3⫻ greater than background
pixels. The fraction of positive pixels for CFP-htt IBs that overlapped positive pixels of ubiquitin staining or LMP-YFP fluorescence was calculated with Metamorph for (n ⫽ 20 neurons).
Live-Cell Imaging and Analysis—Images of cells were
obtained with a robotic microscope system as described (2, 32).
Briefly, the imaging was performed with a Nikon TE300
inverted microscope with a long working-distance Nikon 20⫻
(NA 0.45) objective. Stage movements and focusing were performed using a Proscan II stage controller (Prior Scientific,
Rockland, MA). Samples were illuminated with a 175 watt
Xenon Lambda LS illuminator (Sutter Instruments, Novato,
CA). Blue, green, and red fluorescent protein (BFP, GFP, and
RFP, respectively) images were captured using an 86014 beamsplitter and 350/50⫻; 465/30m, 480/40⫻; 517/30m and 580/
20⫻; 630/60m fluorescence filters respectively. CFP, Venus,
and RFP images were captured using a 86006 beamsplitter and
420/35⫻; 470/30m, 500/20⫻; 535/30m, and 580/20⫻;
630/60m fluorescence filters (Chroma Corp, Rockingham, VT).
Algorithms for plate registration, stage movements, filter
movements, focusing, and acquisition were generated with
Metamorph imaging software (Molecular Devices, Sunnyvale,
CA). Images were analyzed manually using Metamorph software. Fully automated acquisition and analysis algorithms have
been created (Media Cybernetics, Bethesda, MD). Survival
analysis was performed with the Statview software package
(SAS Institute, Cary, NC); t tests for comparisons of means and
two-sample Kolmogorov-Smirnov tests for comparisons of distributions were performed with Prism (Graphpad Software,
San Diego, CA).
RESULTS
Longitudinal Live-Cell Monitoring of UPS Function in Primary Neurons—To monitor dynamic changes in protein degradation in live cells, we used a unique high-throughput image
acquisition platform (2, 32) and fluorescent protein substrates
of UPS degradation. We used fluorescent proteins with the CL1
peptide fused to the C terminus (34) or a non-hydrolyzable
ubiquitin moiety (UbG76V) fused to the N terminus (35) to target them to the UPS for degradation. These destabilized fluorescent proteins were transfected into primary neurons and
fluorescence in individual cells was monitored for hours or days
to detect changes in the degradation of UPS substrates. To control for nonspecific changes in transcription and protein handling while monitoring cell survival (2), we co-transfected and
tracked the fluorescence of unmodified fluorescent proteins in
the same cells.
Destabilized Fluorescent Proteins Accumulate after Proteasome Inhibition in Primary Neurons—Fluorescence intensity in
live cells is an accurate indicator of the amount of fluorescent
protein within the cell (2). Fluorescence levels in primary striatal neurons of a destabilized form of enhanced GFPu (5) (Fig.
1A), monomeric mRFPu (27) (Fig. 1C), or two forms of the
4399
FIGURE 1. Levels of proteasome reporters increase after inhibition of proteasome. A, after transfection with GFPu and mRFP, striatal neurons were
treated with 50 ␮M MG132 for 12 h. GFP fluorescence (A) and the ratio of
GFPu/mRFP fluorescence (B) both increase after treatment relative to control.
C, after transfection with mRFPu and GFP, striatal neurons were treated with
50 ␮M MG132 for 12 h. The mRFPu/GFP ratio is significantly greater than the
control (p ⬍ 0.02). D, after transfection with Venusu and CFP, striatal neurons
were treated with 2 ␮M epoxomycin (solid lines) or vehicle (broken lines) for
10 h. Both mean change in Venusu/CFP fluorescence (D) and single-cell distributions of Venusu/CFP fluorescence (E) are increased relative to control
(p ⬍ 0.05, p ⬍ 0.05). F, after transfection with UbG76V-Venus and CFP, striatal
neurons were treated with 2 ␮M epoxomycin for 10 h. Both mean change in
UbG76V-Venus/CFP fluorescence (F) and single-cell distributions of UbG76VVenus/CFP fluorescence (G) are increased (p ⬍ 0.05, p ⬍ 0.01). Experiments
were repeated twice with over 50 cells analyzed in each condition.
enhanced yellow fluorescent protein variant Venus (UbG76VVenus and Venusu) (28) (Fig. 1, D–G) increased after treatment
with proteasome inhibitor, even when changes in fluorescence
of unmodified spectrally distinct fluorescent proteins in the
same cells was controlled for (Fig. 1, B, C, E, G). The significant
and rapid increase in fluorescence of these reporters from low
baseline levels after proteasome inhibition in primary neurons
is in agreement with previous work in cell lines (5, 6, 26). Addition of the CL1 peptide or UbG76V degron to fluorescent proteins did not cause the proteins to aggregate when they were
expressed in neurons, unlike observations from cell lines (36).
Inhibiting Autophagy Does Not Result in Accumulation of
UPS Reporters in Primary Neurons—To ensure that these
destabilized proteins were targeted primarily to the UPS for
degradation, we used Bafilomycin A1 (BafA1) to inhibit autophagy. BafA1, a vacuolar ATPase inhibitor, prevents autophagosome-lysosome fusion and causes the accumulation of substrates targeted for macroautophagy (37). BafA1 caused a rapid
accumulation of the membrane-bound form of microtubuleassociated protein 1 light chain 3 (LC3-II) and was toxic to
primary neurons (Fig. 2, A and D), but BafA1 did not increase
levels of UPS reporters (Fig. 2, B and C).
FIGURE 2. Limited interaction between the UPS and autophagic pathways in neurons. A, 24 h after cotransfection with UbG76V-Venus and CFP,
striatal neurons were treated with vehicle or 50 nM BafA1. BafA1 treatment
caused a significant amount of toxicity above control (p ⬍ 0.03, top line).
Mean UbG76V-Venus/CFP ratio (B) and the distribution of the single-cell
changes in UbG76V-Venus/CFP (C) in these cells did not increase above control
in 20 h after BafA1 addition. D, neurons or HEK293 cells (E) were treated with
BafA1 or epoxomycin, followed by Western blotting with an LC3 antibody.
While BafA1 caused accumulation of LC3-II in both neurons and HEK293 cells,
epoxomycin increased LC3-II levels only in HEK293 cells. Unlabeled lanes in E
are lysates from cells transfected with LC3.
Proteasome Inhibition Does Not Change LC3-II Levels in Primary Neurons—Proteasome inhibition can increase flux
through the autophagic pathway in some cells (13). To determine if autophagic activity could be confounding fluorescent
reporter measurements of UPS function, we examined the
activity of the autophagic pathway after proteasome inhibition.
The level of LC3-II is commonly used as a surrogate for the
number of autophagosomes and flux through the macroautophagic pathway. After treatment with epoxomicin, primary
neurons showed no change in LC3-II levels (Fig. 2D), though as
seen in previous reports, LC3-II accumulated in HEK293 cells
(Fig. 2E).
UPS Reporter Fluorescence Demonstrates a Graded Response
to Proteasome Inhibition—Having validated the use of destabilized fluorescent proteins as reporters of UPS function in primary neurons, we then examined the nature of their response to
varying levels of proteasome impairment. We co-transfected
mRFPu and GFP into primary striatal neurons and treated the
cells with increasing doses of the proteasome inhibitor MG132.
Though fluorescent UPS reporters have been reported to relocalize to IBs, we found that mRFPu fluorescence remained diffuse in striatal neurons after proteasome inhibitor treatment
(6). As early as 2.5 h after addition of MG132, reporter fluorescence increased in proportion to the amount of MG132 added
(Fig. 3A), and reporter fluorescence continued to increase over
time (Fig. 3B). Thus, in primary neurons, the increase in fluorescence of these proteins faithfully reports the extent of proteasome impairment (5, 6).
By monitoring individual cells treated with MG132 over
days, we determined the effect of increasing proteasome inhibition on the survival of primary striatal neurons. When the
dose of MG132 increased, neurons died faster (Fig. 3C). These
VOLUME 284 • NUMBER 7 • FEBRUARY 13, 2009
UPS Impairment Decreases after
IB Formation—To determine if IB
formation improves or worsens
UPS function, we examined UPS
reporter fluorescence in neurons
during and after IB formation. We
compared these measurements to
UPS reporter fluorescence in an
otherwise matched cohort of neurons that did not form IBs over the
same interval. To again reduce
FIGURE 3. Inhibition of proteasome activity is toxic in a dose-dependent fashion. A, UPS reporter fluores- potential biases introduced by using
cence shows a dose-dependent response to MG132 treatment. MG132 at the indicated doses was added to
striatal neurons 24 h after transfection with mRFPu and GFP. The change in mRFPu/GFP ratio over the first 2.5 h IB formation as a selection criterion,
after MG132 administration is shown. B, UPS reporter fluorescence continues to increase up to 12 h after the neurons from each cohort were
addition of MG132. Note the difference in scale with A. Measurements from 80 ␮M were excluded due to
noticeable toxicity. C, MG132 is toxic to neurons in a dose-dependent fashion. The same neurons shown in A matched for the length of time they
and B were observed with the risk of death as shown. Longitudinal analysis was repeated twice on different lived in vitro. We found that neutransfections, with n ⬎ 50 for each treatment in each experiment.
rons that formed IBs had significantly smaller increases in UPS
reporter fluorescence (Fig. 4, E and
neurons demonstrated a proportional relationship between F), indicating that less UPS impairment occurs in cells after IB
proteasome impairment and the accumulation of UPS sub- formation than in cells that did not form IBs.
strates; similarly, there was a proportional relationship between
IB Formation Improves Neuronal Survival—To determine if
proteasome impairment and neuronal toxicity.
this reduced UPS impairment changes neuronal survival, we
Longitudinal Live Cell Detection of UPS Function in a Pri- compared the survival of neurons that we analyzed for UPS
mary Neuronal Model of HD—We then examined a primary function. When we examined matched cohorts of neurons
striatal model of HD (2, 30) and prospectively followed visual transfected with httex1-Q72-GFP, mRFPu, and BFP that formed
markers of UPS function, IB formation, and neuronal viability or did not form IBs, those cells that formed IBs survived longer
in single cells. This model reproduces key features of HD, (Fig. 4, G and H). This finding agrees with previous results
including neuronal subtype specificity (30) and polyQ length- showing that neurons survive longer if they form IBs (2).
dependent toxicity (2, 30). To induce the HD disease phenotype
in this model, we transiently transfected an N-terminal htt frag- DISCUSSION
ment with 72 glutamines fused to GFP (httex1-Q72-GFP). We
By applying a high-throughput single-cell longitudinal imagsimultaneously introduced mRFPu and BFP into the same neu- ing platform to a neuronal model, we were able to examine the
rons to monitor UPS impairment and cell viability, respectively. events in the cellular pathogenesis of HD with improved sensiVirtually all IBs in this model stain with ubiquitin and colocalize tivity and temporal resolution. Through the use of spectrally
with proteasome subunits (supplemental Fig. S1). From series distinct fluorescent species, we simultaneously monitored neuof images of individual neurons, we quantified single-cell ronal viability, htt IB formation, and intracellular protein degchanges in UPS reporter fluorescence over the lifetimes of cells radation. We found that neurons that form IBs have increased
expressing the httex1-Q72-GFP protein (Fig. 4A).
UPS impairment preceding IB formation and less UPS impairWould UPS function differ in neurons that do and do not ment after IB formation than cells that do not form IBs. Though
form IBs? By reviewing images from our longitudinal analy- tonic UPS inhibition is toxic to primary striatal neurons, neusis experiments, we identified neurons that had or had not rons that formed IBs survived better than those that did not.
formed an IB at some point over the course of the experi- These results support a model in which IB formation reflects a
ment. From these two groups, we then chose neurons that beneficial cellular response to mutant protein, mediated in part
were from the same well of the culture dish to form two by restoring UPS function.
cohorts based on IB formation. To reduce potential biases
Though multiple pathways of intracellular protein degradaintroduced by using IB formation as a selection criterion, we tion may handle aggregation-prone protein, we found that
included only neurons that had already lived the same length some proteins are likely targeted primarily to the UPS for degof time in vitro. We then monitored UPS reporter fluores- radation. In our experience with fluorescent UPS reporters, we
cence in neurons before, during, and after IB formation and found little evidence that they are routinely degraded by autocompared it to that in the cohort of age-matched neurons phagy. Though it is clear that autophagy modulates the turnthat did not form IBs.
over and toxicity of aggregation prone-proteins, the addition of
UPS Impairment Precedes IB Formation—Those cells that the CL1 or UbG76V degron does not cause fluorescent proteins
would go on to form IBs had significantly larger increases in to aggregate in neurons. This discrepancy with other reports in
UPS reporter fluorescence before IB formation, both in the sin- cell lines may be due to lower expression levels in neurons after
gle-cell distribution of reporter fluorescence (Fig. 4B) and in transient transfection.
mean reporter fluorescence (Fig. 4C). This relationship was
The finding that proteasome inhibition is not sufficient to
independent of the time at which IBs formed (Fig. 4D).
change the flux through the autophagic pathway in primary
4401
proteins normally targeted to the
UPS to other pathways of intracellular protein degradation. In both
yeast and mammalian cells, misfolded and aggregation-prone proteins may be targeted to different
intracellular compartments depending on the availability of ubiquitin
(31). Differential localization may
be one component of targeting proteins to the autophagic pathway of
protein degradation, which has
been implicated in the clearance of
aggregation-prone protein, including mutant htt (17, 21). If expanded
polyQ tracts impair the ability of the
proteasome to degrade other cellular proteins (9) or if ubiquitination is
inadequate due to ubiquitin sequestration by IBs, shifting polyQ degradation from the UPS to the autophagic pathway could effectively
increase the flux of other proteins
through the UPS.
A second possibility that is not
mutually exclusive is that IB formation is part of a cellular program to more efficiently degrade
protein through the UPS. The
recruitment of chaperones and
proteasomal machinery to intracellular inclusions varies based on
protein and cell type (19, 25).
Though the IBs in our primary
FIGURE 4. IB formation and UPS function in primary neurons. A, GFP-htt, BFP, and mRFPu were imaged over neuronal model are long-lived,
the course of days to follow htt IB formation, UPS impairment, and neuronal survival. Single-cell distributions with fewer than 2% disappearing
(B) or population means (C) of the change in mRFPu/GFP fluorescence in the interval preceding IB formation at
54 h. The increase in mRFPu/GFP ratio was higher in neurons that went on to form IBs (p ⬍ 0.05, p ⬍ 0.05). D, in before the neuron that contains
a parallel experiment, single-cell distributions of the change in mRFPu/GFP fluorescence in the interval preced- them dies (2), a small proportion
ing IB formation at 76 h also show higher UPS impairment in those neurons that will go on to form IBs (p ⬍ 0.05). of cells can clear IBs, and a detailed
After 54 h, single-cell distributions (E) or population means (F) show a greater increase in mRFPu/GFP fluorescence in those cells that did not form IBs (p ⬍ 0.05, p ⬍ 0.05). The survival of those neurons that formed htt IBs longitudinal analysis of these cells
at 18 h (G) or 27 h (H) was better than the survival of neurons that survived at least that long but never formed will likely be informative.
IBs (p ⬍ 0.01, p ⬍ 0.03). Longitudinal analysis was repeated twice in different experiments with over 300 cells
Previous work suggested that IB
analyzed in each experiment, with n ⬎ 30 for each cohort.
formation safely sequesters more
neurons also highlights possible differences between mamma- toxic forms of mutant htt to improve neuronal survival. This
lian neurons and other model systems. The difference in behav- study suggests two additional mechanisms by which IB formaior of the autophagic pathway in mammalian neurons may be tion might contribute to improved cell survival after IB formadue to a difference in constitutive activity (39). While most tion. First, we found that tonic UPS inhibition is toxic and that
non-neuronal cells upregulate autophagy after 24 h of starva- IB formation is associated with a relative improvement in UPS
tion, neurons do not in vivo (40) or in vitro5 even after longer function. Thus, IB formation may partially restore longevity by
starvation periods. The finding that the deletion of essential improving UPS throughput and consequently lowering the
autophagic machinery results in a neurodegenerative pheno- overall cellular burden of misfolded proteins. A second but
type points to a critical role in neuronal function and survival related possibility is suggested by reports that transient suble(38, 41).
thal proteasome inhibition can induce cells to adapt in ways
Though it remains unclear how IB formation is functionally that protect them against further insults (33). Transient protealinked to an improvement in UPS function, one possibility is some inhibition might trigger a cell-wide adaptive response in
that IB formation is a step toward shunting aggregation-prone neurons that may involve coordinated changes in molecular
chaperones and protein turnover pathways. If so, such an
5
adaptive response may be important in a variety of neurodegenA. Tsvetkov and S. Finkbeiner, unpublished observations.
VOLUME 284 • NUMBER 7 • FEBRUARY 13, 2009
erative diseases that result from misfolded intracellular
proteins.
Acknowledgments—We thank R. Kopito for pEGFP-CL1, V. Rao for
pGW1-GFPu, A. Miyawaki for pCS2-Venus, M. Mancini for pYFPLMP2, and N. Dantuma for UbG76V-GFP; members of the Finkbeiner
laboratory for insightful discussions; S. Ordway and G. Howard for
editorial assistance; and K. Nelson for administrative assistance. The
animal care facility was supported in part by a National Institutes of
Health Extramural Research Facilities Improvement Project (C06
RR018928).
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4403
FIGURE S1. Inclusion bodies are ubiquitinated and co-localized with proteasomes. (A)
Striatal neurons were transfected with CFP-htt (middle panel), LMP2-YFP (proteasome subunit)
(left panel), fixed after 48 h, and stained with an antibody against ubiquitin (right panel). (B)
Colocalization of CFPhtt fluorescence with ubiquitin staining and with proteasomes indicated by
LMP2-YFP fluorescence. Colocalization for IBs/ubiquitin: 92.7%+/-7.6; for IBs/proteasomes
73.8%+/-15.8. The bar is 50 ⎧m.
14
[Autophagy 5:7, 1-2; 1 October 2009]; ©2009 Landes Bioscience
Autophagic Punctum
Protein turnover and inclusion body formation
This manuscript has been published online, prior to printing. Once the issue is complete and page numbers have been assigned, the citation will change accordingly.
Siddhartha Mitra,1,4 Andrey S. Tsvetkov1-3 and Steven Finkbeiner1-4,*
1Gladstone Institute of Neurological Disease; San Francisco, CA USA; 2Taube-Koret Center for Huntington’s Disease Research; San Francisco, CA USA; 3Neuroscience
Program; Departments of Neurology and Physiology; 4Biomedical Sciences Program and Medical Scientist Training Program; University of California; San Francisco, CA
USA
Key words: huntington disease, huntingtin, polyglutamine, autophagy, neurodegeneration, ubiquitin, proteasome
In a recent study, we investigated the relationship between
inclusion body (IB) formation and the activity of the ubiquitin-proteasome system (UPS) in a primary neuron model of
Huntington disease. We followed individual neurons over the
course of days and monitored the level of mutant huntingtin
(which causes Huntington disease), IB formation, UPS function,
and neuronal toxicity. The accumulation of UPS substrates and
neuronal toxicity increased with increasing levels of proteasome
inhibition. The UPS was more impaired in neurons that subsequently formed IBs than in those that did not; however, after IBs
formed, UPS function improved. These findings suggest that IB
formation is a protective cellular response mediated in part by
increased degradation of intracellular protein.
Many aggregation-prone proteins responsible for neurodegeneration inhibit the UPS, but the effect of IB formation on UPS
function has been difficult to study. IBs form asynchronously
in only a subset of cells that express aggregation-prone proteins.
Some of this variation likely arises from cell-to-cell differences in
the balance between protein production and protein degradation.
Unfortunately, traditional biochemical and imaging approaches
give a static picture of different populations of cells and combine
measurements from cells with and without IBs. A single-cell longitudinal approach has been invaluable in elucidating the physiology
of stochastic cellular events. Using this approach previously, we
showed that the amount of intracellular mutant protein predicts
IB formation. In this study, we found that cells that eventually
formed IBs had higher levels of UPS impairment than cells that
did not. After IBs formed, UPS impairment improved relative to
that in cells without IBs.
*Correspondence to: Steven Finkbeiner; Gladstone Institute of Neurological Disease;
1650 Owens Street; San Francisco, CA 945158 USA; Tel.: 415.734.2000; Fax:
415.355.0824; Email: [email protected]
Submitted: 06/11/09; Revised: 06/15/09; Accepted: 06/16/09
Previously published online as an Autophagy E-publication:
http://www.landesbioscience.com/journals/autophagy/article/9291
Punctum to: AUTHOR: please provide the citation information for the
paper to which this paper is commenting
1
UPS impairment is toxic to many cell types, including neurons.
In our study, neuronal toxicity increased with increasing levels of
pharmacological UPS inhibition. Yet cells expressing mutant htt
that formed IBs—those with higher levels of UPS inhibition—
survived longer than cells that did not form IBs and had lower levels
of UPS impairment after IB formation. One explanation is that a
compensatory process accompanies IB formation. Alternatively,
the IB itself may afford protection, perhaps by sequestering toxic
hard-to-degrade intracellular proteins. The improvement in UPS
function after IB formation is consistent with both hypotheses
(Fig. 1).
Increasing evidence has implicated the autophagic pathway
in Huntington disease and other neurodegenerative disorders.
To determine whether concurrent changes in autophagy affected
our measurement of UPS activity, we examined the activity of
the autophagic pathway after treatment with the UPS inhibitor
epoxomicin. LC3-II levels, a surrogate marker of macroautophagic
flux, are unchanged in primary striatal neurons. In HEK293 cells,
however, proteasome inhibition leads to LC3-II accumulation,
consistent with previous reports.
What might account for this surprising difference between
neuronal and non-neuronal cells? One possibility is the death
of neurons that upregulate autophagy; however, the inhibitor
treatment did not cause significant toxicity, a finding supported
by the similar levels of LC3-I in the two cell types. The absence
of increased flux through the autophagic pathway may reflect
the inability of neurons to upregulate autophagy. Alternatively,
autophagosome-lysosome fusion may not be rate-limiting in some
cell types and, as a result, LC3-II levels may be an insensitive
marker of autophagic flux in neurons. Autophagy has been characterized mostly in yeast and mammalian non-neuronal cells, and
the few studies in neurons reached different conclusions. Further
characterization of neuronal responses to autophagy-inducing
stimuli will be helpful.
Why do some cells form IBs and survive longer? Although intracellular mediators of IB formation have been identified, answering
this question will require knowledge about how the UPS and the
autophagic pathway interact in handling toxic aggregation-prone
proteins. Particular substrates are often preferentially targeted to
one of the two pathways. After cell stress, the concerted action of
both pathways is clearly important for cellular homeostasis. Since
Autophagy
2009; Vol. 5 Issue 7
Protein turnover and inclusion body formation
Figure 1. The effect of IB formation on UPS function and neurodegeneration. Mutant aggregation-prone protein leads to toxic UPS impairment. A subset of
neurons with higher levels of UPS impairment form IBs. UPS function subsequently improves, and these cells survive longer than cells that do not form IBs.
UPS inhibition alone does not increase autophagy in neurons,
IB formation may be necessary to induce autophagy in certain
cell types. Further investigation of both the molecular mediators
of autophagy and the dynamic changes in autophagic activity
during IB formation will help to reveal the roles of the UPS and
the autophagic pathway in preventing cell death. Much of the
machinery and physiology may vary with the cell type and, in the
case of neurodegenerative disease, the neuronal subtype. Without
a better understanding of cell-type-specifc variations in the UPS
and autophagic activity, it will be difficult to determine the role
of protein degradation in the pathogenesis of neurodegenerative
disease.
Acknowledgements
This work was supported by R01 2NS039746 and 2R01
NS045191 from the National Institute of Neurological Disease
and Stroke, P01 2AG022074 from the National Institute on
Aging, the Taube-Koret Center for Huntington’s Disease Research,
and the J. David Gladstone Institutes (S.F.); a Milton Wexler Award
and a fellowship from the Hereditary Disease Foundation (A.T.);
NIH-NIGHMS UCSF Medical Scientist Training Program and a
fellowship from the UC-wide adaptive biotechnology (GREAT)
program (S.M.); and RR018928 from the National Center for
Research Resources. Kelley Nelson provided administrative assistance, and Gary C. Howard edited the manuscript.
www.landesbioscience.com
Autophagy
2
Human Molecular Genetics, 2009, Vol. 18, No. 11
doi:10.1093/hmg/ddp115
Advance Access published on March 11, 2009
1937–1950
Cytoplasmic retention of polyglutamine-expanded
androgen receptor ameliorates disease via
autophagy in a mouse model of spinal
and bulbar muscular atrophy
Heather L. Montie1, Maria S. Cho1, Latia Holder1, Yuhong Liu1, Andrey S. Tsvetkov2, Steven
Finkbeiner2,3,4,5 and Diane E. Merry1,
1
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA,
Gladstone Institute of Neurological Disease, San Francisco, CA, USA, 3Taube-Koret Center for Huntington’s Disease
Research, San Francisco, CA, USA, 4Department of Neurology and 5Department of Physiology, University of
California, San Francisco, CA, USA
2
Received January 13, 2009; Revised February 19, 2009; Accepted March 9, 2009
The nucleus is the primary site of protein aggregation in many polyglutamine diseases, suggesting a central
role in pathogenesis. In SBMA, the nucleus is further implicated by the critical role for disease of androgens,
which promote the nuclear translocation of the mutant androgen receptor (AR). To clarify the importance of
the nucleus in SBMA, we genetically manipulated the nuclear localization signal of the polyglutamineexpanded AR. Transgenic mice expressing this mutant AR displayed inefficient nuclear translocation and
substantially improved motor function compared with SBMA mice. While we found that nuclear localization
of polyglutamine-expanded AR is required for SBMA, we also discovered, using cell models of SBMA, that it
is insufficient for both aggregation and toxicity and requires androgens for these disease features. Through
our studies of cultured motor neurons, we further found that the autophagic pathway was able to degrade
cytoplasmically retained expanded AR and represents an endogenous neuroprotective mechanism.
Moreover, pharmacologic induction of autophagy rescued motor neurons from the toxic effects of even
nuclear-residing mutant AR, suggesting a therapeutic role for autophagy in this nucleus-centric disease.
Thus, our studies firmly establish that polyglutamine-expanded AR must reside within nuclei in the presence
of its ligand to cause SBMA. They also highlight a mechanistic basis for the requirement for nuclear localization in SBMA neurotoxicity, namely the lack of mutant AR removal by the autophagic protein degradation
pathway.
INTRODUCTION
Nuclear residing proteins are normally directed to the nucleus
by a signaling sequence, a particular folding pattern and/or a
post-translational modification. After they have served their
function, nuclear proteins are either degraded by nuclear proteasomes or exported to the cytoplasm for degradation. A
mutation within a protein, such as the expansion of a polyglutamine tract, causes it to accumulate within particular cellular
compartments, as it is refractory to degradation. Nuclear
accumulation of misfolded proteins is most likely due to the
lack of a secondary degradation mechanism within nuclei
and this accumulation of mutant protein is toxic to neurons.
Spinal and bulbar muscular atrophy (SBMA, Kennedy’s
disease) is an X-linked neurodegenerative disease resulting
from the expansion of a polyglutamine (polyQ)-encoding
CAG tract in the 50 end of the androgen receptor (AR) gene
(1). When containing more than 40 CAG repeats, the AR
causes slowly progressive proximal limb and bulbar
muscle weakness, fasciculations and atrophy in men (2,3).
To whom correspondence should be addressed at: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 228 Bluemle Life
Sciences Building, 223 S. 10th Street, Philadelphia, PA 19107, USA. Tel: þ1 2155034907; Fax: þ1 2159239162; Email: [email protected]
# The Author 2009. Published by Oxford University Press. All rights reserved.
For Permissions, please email: [email protected]
1938
Patients may also suffer some sensory loss (4,5) and display
slight androgen insensitivity (2). While partial loss of AR
function exists in SBMA, this does not represent the primary
disease etiology (6,7); rather accumulation of toxic AR
protein species leads to motor neuron dysfunction and death
(8 – 10).
SBMA is one of a family of nine polyQ-expansion diseases
(reviewed by 11), with a common pathological hallmark; the
accumulation of misfolded and aggregated species of mutant
protein in the cytoplasm or nuclei of vulnerable neurons.
Although there are conflicting views in the field concerning
the correlation of aggregates with disease, considerable data
indicate that inclusions themselves are not toxic (12,13).
Instead, species that are produced in early stages of the aggregation cascade (likely proteolyzed AR monomer and oligomer) induce toxicity. Nonetheless, the presence of inclusions
in a population of neurons reveals the late stage of a pathogenic process.
The common finding of nuclear inclusions in polyQ diseases suggests a central role for the nucleus in pathogenesis.
While inclusions of polyQ-expanded huntingtin are found in
both the cytoplasm and nucleus, the accumulation of nuclear
mutant huntingtin causes the greatest neuronal toxicity
(13,14). In SCA-1 and SCA-3, inclusions of the mutant
protein are found only within nuclei (15,16) and mutation of
the endogenous nuclear localization signal (NLS) within
each of these respective proteins, to sequester them within
the cytoplasm, has proved to be neuroprotective (17,18).
These findings highlight an important role for the nucleus in
the toxicity induced by polyQ-expanded proteins, although
the mechanistic basis for this role has remained elusive.
In SBMA, inclusions of aberrantly cleaved polyQ-expanded
AR are also present primarily in nuclei (19), although neuropil
accumulation of 1C2-positive material has been observed (20).
In cell and rodent models of SBMA, nuclear aggregation and
disease are dependent on the presence of AR ligands [testosterone or dihydrotestosterone (DHT)] (10,21– 27), which drive
nuclear translocation of the AR. As a type I nuclear hormone
receptor transcription factor, the unliganded AR resides primarily within the cytosol, where it is associated with heat shock and
accessory proteins (28,29). Upon hormone binding, the AR
undergoes a conformational change that exposes its bipartite
NLS, directing it to the nucleus, where it regulates transcription
of its target genes.
The localization of inclusions within nuclei and the dependence of disease on androgens suggest a central role for the
nucleus in SBMA. Moreover, the finding that some alternative
ligands that direct AR to the nucleus also cause disease supports this idea (24,30). In a Drosophila model of SBMA,
retention of a polyQ-expanded AR fragment in the cytoplasm
ameliorated disease (26). However, in contrast to these results,
in a cell model of SBMA, fast axonal transport was reduced
by expanded AR in a hormone-independent manner (31),
suggesting a cytoplasmic site of pathology, and making uncertain the role of the nucleus in disease. In this study, we sought
to determine, using transgenic mouse and cell models, whether
mammalian systems reveal a necessity for nuclear localization
and whether nuclear localization is sufficient for disease.
Our results firmly establish that nuclear localization of polyQexpanded AR is necessary, but not sufficient for nuclear
Figure 1. Protein expression of ARdNLS mice. Five-week-old male mice
were sacrificed and whole brain and spinal cord lysates evaluated for AR by
western blot. AR protein was detected with antibody AR(N-20). GAPDH
was used as a loading control. nTG, non-transgenic.
aggregation and toxicity. Furthermore, we present evidence
that the lack of access to the autophagic degradation pathway
represents one explanation for the enhanced toxicity of nuclearconfined mutant AR.
RESULTS
ARdNLS112Q transgenic mice express greater levels
of AR than AR112Q mice
In order to understand the role of the nucleus in disease, we
created transgenic mice bearing an AR with a mutation in the
NLS. We previously created a transgenic mouse model of
SBMA expressing full-length (human) polyQ-expanded AR
(112Q) driven by the prion protein promoter (PrP) (22). The
new transgenic mice (ARdNLS112Q and ARdNLS24Q) were
created to express transgenic AR with a deletion of amino
acids 628– 640 within the bipartite NLS of the AR. A line of
ARdNLS112Q was established which expresses over 2-fold
more AR protein, in brain and spinal cord, than AR112Q
mice (Fig. 1). In addition, ARdNLS24Q mice expressed equivalent levels of AR as ARdNLS112Q mice (Fig. 1), and both
lines were used for behavioral analysis.
Motor deficits associated with SBMA are ameliorated
in ARdNLS112Q male mice
We previously determined that the rotarod assay is a sensitive
measure of motor function in SBMA mice (22). Therefore, we
measured latency to fall from an accelerating rotarod, as well
as grip strength, every 4 weeks beginning at 8 weeks of age.
This behavioral analysis of a large age-matched cohort of
ARdNLS112Q transgenic males (n ¼ 18) and AR112Q males
(n ¼ 10) revealed delayed onset and reduction of motor deficits
associated with SBMA, when compared with non-transgenic littermates (n ¼ 18). While AR112Q males showed significant
and progressive deficits in maintaining themselves on an accelerating rotarod at 8, 12 and 16 weeks of age, ARdNLS112Q
males performed as well as non-transgenics (Fig. 2A). At 20,
24 and 28 weeks of age ARdNLS112Q males had significantly
reduced rotarod function compared with non-transgenics, but
still performed substantially better than AR112Q males
(Fig. 2A). Female ARdNLS112Q (n ¼ 10) performed as well
as non-transgenic littermates (n ¼ 15) until 24 weeks of age,
when they had a slight reduction in rotarod performance
(Fig. 2B). ARdNLS24Q (n ¼ 15) males did not develop any
1939
Figure 2. Motor deficits associated with SBMA are ameliorated in ARdNLS112Q mice. Latency to fall from an accelerating rotarod was measured every 4
weeks from 8 to 28 weeks of age in male (A) [# ¼ P , 0.05 between AR112Q males and both non-transgenic (nTG) and ARdNLS112 males only; ¼ P ,
0.05 between all groups] and female (B) transgenic mice. 8 to 28 ¼ age in weeks; Tick numbers on x-axis represent trials 1 –4 for each age. Error bars represent
standard deviation for each trial for each group. (C) Forepaw and all paw grip strength were measured every 4 weeks from 8 to 28 weeks of age. Error bars
represent standard deviation. ¼ P 0.05.
rotarod deficits up to 28 weeks of age (Supplementary Material,
Fig. S1).
Both forepaw and all paw grip strength was significantly
reduced in AR112Q male mice beginning at 12 weeks of
age, while both measures of grip strength of ARdNLS112Q
mice were similar to non-transgenic males (Fig. 2C). At 16,
20 and 24 weeks of age (data not shown), grip strength
results resembled those shown at 12 weeks of age. At 28
1940
weeks of age, results of grip strength reflected those of rotarod
analysis in that AR112Q males had significantly reduced grip
strength compared with both ARdNLS112Q and nontransgenic males and ARdNLS112Q males were somewhat
weaker than non-transgenic males. Female ARdNLS112Q
mice showed grip strength similar to non-transgenic females
up to 28 weeks of age (Fig. 2C).
AR112Q male mice fail to gain weight after 6 months of
age (22). Analysis of the present cohort showed a failure of
AR112Q male mice to gain weight after 28 weeks of age,
while ARdNLS112Q males continued to gain weight in the
same manner as non-transgenic male littermates (Supplementary Material, Fig. S2A). Female ARdNLS112Q mice had
slightly greater weight gain over time compared with nontransgenic littermates (Supplementary Material, Fig. S2B).
ARdNLS24Q male mice also showed no decrease in weight
gain (Supplementary Material, Fig. S2C).
Additional tests of motor function, including footprint and
rearing analysis, revealed similar results as rotarod and grip
strength analysis (data not shown). From 8 to 16 weeks of
age, ARdNLS112Q male mice demonstrated a normal gait,
while AR112Q males exhibited a wider and shorter gait.
From 20 to 28 weeks of age, ARdNLS112Q males had a slightly
lower and wider stance compared with nTG males, but their gait
was substantially better than that of AR112Q males. Analysis of
vertical activity (during a 5-min period) was performed using a
Versamax activity monitor (AccuScan Instruments, Columbus,
OH). At ages when ARdNLS112Q males performed as well as
nTG males on the rotarod, their vertical activity was also
normal, while AR112Q males showed significant deficits.
After 20 weeks of age, ARdNLS112Q males had decreased vertical activity compared with nTG males but increased vertical
activity compared with AR112Q males. Female AR112Q
mice had normal gait and vertical activity at all ages evaluated.
As previously described, survival of the AR112Q mice was not
substantially compromised; survival of ARdNLS112Q mice
was also unchanged (data not shown).
ARdNLS112Q has delayed nuclear accumulation
and forms oligomers later than AR112Q
Neuropathological analysis of AR112Q, ARdNLS112Q and
non-transgenic male mice was performed at 8, 16 and 24
weeks of age. In AR112Q males at 8 weeks of age, AR112Q
protein was localized primarily within nuclei of spinal motor
neurons (Fig. 3A and Supplementary Material, Fig. S4) and
these males had significant deficits in motor function
(Fig. 2A). In contrast, at this age in ARdNLS112Q mice, transgenic AR protein was observed primarily within the
cytoplasmic compartment, as observed both by immuofluoresFigure 3. Analysis of AR aggregation in spinal cord. (A) Anterior horn from
lumbar spinal cord of 8, 16 and 24 week old mice immunostained for AR
(ARH280) and stained with Hoechst to reveal nuclei. Left panel, immunostaining of AR; middle panel, Hoechst staining; right panel, merged image. Arrow
in image of 16 week ARdNLS112Q male indicates a small intranuclear
inclusion. Arrow in image of 37 week ARdNLS112Q female indicates cytoplasmic AR. (B) Protein lysates from the same spinal cords as in (A) were prepared to evaluate oligomeric species of AR (ARH280) by western blot.
cence and fractionation (Fig. 3A and Supplementary Material,
Fig. S4) and males had normal motor function (Fig. 2A). In
addition, western blot analysis of brain and spinal cord revealed
substantially more SDS-insoluble, high-molecular weight oligomeric species of AR112Q than ARdNLS112Q at this age
(Fig. 3B). At 16 weeks of age oligomeric species of AR112Q
were increased (Fig. 3B), although inclusions were not detected
(Fig. 3A). Sixteen week-old male ARdNLS112Q revealed
accumulated mutant AR within nuclei (Fig. 3A) and a small
proportion of neurons contained small punctate intranuclear
inclusions (Fig. 3A). Western analysis revealed oligomeric
forms of ARdNLS112Q, although these were substantially
less abundant than those from AR112Q mice (Fig. 3B),
despite the abundance of AR protein. By 24 weeks of age,
AR112Q male mice had a considerable proportion of neurons
in the anterior horn of the spinal cord with large intranuclear
inclusions (Fig. 3A); as previously shown, inclusions consisted
of proteolyzed AR (data not shown) (22). At this age,
ARdNLS112Q males also had a significant number of
neurons with large intranuclear inclusions of mutant AR
(Fig. 3A); intranuclear inclusions of ARdNLS112Q were also
composed of fragmented AR, as they lacked the epitopes for
antibodies AR441 and ARC-19 (data not shown). Similar
results were observed in cortical neurons from these animals
(Supplementary Material, Fig. S3A and B). In females,
ARdNLS112Q was found largely within the cytoplasm at all
ages, although by 37 weeks, a small number of neurons with
large nuclear inclusions were observed (Fig. 3A). In males,
ARdNLS24Q had also accumulated within neuronal nuclei by
37 weeks of age, but did not form intranuclear inclusions (Supplementary Material, Fig. S5). Inclusions of ARdNLS112Q
were confirmed by confocal microscopy to be contained
within nuclei (data not shown). In addition, our previous
studies revealed decreased immunoreactivity of unphosphorylated neurofilament heavy chain (NF-H) in soma of both
spinal motor neurons and neurons of the cerebral cortex (22).
This alteration was absent from neurons of ARdNLS112Q
mice (data not shown).
Polyglutamine-expanded ARdNLS fails to cause nuclear
aggregation or toxicity in a cell model of SBMA
We previously created an inducible cell model of SBMA that
expresses full-length human AR with 112 glutamines and
forms intranuclear inclusions in response to DHT (27).
Notably, as in patients’ tissue, nuclear inclusions in this
model consist of proteolyzed N-terminal fragments of AR.
In the present studies, we established a cell line expressing
ARdNLS78Q to evaluate the metabolism of cytoplasmically
retained polyQ-expanded AR in culture. In contrast to
AR112Q-expressing cells, ARdNLS78Q-expressing cells
showed a diffuse cytoplasmic distribution of AR in the presence of hormone (DHT), and failed to form intranuclear
inclusions (Fig. 4A). Over time, these cells formed large cytoplasmic aggregates of full-length AR [detected with antibodies
to the N-terminus (AR(N-20))], an internal epitope (AR441)
(Fig. 4A) and the C-terminus [AR(C-19)] (data not shown).
ARdNLS10Q cells also contained cytoplasmic AR in the presence of DHT and never formed nuclear or cytoplasmic aggregates (data not shown). To confirm that mutant ARdNLS78Q
1941
is capable of forming nuclear inclusions, we targeted ARdNLS
to the nucleus with an exogenous NLS (NLSX3ARdNLS63Q). This resulted in the hormone-dependent formation of nuclear inclusions of full-length AR (Fig. 4B and
data not shown). In these cell lines, AR was expressed at comparable levels and was stabilized by DHT (Fig. 4C).
Hormone treatment of AR112Q-expressing cells resulted
in toxicity (Fig. 4D). However, polyQ-expanded ARdNLS
(ARdNLS78Q)-expressing cells (Fig. 4D) failed to die in
response to hormone, indicating that nuclear localization is
necessary, not only for AR aggregation, but for cell death as
well. Targeting polyQ-expanded ARdNLS to the nucleus
(NLSX3-ARdNLS63Q) resulted in DHT-dependent death
(Fig. 4D), confirming that the deletion in the NLS does not
affect the capacity of the polyQ-expanded AR to confer toxicity
when localized to the nucleus. Thus, the possibility that deletion
of the NLS alters AR conformation and prevents toxicity for
reasons unrelated to its localization is unlikely, due to our
finding that nuclear targeting confers on the mutant AR
protein the capability of forming inclusions and causing toxicity.
Nuclear localization of polyglutamine-expanded AR
is insufficient for aggregation and toxicity
Our results indicate a requirement for nuclear localization in
both the nuclear aggregation and toxicity of polyQ-expanded
AR. We next sought to determine whether nuclear localization
is sufficient for these events. To accomplish this, we created
PC12 inducible cell lines that express an AR targeted to the
nucleus in the absence of hormone (NLSX3-AR). In the
absence of DHT, NLSX3-AR76Q was localized within
nuclei, while AR112Q was diffusely distributed within cytoplasm and nuclei (Fig. 5). No intranuclear inclusions of
NLSX3-AR76Q were observed in the absence of DHT;
inclusions consisting of N-terminal AR fragments were
formed exclusively in response to DHT (Fig. 5). These data
indicate that nuclear localization of the polyQ-expanded AR
is insufficient for nuclear aggregation. Moreover, nuclear
localization is insufficient for toxicity, as NLSX3-AR76Q
PC12 cells failed to die in the absence of DHT, and only
did so in the presence of DHT (Fig. 4C). It was also noted
that NLSX3AR, containing an even shorter polyQ-tract
(76Q) than non-NLS-tagged AR (112Q), induced a greater
level of toxicity, despite equivalent protein levels (Fig. 4C),
consistent with a role for the nucleus in mediating toxicity.
NLSX3-AR35Q cells showed no aggregation or toxicity in
response to DHT treatment (data not shown).
Primary motor neurons from ARdNLS112Q mice are
protected from DHT-dependent toxicity by autophagy
In order to evaluate SBMA motor neuron toxicity in response to
DHT, we initiated spinal cord cultures from non-transgenic,
AR112Q and ARdNLS112Q transgenic mice. Monomeric
levels of both AR112Q and ARdNLS112Q were increased
(stabilized) in the presence of DHT; in addition, ARdNLS112Q
was expressed at significantly higher levels than AR112Q
(Fig. 6A). While DHT caused the loss of 40% of
AR112Q-expressing motor neurons, it failed to cause the death
of ARdNLS112Q-expressing motor neurons (Fig. 6B).
1942
Figure 4. Polyglutamine-expanded ARdNLS fails to produce intranuclear inclusions or toxicity in a cell model of SBMA. (A) Immunofluorescence of stably
transfected tet-inducible PC12 cells treated with doxycycline to express either AR112Q or ARdNLS78Q and DHT for 48 h. Cells were immunostained with
antibodies to the N-terminus (AR(N-20)), and an internal epitope (AR441) of the AR and stained with Hoechst to reveal nuclei. Arrow in AR112Q panel indicates intranuclear inclusions that lack the epitope for AR441. Arrow in ARdNLS78Q panel indicates cytoplasmic inclusion that contains the epitope for AR441.
(B) NLSX3-AR76Q and NLSX3-ARdNLS63Q PC12 cells were treated with doxycycline to express AR in the presence of DHT and immunostained as in (A).
Arrow in NLSX3-AR76Q panel indicates intranuclear inclusions that lack the epitope for AR441. Arrow in NLSX3-ARdNLS63Q panel indicates intranuclear
inclusions that also contain the epitope for AR441. (C) AR112Q, ARdNLS78Q, NLSX3-AR76Q and NLS-ARdNLS63Q PC12 cells were treated with doxycycline (DOX) to induce AR in the absence or presence of DHT. Cells were harvested after 48 h and evaluated for AR protein levels via western blot analysis.
(D) Analysis of PC12 cell toxicity. Cells expressing AR10Q, AR112Q and ARdNLS78Q were treated with doxycycline to express AR in the presence or absence
of DHT for 12 days, and cell death determined by trypan blue uptake. Two-hundred cells were counted and the percentage of trypan blue-positive cells determined. Student’s t-tests were performed. ¼ P 0.05.
Figure 5. Nuclear localization of polyglutamine-expanded AR is insufficient for the formation of nuclear inclusions in a cell model of SBMA. Stably transfected
tet-inducible PC12 cells were treated with doxycycline to express either AR112Q or NLSX3-AR76Q in the presence or absence of DHT for 48 h. Cells were
immunostained with antibodies to the N-terminus (AR(N-20)), and an internal epitope (AR441) of AR and stained with Hoechst to reveal nuclei. White arrow in
AR112Q panel indicates diffuse cytoplasmic AR in the absence of DHT. White arrow in NLSX3AR76Q panel indicates diffuse nuclear AR in the absence of
DHT. Student’s t-tests were performed. ¼ P 0.05.
We next sought to determine the mechanism by which
ARdNLS112Q motor neurons resist DHT-dependent death.
Immunofluorescence staining revealed the presence of cytoplasmic puncta consisting of mutant AR in ARdNLS112Q
motor neurons (Fig. 6C). With the knowledge that
ARdNLS112Q enters the nucleus with reduced efficiency in
the presence of hormone (Figs 3A and 4A), and that it forms
cytoplasmic inclusions (Figs 3A and 6C), we considered
autophagy to be a likely candidate. It is well established that
activation of autophagy is neuroprotective in misfolded
protein diseases (reviewed in 32). Therefore, we evaluated
the essential autophagy marker LC3B (33) in primary motor
neurons. Immunofluorescence analysis of LC3B in
ARdNLS112Q motor neurons revealed punctate cytoplasmic
staining of LC3B following treatment with DHT (Fig. 7A),
indicating the activation of autophagy in these neurons. In
addition, LC3B puncta were found to co-localize with AR
(Fig. 7A). Punctate staining of LC3B was not detected in
nTG or AR112Q motor neurons following DHT treatment
(data not shown).
Figure 6. Primary motor neurons from SBMA mice die in response to
hormone treatment while motor neurons from ARdNLS112Q mice survive.
(A) Primary motor neuron cultures were initiated from AR112Q and
ARdNLS112Q transgenic mouse embryo spinal cords. Cultures were treated
with or without DHT for 7 days and protein lysates evaluated by western
blot for AR and GAPDH. (B) Cultures were treated as in (A) and additional
ARdNLS112Q and AR112Q motor neuron cultures were treated with 3methyladenine to inhibit autophagy. Ten random fields of immunostained
(SMI32) motor neuron cultures were counted under a fluorescent Leica microscope. Counts from three separate wells for each cell line and treatment group
were graphed. Student’s t-tests were performed. ¼P 0.05. (C) Cultures
were treated as in (A) and immunostained for AR (AR(N-20)) and neurofilament heavy chain (SMI32) to reveal motor neurons. Note the presence of cytoplasmic inclusions of ARdNLS112Q. Nuclear ARdNLS112Q protein is also
observed.
Given the suggestion that autophagy was activated in
ARdNLS112Q motor neurons, we determined the role of
autophagy in the resistance of ARdNLS112Q motor neurons
to DHT-dependent death. To this end, we treated spinal cord
cultures with 3-methyladenine (3-MA), a well-known inhibitor
of autophagy (34). 3-MA failed to cause toxicity of nontransgenic motor neurons (data not shown); moreover, it did
not enhance DHT-dependent toxicity of AR112Q-expressing
motor neurons (Fig. 6B). In contrast, 3-MA induced
DHT-dependent death of ARdNLS112Q motor neurons
(Fig. 6B). Biochemical analysis of protein extracts from
these cultures showed a large increase in the monomeric
1943
Figure 7. Autophagy protects ARdNLS112Q motor neurons from
DHT-dependent death. (A) Primary motor neuron cultures were initiated
from ARdNLS112Q transgenic mouse embryo spinal cords. Cells were
treated with or without DHT for 7 days, and immunostained for neurofilament heavy chain (SMI32) to reveal motor neurons, and LC3B (LC3B) to
detect autophagosomes. ARdNLS112Q motor neuron shown was doubleimmunostained for AR (AR-318) and LC3B, then immunostained using
SMI32. (B) Additional ARdNLS112Q motor neurons were treated with
or without 3-methyladenine (3-MA), to inhibit autophagy, for the last 3
days of the 7-day treatment period with DHT. Protein lysates were analyzed by western blot with antibodies to AR (AR(N-20)), LC3B and
GAPDH.
form of ARdNLS112Q following treatment with DHT and
3-MA (Fig. 7B), well above the stabilization of AR seen
with DHT alone. In addition, the active form of LC3B
(LC3B II) was decreased in the presence of 3-MA (Fig. 7B),
validating the inhibitory effects of 3-MA on autophagy.
DHT-dependent death of motor neurons from AR112Q
mice is prevented by activation of autophagy
The observation that endogenous autophagy can protect motor
neurons from DHT-dependent death when polyQ-expanded
AR is retained within the cytoplasm (ARdNLS112Q) confirms
the importance of this degradation pathway in clearing misfolded cytoplasmic proteins. We next sought to determine
whether pharmacologic activation of autophagy could rescue
nuclear polyQ-expanded AR (AR112Q)-expressing motor
neurons from DHT-dependent death. We used an AKT inhibitor (AKTi) to activate autophagy in spinal cord cultures from
our SBMA (AR112Q) mice. Previous studies demonstrated
1944
Figure 8. DHT-dependent death of motor neurons from SBMA mice is prevented by activation of autophagy. (A) Primary motor neuron cultures were initiated
from AR112Q transgenic mouse embryo spinal cords. Cells were treated with or without DHT for 7 days, in the presence or absence of an AKT inhibitor (AKTi)
for the last 3 days. Counts from three separate wells for each cell line and treatment group were graphed. AKTi treatment rescued AR112Q motor neurons from
DHT-dependent death. (B) Cultures were immunostained for neurofilament-heavy chain (SMI32) to reveal motor neurons and immunostained for LC3B to reveal
autophagosomes. (C) Cells treated in parallel to those described in (A) were harvested and protein lysates analyzed by western blot with antibodies against AR
(AR(N-20)), LC3B and GAPDH. (D) AR112Q motor neurons were treated with trehalose for the last 3 days of a 7-day treatment period with or without DHT.
Motor neurons were immunostained for neurofilament-heavy chain (SMI32) and LC3B. (E) Cells treated as in (D) were harvested and protein lysates analyzed by
western blot for AR (AR(N-20)), LC3B and GAPDH. (F) Motor neurons were counted as in (A) following trehalose treatment, and trehalose was found to protect
AR112Q motor neurons from DHT-dependent death. Student’s t-tests were performed. ¼ P 0.05.
the ability of the AKT inhibitor, phenoxazine, to activate
autophagy in primary neurons expressing mutant huntingtin
(Tsvetkov and Finkbeiner, unpublished results). Treatment of
AR112Q motor neurons with AKTi for the last 3 days of a
7-day DHT treatment resulted in a substantial rescue from
DHT-dependent death (Fig. 8A). As expected, AKTi-treated
motor neurons contained cytoplasmic puncta of LC3B
(Fig. 8B). Moreover, western analysis of AKTi-treated
cultures revealed a significant increase in the active form of
LC3B (LC3B II) (Fig. 8C). Non-transgenic motor neuron
cultures also showed an increase in LC3B II following
treatment with AKTi (data not shown). To confirm these
findings, we evaluated another activator of autophagy, trehalose, which was previously shown to activate mTORindependent autophagy (35) and relieve the neurotoxicity of
polyQ-expanded huntingtin (36,37). Treatment of AR112Q
spinal cord cultures with trehalose resulted in the formation
of LC3B-positive cytoplasmic puncta (Fig. 8D), an increase
in LC3B II (Fig. 8E) and rescue from DHT-dependent death
(Fig. 8F). Non-transgenic cultures also showed increased
LC3B II levels following trehalose treatment (data not shown).
No effect on monomeric levels of AR112Q by either
autophagy-inducing regimen was observed (Fig. 8C and E).
DISCUSSION
A critical role for the nucleus in polyglutamine disease has
emerged in recent years. In SBMA, this is evidenced by the presence of inclusions of polyQ-expanded AR within nuclei and
the dependence of disease upon androgens, which enable the
AR to translocate into the nucleus following binding. Previous
studies of Drosophila and mammalian cell culture models to
delineate the role of nuclear versus cytoplasmic AR in SBMA
have raised questions due to conflicting results (26,31). To
clarify the importance of the nucleus in SBMA using mammalian systems, we created transgenic mouse and cell models that
express polyQ-expanded AR with a deletion in a portion of its
bipartite NLS (amino acids D628– 640; ARdNLS112Q), to
reduce its androgen-dependent nuclear transit. We hypothesized that nuclear localization of the mutant AR is essential
for disease and that cytoplasmic retention of mutant AR
would be neuroprotective in these models.
We observed that DHT-dependent polyQ-induced toxicity
was ameliorated in three mammalian models of SBMA.
First, even temporary retention of polyQ-expanded AR
within the cytoplasm ameliorated motor deficits in male transgenic mice. At 8 weeks of age, when ARdNLS112Q was localized within the cytoplasm, male mice were completely
normal, while AR112Q male mice, with exclusively nuclear
AR, displayed substantial motor deficits. With age, older
male mice accumulated nuclear ARdNLS112Q, despite
mutation of the NLS. This nuclear localization was also
observed in male ARdNLS24Q mice, but not in female transgenic mice, demonstrating that ARdNLS is capable of
hormone-dependent nuclear translocation, albeit with substantially reduced efficiency. Only when ARdNLS112Q had accumulated within nuclei and formed both oligomeric and
aggregated species did male mice begin to display signs of
motor deficits, consistent with the previous demonstration
that oligomeric AR species precede disease symptoms (38).
However, despite the eventual nuclear localization and aggregation of mutant ARdNLS112Q protein, male ARdNLS112Q
mice exhibited substantially improved motor function. These
results indicate that (i) retention of a significant portion of
polyQ-expanded AR within the cytoplasm is sufficient to
both delay and ameliorate disease and (ii) nuclear localization
enhances the formation of oligomeric AR species that precede
motor deficits. In addition to the amelioration of motor deficits
in mice by cytoplasmic AR retention, motor neurons from
ARdNLS112Q mice were resistant to DHT-dependent death.
Finally, our studies in PC12 cells indicate that the mutant
AR must enter the nucleus both for nuclear aggregation and
toxicity. Therefore, nuclear localization is essential for
polyQ-expanded AR to elicit its primary toxic effects.
Complete and efficient nuclear localization of polyQexpanded AR (AR112Q) caused early, severe and progressive
motor deficits in male mice; these deficits were significantly
worse than those eventually observed in older ARdNLS112Q
mice, which exhibited aggregated nuclear AR. Nuclear
1945
accumulation ARdNLS112Q in male mice was somewhat surprising, based on our data in PC12 cells, but it was also not completely unexpected. A similar, but more substantial, deletion of
the AR NLS (D628 – 657) allowed partial nuclear entry upon
androgen binding (39). These results suggest that an alternative
hormone-dependent signal may be utilized in the absence of a
functional bipartite NLS. It is also important to note that the
ARdNLS112Q likely translocated to the nucleus as full-length
monomer rather than as a proteolyzed fragment. In support of
this, we observed substantial levels of full-length
ARdNLS112Q by western analysis at ages when this protein
was visualized within nuclei by immunofluorescence. In
addition, we observed the localization of normal
ARdNLS24Q within nuclei of male mice in the absence of
pathologic inclusions, confirming that full-length ARdNLS is
capable of eventual nuclear translocation. It is curious that,
despite higher levels of ARdNLS112Q protein and its eventual
accumulation within nuclei, ARdNLS112Q mice developed
only modest motor impairments with age. Data from PC12
cells expressing expanded ARdNLS demonstrate that this
mutant AR is benign when retained within the cytoplasm, but
causes substantial toxicity when directed to the nucleus with
an exogenous NLS. Thus, in the case of the mice, it may be
that simply delaying onset of disease by reducing nuclear
transit of mutant AR minimizes its overall impact on neuronal
function.
Our finding that ARdNLS112Q formed inclusions earlier than
AR112Q was somewhat surprising. The formation of
ARdNLS112Q inclusions may be due to the higher levels of the
protein, once it has accumulated within nuclei, compared with
AR112Q. We also observed that, although ARdNLS112Q
formed inclusions earlier than AR112Q (at 16 weeks, with oligomers also present at this time), AR112Q formed oligomers much
earlier (8 weeks of age) than ARdNLS112Q. The efficient nuclear
localization of AR112Q likely resulted in the earlier formation
and sustained presence of oligomers and thus earlier and more
substantive disease.
The requirement in SBMA for nuclear mutant AR localization defined by our transgenic mouse and cell culture
studies led us to evaluate if nuclear localization is sufficient
for disease. Our cell studies revealed that nuclear localization
alone is not sufficient for disease, and that androgen binding by
the AR is essential for its aberrant metabolism and ability to
induce toxicity. Targeting of a polyQ-expanded AR with a
shorter polyglutamine tract (76Q) to the nucleus led to toxicity
in a hormone-dependent manner. Moreover, we observed
enhanced toxicity of this protein over normally trafficked
AR112Q, despite the shorter polyglutamine length, confirming
the importance of nuclear localization in toxicity.
In SBMA, nuclear inclusions consist of an N-terminal
fragment(s) of AR (19,22,27). Fragmented polyQ-expanded
proteins have been documented by numerous groups, and
may be a result of normal or aberrant protease cleavage, or
inefficient processing by the proteasome. These fragments
have been shown to be refractory to degradation (40) and
are more toxic than intact, full-length, polyglutamineexpanded proteins (22,41 – 45). In our present studies, the
cytoplasmic retention of polyQ-expanded AR led to the formation of large cytoplasmic inclusions that contained fulllength AR, unlike the nuclear inclusions of patients’ tissue,
1946
which contain only N-terminal AR species (19). When the
mutant expanded ARdNLS was directed to the nucleus with
an exogenous NLS, intranuclear inclusions were detected
that contained the epitope for antibody AR441. It is unclear
whether there is any fragmented AR within these aggregates
or whether complete loss of the AR441 epitope would occur
with more time. Our aggregation studies were carried out
after 2 days of hormone treatment, while toxicity was evaluated after 12 days of hormone treatment. In mice, nuclear
accumulated ARdNLS112Q was found to form intranuclear
inclusions of fragmented AR, and thus we hypothesize that
fragmented AR represents the most toxic species. In all,
these data suggest that nuclear localization of polyQ-expanded
AR is a prerequisite for its proteolysis and nuclear accumulation. This feature places the nucleus at a central point of
pathology, the aberrant cleavage of mutant AR to a form
that is both toxic and aggregation-prone.
While our studies place the location of mutant AR toxicity
in the nucleus, the mechanism by which the polyglutamineexpanded AR confers toxicity within the nucleus is unclear.
While AR transcriptional activity is not required for toxicity
(24), transcriptional dysregulation occurs in the presence of
the mutant AR (46,47). In addition, proteasome function is
impaired in mutant AR-expressing cells (our unpublished
results) and flies (48). Mitochondrial dysfunction has also
been described in the face of nuclear mutant AR (49), concomitant with the altered transcription of genes involved in mitochondrial function. In addition to representing a major site for
the toxic cellular sequelae of expanded-polyglutamine AR, the
nucleus also represents a major site of altered metabolism of
the mutant AR. One of the distinctive features of nuclear
mutant AR when compared with cytoplasmic AR is the host
of AR post-translational modifications, protein – protein interactions and structural AR changes that occur in response to
hormone binding (50,51). Our current and future studies will
address alterations in these pathways and their role in
nuclear polyQ-expanded AR toxicity.
Upon determining that a critical role for the nucleus exists
in SBMA pathogenesis, we investigated the mechanistic
basis for the neuroprotective role of cytoplasmic retention of
polyQ-expanded AR. Previous studies have revealed failure
of the proteasome to efficiently and appropriately degrade misfolded proteins, specifically those containing polyQ expansions (40), unless chaperone-mediated therapies are initiated
(52 – 59). In addition, it has become increasingly clear that
the ubiquitin proteasome system is reduced in neuronal
nuclei compared with the cytoplasmic compartment (60,61),
suggesting one explanation for the differential toxicities conferred by misfolded proteins in these two locations.
However, another important and emerging feature of cytoplasmic localization of misfolded proteins is their availability
to activate a second method of degradation, the autophagic/
lysosomal pathway, which has been shown to degrade
polyQ-expanded proteins (62). When pharmacologically activated, autophagy can effectively degrade misfolded proteins
and is neuroprotective (reviewed by 32,35).
Our studies of cultured, transgenic motor neurons revealed
that ARdNLS112Q motor neurons failed to die in response
to DHT (Fig. 6). The observation of LC3 puncta indicates
that autophagy was activated in these motor neurons. Further-
more, the inhibition of autophagy led to DHT-dependent toxicity, indicating that the cytoplasmic mutant AR is capable of
causing toxicity when autophagy is inhibited. Finally, the
increase in mutant AR protein upon autophagy inhibition supports our conclusion that the mutant AR is, at least in part,
degraded by this pathway. While inhibition of autophagy
caused a substantial increase in DHT-dependent toxicity in
ARdNLS112Q motor neurons, it had no effect on the toxicity
of AR112Q motor neurons (Fig. 7B) or of non-transgenic
motor neurons (data not shown), indicating that endogenous
autophagy does not substantially modulate toxicity in
AR112Q motor neurons, in which AR112Q is confined to
the nucleus. Thus, the results of our studies shown here establish that the differential toxicity of nuclear versus cytoplasmic
mutant AR can be explained, in part, by the differential activation of, and AR degradation by, autophagy.
The data presented here reveal that cytoplasmically retained
polyQ-expanded AR (ARdNLS112Q) can be degraded by
autophagy, protecting motor neurons from DHT-dependent
death. The high levels of ARdNLS112Q protein, even in the
face of robust and efficient autophagic degradation, are consistent with the increased transgene copy number in ARdNLS112Q
mice. Despite this increased protein, however, ARdNLS112Q
mice showed reduced motor symptoms. Thus, the increased
ARdNLS112Q protein in the cytoplasm represents a form that
is less toxic than nuclear-confined AR. Whether this form is
non-toxic due to its lack of amino-terminal fragment-producing
proteolysis or to other aspects of AR metabolism that occur
within the nucleus is an active area of investigation. In all,
our observations indicate one mechanism by which cytoplasmic
retention of polyQ-expanded AR is neuroprotective; the mutant
protein is available to be degraded by autophagy. In accordance,
nuclear localization of polyQ-expanded AR likely limits its
access to the autophagic pathway and thus is one mechanism
by which this localization contributes to its toxic effects
within motor neurons.
The potent neuroprotective effects of autophagy in
ARdNLS112Q motor neurons led us to evaluate whether
enhanced activation of the autophagic pathway would
protect neurons from a nuclear localized polyQ-expanded
protein, AR112Q. Pharmacologic induction of both mTORdependent and -independent pathways of autophagy rescued
AR112Q motor neurons from DHT-dependent death. This
intervention, however, had no effect on monomeric levels of
AR112Q. This lack of an effect on AR112Q levels is similar
to findings of a previous study in which autophagy was ineffective at eliminating nuclear inclusions of mutant protein
(63), but is contrary to results in a fly model of SBMA, in
which HDAC6 over-expression (which enhances autophagy)
led to lower steady-state levels of monomeric and aggregated
polyQ-expanded AR (48). It may be that, while monomeric
AR was unchanged in our study, oligomeric and nuclear
aggregated forms of AR112Q were altered; these species
were not evaluated in our spinal cord culture model due to difficulties with their detection. This would be in keeping with
earlier studies showing that nuclear aggregates may be
dynamic in nature (64 – 66). Alternatively, the effects of autophagy on motor neuron viability may be independent of direct
effects on mutant AR. It may be that activation of autophagy
alleviates proteasomal inhibition induced by mutant AR, in
turn enhancing cell viability, as described by Pandey et al.
(48). It may also be that autophagy plays a more general
role, relieving proteotoxic stress induced by polyQ-expanded
nuclear AR, perhaps by promoting the autophagic degradation
of misfolded metastable proteins (67).
In all, these findings indicate that hormone binding and
nuclear localization are essential for the polyQ-expanded AR
to aggregate and induce toxicity within motor neurons. Therefore, nuclear hormone-dependent AR events will be key in
understanding the specific modifications, interactions and
metabolic products responsible for causing disease. Although
hormone withdrawal has proved neuroprotective in mouse
models of SBMA, its effects in SBMA patients have yet to
be firmly established. Moreover, it is expected that therapies
directed at the specific events that lead to the formation of a
toxic AR species within motor neurons will prove to be
more beneficial to patients and cause less side effects, as
they will allow for normal AR function that is otherwise interrupted by hormone withdrawal. The studies herein highlight a
need to focus on the nucleus in SBMA, as well as on the
autophagic pathway when developing these therapies.
MATERIALS AND METHODS
ARdNLS and NLSX3-AR inducible PC12 cell lines
Site-directed mutagenesis (Quick Change II XL, Stratagene)
was performed on a pTRE plasmid (Clontech, Mountain
View, CA), previously engineered to contain full-length
human AR cDNA, bearing 10 or 112 CAGs, to delete the
nucleic acids encoding amino acids 628– 640 of the AR
(within the NLS) (ARdNLS). Mutation and CAG repeat
length were confirmed by sequence analysis.
NLSX3-AR was created as follows: The SV40 NLS in triplicate (NLSX3) was PCR-amplified from pShooterTM pEF/
myc/nuc (Invitrogen, Carlsbad, CA) vector, and an EcoRI
restriction digest site engineered on both the 50 and 30 ends.
An NheI restriction was also engineered just upstream of the
EcoRI site at the 50 end. The PCR product was cloned into
plasmid pCMVAR (16-CAG)DHA (9) (EcoRI site is just 50
of the CTG start of the AR cDNA). The pCMV-NLSX3-AR
(16-CAG)DHA was then digested with NheI and NarI, and
pTRE-AR (112-CAG) was linearized with NheI and partially
digested with NarI. The NLSX3-AR fragment from
pCMVAR(16-CAG)DHA was ligated to pTRE-AR(112-CAG)
(containing full length AR), resulting in pTRE-NLSX3AR(112-CAG). ARdNLS was then cloned into this construct
using NruI and BstBI. All constructs were sequenced to
verify mutation and CAG length.
Stable transfections of Tet-On PC12 cells (Clontech) were
performed using LipofectAMINE Plus (Invitrogen) with a
plasmid conferring hygromycin resistance (pTK-hygromycin).
Stable transformants were selected with 200 mg/ml hygromycin. Single colonies were isolated and expanded and screened
for doxycycline-inducible AR protein expression by slot blot
and western blot analysis using AR(N-20) antibody (Santa
Cruz, Santa Cruz, CA). AR expression levels were adjusted
with various doxycycline concentrations to achieve protein
levels equivalent to AR112Q PC12 cells. Genomic DNA
was extracted from each clone to verify mutation and CAG
1947
length via sequence analysis. Cells were maintained in
normal growth media [Dulbecco’s modified Eagle’s medium
with 10% heat-inactivated horse serum, 5% fetal bovine
serum, 2 mM L-glutamine, 100 units/ml penicillin/streptomycin, 200 mg/ml hygromycin (Invitrogen) and 100 mg/ml
G418 (Mediatech, Manassas, VA)] at 378C, 10% CO2.
Treatment of inducible PC12 cell lines
Stable Tet-On PC12 cell lines were treated with doxycycline
to express AR for various times and with various concentrations of DHT in charcoal-stripped serum-containing cellculture media.
PC12 cell toxicity assay
Stable Tet-On PC12 cell lines (AR10Q, AR112Q,
ARdNLS78Q, NLSX3-AR76Q and NLSX3-ARdNLS63Q)
were treated with doxycycline to express equivalent levels
of AR, in the absence and presence of 10 nM DHT for 12
days. At the end of treatment, cells were harvested and
stained with trypan blue. Two hundred cells were counted
and the percentage of trypan blue-positive cells determined.
Significance was determined with Student’s t-test.
ARdNLS PC12 cell and transgenic mouse constructs
The human AR gene, bearing either 24 CAGs (normal) or 112
CAGs (expanded), was previously cloned into the prion
protein promoter (PrP) construct deleted of coding sequences
(22). The deleted portion of the nuclear localization sequence
of the AR (deleted of nucleic acids encoding amino acids
628 – 640) was cloned into the NruI and BstBI sites of both
the 24 CAG and 112 CAG containing PrP-AR constructs.
DNAs were linearized and the plasmid backbone (pBS)
removed by digestion with NotI, gel-purified and injected
into fertilized oocytes (C57Bl/6), by the Kimmel Cancer
Center Transgenic Facility at Thomas Jefferson University.
Both ARdNLS and AR112Q mice were maintained on a
C57Bl/6 background (Charles River, Wilmington, MA). Founders were screened by genotyping tail clips and brain and
spinal cord lysates from 5-week-old male mice were analyzed
for ARdNLS protein expression compared with those from
age-matched AR112Q SBMA male mice. Additionally, CAG
repeat length was determined by sequence analysis of PCR
products (Laragene, Inc., Los Angeles, CA).
Genotyping mice
DNA from mice was prepared from tail or ear biopsies using
Red Extract-N-Amp Kit (Sigma). Transgenic animals were
identified by PCR of the human AR: forward primer from
the PrP promoter region (50 -ACTGAACCATTTCAACC
GAGC-30 ) coupled with a reverse primer from the AR
sequence 50 to the CAG repeat (50 AGGTGCTGCGCTCGC
GGCCTCT-30 ).
1948
Western blot analysis
Freshly dissected tissue was flash-frozen in liquid nitrogen.
Frozen sections were pulverized in a mortar and pestle on
dry ice and homogenized in either 10 volumes of Triton-DOC
buffer (1% sodium deoxycholate and 0.5% Triton X-100 in
PBS with protease inhibitors) or RIPA buffer (50 mM Tris –
HCl, pH 8.0, 0.15 M NaCl, 0.1% Nonidet P-40, 0.5%
sodium deoxycholate, 0.1% SDS and protease inhibitors).
PC12 cells and cells from primary spinal cord cultures were
lysed with Triton-DOC buffer. All lysates were sonicated
three times for 10 s using a Branson cup sonifier. A portion
of tissue lysates in RIPA was centrifuged at 15 000g for
5 min at 48C for detection of oligomeric species of AR (38).
A DC protein assay (BioRad, Hercules, CA) was performed
to determine protein concentration and lysates were electrophoresed by SDS – PAGE and transferred to 0.45 mm PVDF
(Immobilon-P). Western hybridization was performed using
the following antibodies: AR(N-20), GAPDH (1:1000)
(Santa Cruz Biotechnology) and LC3B (1:500) (NB6001384) (Novus Biologicals, Littleton, CO). Detection was
performed with ECL (Amersham Biosciences, Arlington
Heights, IL).
Behavioral analysis
Every 4 weeks, beginning at 8 weeks of age, an age-matched
cohort of ARdNLS24Q males, AR112Q males, ARdNLS112Q
males, ARdNLS112Q females, non-transgenic males and nontransgenic females was subject to various measures of motor
function. Mice were tested during the light phase of a 12 h
light/dark cycle for their latency to fall off a steadily accelerating rotarod (4– 40 rpm over 10 min) (Ugo Basile, Comerio,
VA-Italy). During the first week of testing, mice were tested
four times per day for 3 consecutive days. The first 2 days constituted a learning period and data collected on the third day
were used for analysis. For subsequent testing sessions, mice
were only subjected to rotarod for 1 day, as statistical analysis
revealed no loss of statistical power under this regimen
(unpublished data). Mice were allowed a rest period of at
least 15 min between testing sessions. Scores were analyzed
by two-way repeated measures ANOVA with a Tukey post
hoc analysis using SigmaStat 3.0 software (SPSS Inc.,
Chicago, IL). A grip strength meter (Columbus Instruments,
Columbus, OH) was used to measure the force exerted by a
mouse as it was pulled across a grid by its tail. Grip strength
was measured for forepaws only or hindpaws and forepaws
together. Six measures were taken for both measures of grip
strength and the lowest and highest scores for each animal
dropped. An average for each animal was used for statistical
analysis. Significance was determined with two-way repeated
measures ANOVA and a Tukey post hoc analysis (SigmaStat).
ARdNLS112Q or non-transgenic spinal cords were pooled
separately, plated for culture and incubated for 3 weeks in
media conditioned by glial culture from 13.5-day-old nontransgenic brain [MEM, 35 mM NaHCO3, 0.5% dextrose, 1%
N3, 10 nM 2.5 S NGF (added after conditioning)]. During
the development of this culture system, motor neurons were
identified using antibodies to choline acetyltransferase,
neuron-specific enolase and neurofilament heavy chain
(SMI32). Motor neurons were identified to have much larger
cell bodies relative to other spinal neurons and large, tapering,
highly branched dendrites with a fibrillar appearance. In our
experiments presented here, SMI32 immunoreactivity and
morphology were used to identify motor neurons. Three
weeks after initiation, cultures were treated with or without
10 mM DHT for 7 days. Additional reagents/drugs were administered for the last 3 days of the 7 day treatment period [5 mM
3-Methyladenine (3-MA), 100 mM Trehalose (Sigma), 2.5 mM
AKT inhibitor X [10-(40 -N-diethylamino)butyl)-2-chlorophenoxazine] (AKTi) (Calbiochem, San Diego, CA)]. Three
wells of each motor neuron culture line and treatment group
were immunostained as described in what follows. Motor
neurons were determined by SMI32 stain and morphology
and counted. Significance was determined by a Student’s
t-test.
Immunofluorescence
Dissected fresh whole brain and spinal cord were frozen in
OCT, then sectioned with a cryostat (7 mm). Tissue sections,
motor neuron cultures and PC12 cells were fixed with 4% paraformaldehyde for 10 min, washed in PBS, permeabilized with
0.3% Triton X-100 for 15 min (cells only), blocked in 1.5%
goat serum (Jackson ImmunoResearch, West Grove, PA) for
20 min and incubated for 60 min in primary antibody diluted
in 1.5% goat serum. Tissue sections or cells were washed in
PBS and incubated for 30 min with secondary antibodies
(FITC- or Texas Red-conjugated) (Jackson ImmunoResearch,
West Grove, PA), washed in PBS, incubated for 10 min with
Hoechst (2 mg/ml), washed in PBS and mounted in Vectashield (Vector Laboratories, Burlingame, CA). Fluorescence
was visualized with a Leica (Leica Microsystems GmbH,
Wetzlar, Germany) microscope; images were captured with
a Leica camera and compiled with IP Lab software (BD Biosciences, Rockville, MD). Antibodies used include AR(N-20),
ARH280, AR441 (1:100) (Santa Cruz), AR-318 (Vector Laboratories Burlingame, CA), SMI32 (1:1,000) (Sternberger
Monoclonal, Baltimore, MD) and LC3B (NB600-1384)
(1:200) (Novus Biologicals).
SUPPLEMENTARY MATERIAL
Supplementary Material is available at HMG online.
Primary motor neuron cultures
Dissociated spinal cord cultures were established according to
Roy et al. (68). In brief, spinal cords were dissected on ice
from 13.5-day-old embryos in dissection media (0.1% dextrose, 2% sucrose, 1.4 mM NaCl, 5.4 mM KCl, 0.17 mM
Na2HPO4, 22 mM KH2PO4, 9.9 mM HEPES). Genotyping
was performed from a tail biopsy. Transgenic AR112Q,
ACKNOWLEDGEMENTS
We are grateful to Carlisle Landel, Ph.D., Director, Transgenic
and Gene Targeting Facility at Thomas Jefferson University
for creation of transgenic mice and for thoughtful discussions.
We also thank Heather Durham, Ph.D., Montreal Neurological
Institute, McGill University, Montreal, for helpful advice on
the initiation of spinal cord cultures.
Conflict of Interest statement. None declared.
FUNDING
This work was supported by the National Institutes of Health
(NS047381 and NS32214 to D.E.M.); (2NS045191 and
2P01AG022074 to S.F.); (DK07705 supporting H.L.M.);
The Taube-Koret Center for Huntington’s Disease Research
(S.F.); and a Milton Wexler Award and Fellowship from the
Hereditary Disease Foundation (A.T.).
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Published December 28, 2009
JCB: Article
IKK phosphorylates Huntingtin and targets it for
degradation by the proteasome and lysosome
Leslie Michels Thompson,1,2,3 Charity T. Aiken,4 Linda S. Kaltenbach,7 Namita Agrawal,4 Katalin Illes,1
Ali Khoshnan,8 Marta Martinez-Vincente,9,10 Montserrat Arrasate,11 Jacqueline Gire O’Rourke,3 Hasan Khashwji,2
Tamas Lukacsovich,4 Ya-Zhen Zhu,1 Alice L. Lau,1 Ashish Massey,9 Michael R. Hayden,12 Scott O. Zeitlin,13
Steven Finkbeiner,14 Kim N. Green,2 Frank M. LaFerla,2 Gillian Bates,15 Lan Huang,4,5 Paul H. Patterson,8
Donald C. Lo,7 Ana Maria Cuervo,9 J. Lawrence Marsh,4,6 and Joan S. Steffan1
Department of Psychiatry and Human Behavior, 2Department of Neurobiology and Behavior, 3Department of Biological Chemistry, 4Department of Developmental and Cell
Biology, 5Department of Physiology and Biophysics, and 6Department of Pathology and Developmental Biology Center, University of California, Irvine, Irvine, CA 92697
7
Center for Drug Discovery and Department of Neurobiology, Duke University, Durham, NC 27704
8
California Institute of Technology, Pasadena, CA 91125
9
Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, NY 10461
10
Institute of Neuropathology, IDIBELL-Hospital Universitari de Bellvitge, L’Hospitalet de Llobregat, 08907 Barcelona, Spain
11
Division of Neuroscience, Center for Applied Medical Research, University of Navarra, E-31008 Pamplona, Spain
12
University of British Columbia, Vancouver, BC, Canada V6T 1Z4
13
Department of Neuroscience, University of Virginia, Charlottesville, VA 22908
14
Departments of Neurology and Physiology, Gladstone Institute of Neurological Disease, University of California, San Francisco, San Francisco, CA 94158
15
Department of Medical and Molecular Genetics, King’s College London School of Medicine, King’s College London, London SE1 9RT, England, UK
E
xpansion of the polyglutamine repeat within the
protein Huntingtin (Htt) causes Huntington’s disease, a neurodegenerative disease associated with
aging and the accumulation of mutant Htt in diseased
neurons. Understanding the mechanisms that influence
Htt cellular degradation may target treatments designed
to activate mutant Htt clearance pathways. We find
that Htt is phosphorylated by the inflammatory kinase IKK,
enhancing its normal clearance by the proteasome and
lysosome. Phosphorylation of Htt regulates additional
post-translational modifications, including Htt ubiquitination, SUMOylation, and acetylation, and increases Htt nuclear localization, cleavage, and clearance mediated by
lysosomal-associated membrane protein 2A and Hsc70.
We propose that IKK activates mutant Htt clearance until
an age-related loss of proteasome/lysosome function promotes accumulation of toxic post-translationally modified
mutant Htt. Thus, IKK activation may modulate mutant Htt
neurotoxicity depending on the cell’s ability to degrade
the modified species.
Downloaded from jcb.rupress.org on April 8, 2010
THE JOURNAL OF CELL BIOLOGY
1
Introduction
Abnormal accumulation of misfolded and aggregated protein
in affected neurons is a hallmark of many neurodegenerative
diseases associated with aging. The major pathways of protein
clearance in the cell are performed by the proteasome and the
lysosome, which both become compromised with age (Cuervo
et al., 2005; Martinez-Vicente and Cuervo, 2007; Chondrogianni
and Gonos, 2008; Tonoki et al., 2009). Parallel with reduced
turnover, proteins mutated in familial neurodegenerative
diseases accumulate and cause dysfunction and death, and
accompanying symptoms. Examples include the polyglutamine
(polyQ) disease protein Huntingtin (Htt) in Huntington’s
disease (HD), tau in frontotemporal dementias (FTD), -synuclein
in Parkinson’s disease (PD), ataxin-1 in spinocerebellar ataxia
1 (SCA1), and SOD1 in amyotrophic lateral sclerosis (ALS).
Post-translational modification of target proteins can
regulate their clearance from cells. Phosphorylation regulates
protein degradation, alters subcellular localization, and/or creates
phosphodegrons/binding motifs for interactors that regulate
secondary modifications such as ubiquitination, SUMOylation,
and acetylation. For instance, phosphorylation of HSF1, MEF2,
Correspondence to Joan S. Steffan: [email protected]
© 2009 Thompson et al. This article is distributed under the terms of an Attribution–
Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication
date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a
Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license,
as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
Abbreviations used in this paper: CMA, chaperone-mediated autophagy; HD,
Huntington’s disease; Htt, Huntingtin; LAMP-2A, lysosomal-associated membrane
protein 2A; polyQ, polyglutamine; wt, wild type.
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JCB • VOLUME 187 • NUMBER 7 • 2009
Results
The IKK complex directly
phosphorylates Htt
The N-terminal 17 amino acids of Htt contain a number of
potentially modifiable residues (Fig. 1 A). In addition to the
lysines at 6, 9, and 15, which can be SUMO modified and ubiquitinated (Steffan et al., 2004), three residues are possible phosphorylation targets (T3, S13, and S16).
Sequence evaluation for conserved motifs revealed that
Htt residues 11–18 share sequence similarity with residues
642–649 of FOXO3a (Fig. 1 B), a substrate of IKK at S644
analgous to Htt S13 (Hu et al., 2004). Because expanded polyQ
Httex1p activates the IKK complex in cell culture and transgenic
mice, and interacts with IKK- in vitro (Khoshnan et al., 2004),
IKK emerged as a candidate kinase that might target Htt S13.
IB kinase (IKK) is composed of three subunits: IKK- and
IKK- are homologous catalytic subunits, and IKK- is a regulatory subunit. As a first step to evaluate phosphorylation of this
domain and a potential role for IKK, mass spectrometry was
used. 25QP-HBH, a His-tagged unexpanded form of Httex1p
was transiently cotransfected with IKK- into ST14A cells,
purified by nickel enrichment under denaturing conditions, digested with chymotrypsin, and analyzed by reverse-phase liquid
chromatography coupled to tandem mass spectrometry. Phosphorylation of both S13 and S16 was observed in the presence
of IKK (Fig. 1 C).
Using Htt peptides phosphorylated at either S13 or S16 as
antigens, affinity-purified rabbit polyclonal antisera, designated
anti–S13-P and anti–S16-P, were generated (Fig. S1). Overexpression of IKK- or IKK- but not IKK- increased phosphorylation of both unexpanded (25QP) and expanded (46QP)
forms of Httex1p with a C-terminal His-HA-HA-His (H4)
tag in ST14A cells cotransfected with Httex1p-H4 and IKK
subunits (Fig. 1 D). Phosphorylation of 46QP-H4 appears less
efficient than 25QP-H4 and phosphorylation of 25QP-H4 is
associated with its reduced abundance. To first determine
whether IKK can directly phosphorylate Htt and determine the
specific residue involved, recombinant IKK phosphorylation of
purified Htt was tested in vitro. S13 of both purified unexpanded
(25QP) and expanded (46QP) Httex1p was phosphorylated by
both IKK- and IKK- (Fig. 1 E), whereas phosphorylation of
S16 was not observed (not depicted). These results suggest that
S13 is a direct target of IKK and that the phosphorylation of
S16 observed with IKK overexpression in cell culture may be
primed by phosphorylation of S13 by IKK. Alternatively, the
sensitivity of anti–S16-P antisera may be inadequate to detect
the in vitro modification.
IKK-activated phosphorylation of
Httex1p regulates its post-translational
modification and subcellular localization
As described above, phosphorylation can regulate posttranslational modification of adjacent lysine residues (Hunter,
2007). The role of Htt S13 and S16 phosphorylation on Htt ubiquitination, SUMOylation, and acetylation of Httex1p was tested.
We previously found that 97QP Httex1p is polyubiquitinated
and GATA-1 activates their SUMOylation (Hietakangas et al.,
2006), phosphorylation of p53 and RelA activates their acetyl
ation (D’Orazi et al., 2002; Hofmann et al., 2002; Chen et al.,
2005), and phosphorylation of IB and FOXO3a activates
their ubiquitination (Karin and Ben-Neriah, 2000; Karin et al.,
2002; Hu et al., 2004). In turn, these modifications may ultimately target the protein for degradation (Hernandez-Hernandez
et al., 2006; Hietakangas et al., 2006; Hunter, 2007; Wu
et al., 2007; Zuccato et al., 2007; Jeong et al., 2009). As protein
clearance mechanisms become impaired upon aging, modified proteins normally targeted for degradation by posttranslational modification may accumulate and disease-causing
proteins take on toxic functions (Orr and Zoghbi, 2007; Shao
and Diamond, 2007).
HD is a member of a family of polyQ repeat expansion
diseases characterized by the accumulation and aggregation of
mutant Htt protein in diseased neurons (Orr and Zoghbi, 2007).
In HD, when the repeat expands above 40, disease will manifest, typically striking in mid-life (Walker, 2007). Above 65
repeats, a juvenile form of the disease occurs. The polyQ
expansion exists within the context of a large 350-kD protein;
however, expressing just the N-terminal fragment of Htt encoded by exon 1 (Httex1p), which contains a highly expanded
polyQ repeat, can precipitate an aggressive HD-like disease in
transgenic mice and flies (Mangiarini et al., 1996; Steffan
et al., 2001). The first 17 amino acids of Htt can mediate aggregation, subcellular localization and membrane association, stability, and cellular toxicity, each of which are implicated in HD
pathogenesis (Steffan et al., 2004; Luo et al., 2005; Warby
et al., 2005, 2009; Anne et al., 2007; Rockabrand et al., 2007;
Atwal and Truant, 2008). The potential for Htt post-translational
modification to have a disease-modifying role has recently
emerged as a consistent theme, with regulatory functions implicated for other sites within the full-length protein as well,
including phosphorylation at S421 by Akt and S434, S1181,
and S1201 by Cdk5 (Humbert et al., 2002; Luo et al., 2005;
Warby et al., 2005; Anne et al., 2007), SUMOylation and ubiquitination at K6, K9, and K15 (Steffan et al., 2004), palmitoylation
at C214 (Yanai et al., 2006), and acetylation at K444 (Jeong
et al., 2009). The regulatory properties of post-translational
modifications extend to other polyQ repeat diseases, most
notably phosphorylation of S776 in expanded ataxin-1, the
mutant protein in SCA1 (Orr and Zoghbi, 2007).
We evaluated the effect of phosphorylation within the
first 17 amino acids of Htt on its subcellular localization, downstream post-translational modifications, and protein clearance.
This domain contains two serines at positions 13 and 16, which
are adjacent to the lysines found to be modified by SUMO and
ubiquitin (Steffan et al., 2004). We demonstrate that the IKK
complex, previously shown to directly interact with Htt (Khoshnan
et al., 2004), phosphorylates Htt S13 and may activate phosphorylation of S16. Phosphorylation of these residues promotes
modification of the adjacent lysine residues and activates Htt
clearance in a manner requiring both the proteasome and
lysosome. We find that expansion of the Htt polyQ repeat
may reduce the efficiency of this phosphorylation, potentially
contributing to the accumulation of mutant Htt.
(Steffan et al., 2004). Making a mutant that cannot be phosphorylated on S13 (S13A) reduces this polyubiquitination,
whereas mimicking phosphorylation of S13 (S13D) retains its
polyubiquitination (Fig. 2 A). If the dual phosphorylation we
see activated by IKK is mimicked (S13,16D), a reduction in
Htt polyubiquitination is again observed (Fig. 2 A). Consistent
with this, overexpression of IKK reduces polyubiquitination of
97QP Httex1p (Fig. S2 A). The S13A and S13,16D mutants
also demonstrate reduced mono-SUMOylation of 97QP
Httex1p, whereas S13D retains its SUMOylation (Fig. 2 B).
Overexpression of IKK leads to a reduction in 97QP monoSUMOylation and an increase in its poly-SUMOylation
(Fig. S2 B). Therefore, IKK may modulate ubiquitin and
SUMO addition to Httex1p, two modifications globally tied to
protein clearance mechanisms.
Figure 1. IKK directly phosphorylates Htt. (A) The first 17 amino acids of the Htt protein contain three residues that may be phosphorylated (red) and
three modifiable lysine residues (blue). (B) Htt S13 is within a domain similar to FOXO3a S644. (C) Mass spectrometry analysis shows that Htt serines
13 and 16 can be phosphorylated. 25QP-HBH was purified under denaturing conditions from St14A cells cotransfected with IKK-. ESI-MS/MS spectra
were obtained after chymotryptic digestion and collision-induced dissociation (CID) for N-terminally acetylated and diphosphorylated peptide on S13 and
S16 Ac-ATLEKLMKAFEpSLKpSF, with the parent ion, [MH2]+2, at m/z 1023.03+2 (M = 2044.06 D). (D) Htt phosphorylation of S13 and S16, detected with
phospho-specific antibodies, is activated with coexpression of IKK- or IKK-. Httex1p was purified from St14A cells transiently transfected with 25QP-H4
or 46QP-H4 with vector control or plasmids encoding subunits of IKK. Total Htt was detected with CAG53b antibody, and myc-actin transfection control
detected with anti-myc antibody. (E) IKK- and IKK- directly phosphorylate Htt S13 in vitro. An in vitro kinase assay was performed with 75 ng recombinant
IKK- or IKK- protein, and wt (SS) or S13,16A (AA) mutant 25Q or 46Q purified Httex1p-H4. Htt was detected with CAG53b or anti-S13-P.
In addition to the detection of phosphorylation at both
S13 and 16, acetylation of lysine K9 was detected together
with modification of S13 by mass spectrometry upon exogenous IKK overexpression (Fig. 2 C). To evaluate this acetyl
ation further and the possible influence of phosphorylation,
affinity-purified polyclonal antiserum was generated against a
K9-acetyl, S13-phospho, S16-phospho Htt peptide (anti–K9-Ac;
Fig. S1). The antibody recognizes Htt 25 or 46QP in the
presence of exogenous IKK-; however, it shows little to no
immunoreactivity without IKK coexpression (Fig. 2 D). Acetyl
ation can be mimicked by a lysine (K) to glutamine (Q) substitution and phosphorylation mimicked by either a serine (S) to
aspartic acid (D) or to glutamic acid (E) substitution. Using a
K9Q, S13,16E (QEE) mimic, immunoreactivity was observed
in the absence of IKK, confirming the specificity of the antibody
Phosphorylation activates Htt degradation • Thompson et al.
1085
Figure 2. Phosphorylation of Httex1p regulates its ubiquitination, SUMOylation, acetylation, and nuclear localization. (A and B) Phosphorylation of
serines 13 and 16 regulates mutant Httex1p ubiquitination and SUMOylation. St14A (A) or HeLa (B) cells were transiently transfected with vector or
HIS-ubiquitin (A) or HIS-SUMO-1 (B) and control and mutant 97QP VL* Httex1p. Conjugated proteins were purified under denaturing conditions by Ni-NTA
magnetic nickel beads and Htt detected with anti-Htt CAG53b by Western analysis. (C) Mass spectrometry analysis shows that Htt S13 phosphorylation
can occur with K9 acetylation. 25QP-HBH was purified from St14A cells cotransfected with IKK- and CBP and treated for 2 h with histone deacetylase
inhibitors 200 mM Trichostatin A/5 mM Nicotinamide. ESI-MS/MS spectra were obtained after chymotryptic digestion and collision-induced dissociation
(CID) for a peptide acetylated at K9 and phosphorylated at S13 MAcKAFEpSLKSF, [MH2]+2 at m/z 655.30+2 (M = 1308.60 D). (D) IKK- overexpression
increases phosphorylation of Htt S13 and acetylation of K9. St14A cells were transiently transfected with Httex1p-H4 with 25 or 46Qs, +/ IKK- or with
46QP QEE-H4. Htt was purified and subjected to Western analysis with anti-K9-Ac, anti-S13-P, and CAG53b. (E) Mimicking phosphorylation significantly
increases nuclear localization in primary neurons. Primary cortical neurons were cotransfected with pcDNA3.1-mRFP and 97QP-GFP or 97QP-DD-GFP
plasmids. The subcellular distribution of these polypeptides was examined by measuring the fluorescence intensity of GFP, to which they are fused, in
regions of the nucleus and cytoplasm for each cell. The extent to which these polypeptides localized preferentially to the nucleus or the cytoplasm was
determined by calculating the ratio of nuclear/cytoplasmic GFP fluorescence intensity and comparing the distribution of the two polypeptides by t test. Error
bars indicate SEM in arbitrary units.
1086
Phosphorylation of Htt by
IKK activates Httex1p and 586aa Htt
fragment clearance
We then asked whether phosphorylation by IKK might also regulate Htt stability. Unexpanded (25QP) and expanded (46QP)
polyQ Httex1p were cotransfected into ST14A cells with IKK-,
as this subunit of IKK had the greatest Htt phosphorylation
activity in cell culture (Fig. 1 D), and levels of Htt evaluated.
Unexpanded Htt showed a dependence on IKK- for enhanced
clearance relative to a myc-actin transfection control, and this
effect persisted in the presence of either the specific proteasome
inhibitor epoxomicin or the lysosome inhibitors ammonium
chloride/leupeptin (Fig. 3, A and B). Proteasome inhibitors
were able to block basal and IKK-–induced degradation of
25QP; however, they were not able to abolish the differences
between both types of degradation, suggesting that the reduction in cellular levels of unexpanded 25QP Httex1p mediated
by IKK involves proteasome-dependent and -independent
degradation of the protein. Inhibition of the lysosome reduced
the effect of IKK- on 25QP clearance, implicating lysosomal
involvement. Interestingly, the IKK-–induced degradation
of Httex1p was largely reduced for the expanded form of the
protein, although the basal degradation of the protein was unperturbed and still dependent on the proteasome system. Phosphorylation of unexpanded polyQ Httex1p may therefore target
it for degradation by both the proteasome and lysosome. Expansion of the polyQ repeat to 46Qs inhibited this IKK-–mediated
reduction in Httex1p abundance (Fig. 3 A), which may at least
partially be the result of its less efficient phosphorylation by
IKK- (Fig. 1 D).
The abundance of Httex1p AA, EE, and QEE mutants
were next compared with control Httex1p or to Httex1p in
which all the lysines were mutated to arginine (K6,9,15R or 3R),
also in the presence of epoxomicin or ammonium chloride/
leupeptin. Consistent with the potential destabilization of unexpanded polyQ by phosphorylation, unexpanded (25QP)
EE and QEE Httex1p levels were lower than control and AA
(Fig. 3 C). In contrast, elimination of modifiable lysines (3R
25QP) had no effect on soluble Htt levels, but did increase levels of insoluble Htt. This result is in contrast to what we previously reported for a highly expanded 3R mutant (3R97QP;
Steffan et al., 2004); however, we did not use filter retardation
assays together with Western analysis to examine levels of insoluble Htt in those studies, and now understand that this 97QP
3R mutant is detectable, however mostly in the insoluble fraction. Inhibition of the proteasome or the lysosome increased
levels of the unexpanded phosphomimetics (25QP EE and
QEE), suggesting that the proteasome and lysosome may both
be involved in clearance of phosphorylated and acetylated
forms of unexpanded Htt fragments.
In contrast to results with unexpanded Htt, expanded control and mutant 46QP proteins accumulated in both soluble and
insoluble fractions (Fig. 3 C, bottom); this accumulation was
also influenced by proteasomal and lysosomal inhibition. The
46QP EE phosphomimetic consistently ran on SDS-PAGE as a
doublet, suggesting the presence of a phosphorylated intermediate that is not well cleared in the presence of the expanded repeat. The doublet was not observed with the acetylation mimetic
QEE, implicating lysine 9 as a critical residue in this clearance
mechanism and possibly suggesting an alternative lysine 9 posttranslational modification other than acetylation in doublet formation. We conclude that expansion of the polyQ repeat in
Httex1p reduces the efficiency of phosphorylation-activated
IKK-mediated clearance.
To further characterize IKK-mediated Htt phosphory
lation and clearance, a larger Htt fragment comprised of 586
amino acids (aa) with and without IKK- (Fig. 3 D) was expressed in ST14A cells. Co-expression of IKK- significantly
reduced levels of unexpanded Htt (586 aas with 15Qs). Although there was a reduction in levels of total unexpanded 586
(15Q) with IKK- overexpression, as determined using anti-Htt
3B5H10 (Fig. 3 D) or anti-Htt EM48 (Fig. S4 A), a significant
increase in an immunoreactive species was observed when the
anti–S13-P antibody was used for detection, particularly upon
longer-term lysosomal inhibition. Expanded repeat 128Q 586aa
fragment levels were not reduced with overexpression of IKK-,
and S13-phosphorylated, expanded 128Q fragment was not
detectible above background levels, again suggesting a reduced
ability of the mutant Htt protein to be phosphorylated and
cleared. Collectively, these data show that IKK- can increase
phosphorylation and reduce levels of unexpanded polyQ
Htt fragments in a manner dependent on both the proteasome
and the lysosome, and that expansion of the polyQ repeat
inhibits this effect.
for this Htt species (Fig. 2 D). Collectively, these data suggest
that IKK-mediated phosphorylation may regulate ubiquitination, SUMOylation, and acetylation within the first 17 amino
acids of Htt.
Because the first 17 amino acids of Htt can function as
a cytoplasmic retention signal and regulate the association
of Htt with mitochondria, Golgi, endoplasmic reticulum, late
endosomes, and autophagic vesicles (Steffan et al., 2004;
Atwal et al., 2007; Rockabrand et al., 2007), and because
overexpression of IKK- was previously demonstrated to promote expanded polyQ Httex1p nuclear localization (Khoshnan
et al., 2004), we tested whether phosphorylation of this
domain could influence its cellular localization. Either phosphomimetic (S13,16D/S13,16E or DD/EE) or phosphoresistant
(S13,16A, or AA) forms of Httex1p were used to assess the
consequence of phosphorylation on soluble cellular localization. Fluorescence of mutant Httex1p with 97Qs fused to
GFP was first assessed in NIH-3T3 cells (Fig. S3 A), demonstrating largely cytoplasmic localization for both 97QP-GFP
and 97QP-AA-GFP. In contrast, phosphomimetic 97QPDD-GFP and 97QP-EE-GFP displayed increased nuclear
localization. Similarly, a statistically significant increase in
nuclear localization of expanded 97QP-DD-GFP over control
97QP-GFP was observed upon transfection into primary
cortical neurons (Fig. 2 E). Nuclear localization was also
enhanced using phophomimetics of unexpanded Httex1p
(25QP-EE-GFP) compared with wild type (wt) or AA in 3T3
cells, suggesting this process may extend to normal Htt or Htt
fragments (Fig. S3 B).
1087
Figure 3. Phosphorylation of unexpanded polyQ Httex1p and 586aa Htt is associated with its reduced abundance in cell culture. (A) Levels of un
expanded polyQ Httex1p are reduced with overexpression of IKK-; this effect is inhibited with expansion of the polyQ repeat. 25QP-H4 or 46QP-H4
was cotransfected with myc-actin and with vector or IKK- into St14A cells. Cells were treated for 16 h with DMSO, 100 nM epoxomicin in DMSO, or
20 mM ammonium chloride/100 µM leupeptin in DMSO. Lysates were subjected to filter-retardation assay and Western analysis using anti-myc to detect
myc-actin, and anti-HA to detect Httex1p. (B) IKK- overexpression reduces levels of unexpanded polyQ Httex1p in the presence of proteasome or lysosome
inhibition. Scion software was used to quantitate triplicate levels of 25QP-H4 from the experiment represented in A, normalized to levels of myc-actin transfection control, within each treatment group: control, epoxomicin, or ammonium chloride/leupeptin. (C) Mimicking phosphorylation of unexpanded polyQ
Httex1p reduces its abundance in cell culture; this effect is reduced with expansion of the polyQ repeat. 25QP-H4 or 46QP-H4, wt control or QEE, EE, AA,
or 3R were cotransfected with myc-actin into St14A cells. Cells and lysates were treated as in A. (D) Levels of phosphorylated unexpanded polyQ 586aa
Htt accumulate with inhibition of the proteasome or the lysosome; phosphorylation is reduced with expansion of the polyQ repeat. 15Q or 128Q 586aa
Htt constructs were cotransfected into St14A cells with myc-actin and with vector or IKK-. Cells were treated for 4 h with DMSO or 100 nM epoxomicin in
DMSO (to eliminate any possible effect on the lysosome by epoxomicin), or for 16 h with water or 20 mM ammonium chloride/100 µM leupeptin in water.
Lysates were subjected to filter-retardation assay and Western analysis using anti-myc, anti–S13-P, and anti-Htt 3B5H10.
1088
Endogenous wt full-length Htt is
phosphorylated by IKK-
The above results show that Htt fragments can be phosphorylated by IKK in cells and in vitro. We next examined whether
exogenous expression of IKK subunits could phosphorylate
full-length, endogenous wt Htt in ST14A cells to determine
whether this type of regulation may be involved in an endogenous clearance mechanism. IKK- overexpression increased the
levels of acetylated and S13-phosphorylated full-length Htt
and Htt fragments in ST14A cells (Fig. 4 A). To ensure that this
effect is specific to IKK, the ability of IKK- to enhance immuno
reactivity to both antibodies was eliminated in the presence
of an shRNA pool against IKK- (Fig. 4 A). As a control, levels
of phosphorylated IB, a defined IKK substrate targeted for
degradation by its phosphorylation (Karin and Ben-Neriah,
2000), were also evaluated in this experiment, and levels (Fig. 4 A,
boxed) paralleled that of phosphorylated and acetylated Htt.
Full-length acetylated and phosphorylated Htt, as well as
phosphorylated Htt fragments, were again increased by inhibition of either the proteasome or the lysosome (Fig. 4 B), suggesting that they may serve as intermediates in the wt Htt
degradation process. High molecular weight full-length S13phosphorylated Htt species were observed upon IKK-
overexpression (Fig. 4 B), which may reflect full-length Htt
post-translational modification by SUMO or ubiquitin as we
observed for Httex1p (Fig. 2, A and B). Using a well-described
anti-Htt antibody, MAB2166 (Millipore), which is uniquely
sensitive enough to detect endogenous rat Htt in ST14A
whole-cell lysate, we did not observe either a loss of endogenous Htt in cells overexpressing IKK- or an accumulation
with inhibition of the proteasome or lysosome, possibly suggesting that the levels of modified Htt that are modulated
represent a small percentage of the total. However, MAB2166
may not interact well with the phosphorylated Htt species.
Within the epitope recognized by MAB2166, Htt residues
414–503, S421 (Humbert et al., 2002), and S434 (Luo et al.,
2005) are phosphorylated by Akt and Cdk5, respectively, and
K444 is acetylated (Jeong et al., 2009). This acetylation
at K444, involved in Htt lysosomal clearance, destroys the
MAB2166 epitope (Krainc, D., personal communication),
supporting the possibility that modifications eliminate reactivity with MAB2166. Although we have not determined whether
these modifications occur at the same time as phosphorylation
of S13 by IKK, Akt acts upstream of IKK activation (Dan
et al., 2008) and it is possible that modification of this epitope
concurrent with S13 phosphorylation may reduce the ability
of MAB2166 to recognize the Htt species being cleared.
We find that MAB2166 does not strongly recognize immunoprecipitated S13/S16-phosphorylated or K9-acetylated Htt
species in cells overexpressing IKK-, whereas Ab1 and
MAB5490/1H6, antibodies that recognize the N-terminal
domain of Htt, do recognize these modified forms (Fig. S4 B).
In addition, Htt antibodies EM48 (recognizing the first 256 aa
of human Htt with a deletion of the polyQ stretch [Gutekunst
et al., 1999]) and 3B5H10 (raised against GST-human Htt
171aa-66Q [Peters-Libeu et al., 2005]) detect reduced abundance
of unexpanded human 15Q 586aa fragment with exogenous
expression of IKK-, whereas MAB2166 does not, supporting
the idea that the cleared Htt species may not be not recognized
well by this antibody (Fig. S4 A).
In addition to genetic modulation of IKK, pharmacological activation of IKK with IL-1 or TNF- was tested for activation of Htt S13 phosphorylation. ST14A cells were treated
with these standard IKK-activating cytokines for 15, 30, 60, and
120 min (Fig. 4 C). Both Il-1 and TNF- increased levels
of phosphorylated full-length and fragmented Htt at 60 and
120 min, similar to phosphorylated IB. S13-phosphorylated
Htt and phosphorylated IB were both reduced at 15 and 30 min,
paralleling the increased clearance of total IB at these time
points. Total full-length Htt levels were not reduced as assessed
by MAB2166 on Western analysis, but an 180-kD-sized set
of N-terminal fragments recognized by anti-Htt Ab1 showed a
possible decrease in abundance at 15 and 30 min and increased
abundance at 60 and 120 min, similar in trend to robust effects
on total IB. S13-phosphorylated Htt and Htt fragments accumulated in cells treated for 60 and 120 min with Il-1. These
phosphorylated Htt species accumulated even further at 120 min
of IL-1 treatment with either epoxomicin or ammonium
chloride/leupeptin, suggesting both proteasomal and lysosomal
involvement (Fig. 4 D).
Endogenous S13-phosphorylated Htt was next analyzed
by immunofluorescence. Without exogenous IKK-, only mitotic cells stained with anti–S13-P (Fig. 5 A), representing
a small fraction of the cell population. In cells that were transiently transfected with FLAG–IKK-, the phosphorylated
species of Htt was detected in cells with exogenous IKK-
expression in a variety of localization patterns (Fig. 5 B and
Fig. S3 C). This staining was specific for phosphorylated S13, as
it could only be competed away with a 1–17aa peptide phosphory
lated on serine 13, but not with the corresponding unmodified
peptide (not depicted). S13-phosphorylated Htt and K9-acetyl
Htt immunoreactivity was also detected in FLAG–IKK-
nucleofected mouse striatal progenitor cells, Hdh7/7 (Fig. 5 B;
unpublished data).
Proteins involved in lysosomal and
proteasomal clearance mechanisms modify
levels of phosphorylated Htt
The pronounced effect of the lysosomal inhibitors on the intracellular levels of phosphorylated S13 Htt compared with the
unmodified protein (Fig. 3 D and Fig. 4, B and D), and the
distinctive punctuate pattern observed in the immunofluorescence studies with the anti–S13-P antibody compatible with
lysosomal association of the modified protein (Fig. 5 and Fig. S3 C),
led us to further characterize the mechanism mediating the
lysosomal degradation of S13-phophorylated Htt. Because
lysosomal inhibition increases levels of the S13 phosphospecies, the expectation is that proteins involved in regulating
lysosomal activity could also influence levels of phosphorylated
Htt. The lysosomal-associated membrane protein 2A (LAMP-2A)
mediates selective autophagy of proteins that contain KFERQlike Hsc70 binding sequences in mammalian cells through
a process known as chaperone-mediated autophagy (CMA;
Massey et al., 2006b). Similarly, Atg7 is essential for autophagy
1089
Figure 4. Phosphorylated and acetylated endogenous wt Htt accumulates with inhibition of the proteasome and lysosome. (A) Overexpression of IKK-
increases phosphorylation of endogenous Htt and IB in cell culture. St14A cells were transiently transfected with vector, IKK-, or IKK-, together with
vector or two different pools of anti–IKK- shRNAs; a control pool that did not silence IKK well (pool 1) and one pool that was effective in silencing IKK
(pool 2), as assessed by levels of FLAG-IKK- and - by Western. Lysates were subjected to Western analysis with anti-Htt MAB2166, anti–S13-P, anti–K9-Ac,
anti-phosphoserine 32 IB (IB-P), anti–IB, anti–-tubulin, and anti-FLAG to detect FLAG-tagged IKK- and IKK-. Bands the size of full-length
endogenous Htt (350 kD) are shown by the arrow on the left. Boxed bands show the reduction in phosphorylated IB with IKK shRNA. (B) Phosphorylated and acetylated Htt accumulate with inhibition of the proteasome or the lysosome. St14A cells were transiently transfected with IKK- or vector, and
were treated as in Fig. 3 D. Lysates were subjected to Western analysis with anti–-tubulin, and anti-Htt antibodies MAB2166, anti–S13-P or anti–K9-Ac.
(C) Pharmacological activation of the IKK complex modulates levels of phosphorylated Htt and IB. St14A cells were incubated with 20 ng/ml TNF- or
IL-1 over a time course. Lysates were subjected to Western analysis as in A with additional detection by anti-Htt Ab1. (D) IL-1–induced Htt S13-phosphorylated
species accumulate with inhibition of the proteasome or lysosome. St14A cells were treated for 2 h with 20 ng/ml Il-1 before lysis. The proteasome and
lysosome were inhibited, and Western analysis performed as in B.
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and a loss of Atg7 function in mouse brain causes neuro
degeneration (Mizushima et al., 1998; Komatsu et al., 2006).
We therefore examined whether LAMP-2A or Atg7 might be involved in the clearance of phosphorylated Htt by the lysosome.
When either endogenous LAMP-2A or Atg7 is knocked down
by shRNA, accumulation of endogenous S13-phosphorylated
Htt and Htt fragments is observed (Fig. 6 A), suggesting that
both proteins may be involved in lysosomal clearance of phosphorylated Htt. Likewise, shRNA against rat LAMP-2A increased
Httex1p levels and aggregation, whereas overexpression of
human LAMP-2A had the opposite effect (Fig. 6 B). These
combined data are consistent with a role for LAMP-2A in the
lysosomal clearance of Htt.
Although Httex1p does not contain a bonafide “KFERQ”like Hsc70-binding motif in its sequence, phosphorylation of
Htt serine 16 (14-LKpSFQ-18) could provide the negative
charge required to convert this sequence to an Hsc70-binding
motif (mimic 14-LKEFQ-18, where the phosphorylated serine
at residue 16 resembles a glutamic acid [E]). We therefore tested
the ability of phosphomimetic Httex1p to interact with GSTHsc70 in vitro and found that mimicking phosphorylation of
Htt serines 13 and 16 on unexpanded polyQ Httex1p (25QP-EE)
increased the in vitro binding of Httex1p to Gst-Hsc70 by a
specific ADP-dependent mechanism (Fig. 6 C, left), whereas
expansion of the polyQ repeat to 46Qs reduced this interaction
(Fig. 6 C, right).
Because an interaction with Hsc70 could regulate clearance
of phosphorylated mutant Htt, the ability of Hsc70 to reduce
Htt-mediated toxicity (Fig. 6 D) was tested. Overexpression of
Hsc70 increased the survival of HdhQ111/Q111-expressing cells
more than overexpression of Hsp70, the latter being 85%
identical to Hsc70. Exogenous expression of Hsp70 or Hsc70
showed that Hsc70, but not Hsp70, increased levels of Htt
acetylation and S13 phosphorylation, suggesting that Hsc70
may specifically activate an IKK-regulated Htt degradation
process (Fig. S5). Minor differences in heat-shock proteins
have previously been demonstrated to define their function.
For instance, in yeast, the homologues of Hsp70/Hsc70, Ssa1p,
and Ssa2p are 97% identical and yet only Ssa2p is required to
target protein substrates to the yeast vacuole, the functional
equivalent of the mammalian lysosome (Brown et al., 2000).
We propose that Hsc70 may increase Htt clearance by the proteasome and the lysosome by activating S13 phosphorylation
and K9 acetylation.
Figure 5. Phosphorylated Htt can be detected by immunofluorescence in cell culture. (A) S13-phosphorylated Htt is present in untransfected mitotic rat
S14A cells. (B) S13-phosphorylated Htt colocalizes with FLAG immunoreactivity in rat St14A cells lipofected with FLAG–IKK-, and S13-phosphorylated Htt
colocalizes with FLAG immunoreactivity in mouse Hdh7/7 cells nucleofected with FLAG–IKK-.
Mimicking phosphorylation of mutant Htt in
rat brain slice cultures reduces its toxicity
The data presented show that IKK- can enhance the level of
a phosphorylated form of Htt that appears to be more readily
cleared. To test whether this phosphorylation is functionally
significant, toxicity of Htt phosphomimetics was compared
with control expanded repeat Htt in an acutely transfected rat
cortico-striatal slice culture model where toxicity is dependent
on expansion of the polyQ repeat (Khoshnan et al., 2004).
Phosphomimetic (DD) or phosphoresistant (AA) forms of
expanded polyQ Httex1p (97QP) were tested for their effects
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Figure 6. LAMP-2A, Atg7, and Hsc70 may modulate Htt clearance and toxicity. (A) Reducing levels of LAMP-2A or Atg7 in cell culture increases abundance
of S13-phosphorylated Htt. St14A cells were transiently transfected with shRNA for rat LAMP-2A or Atg7, or pSUPER vector control; antibodies used for
Western analysis are shown to the left of the Western panels. (B) LAMP-2A levels modulate Httex1p abundance. St14A cells were transiently cotransfected
with LAMP-2A shRNA, human HA-tagged LAMP-2A or vector control, and myc-actin transfection control. Lysates were subjected to Western analysis and
filter-retardation assay, detected with anti-Htt CAG53b, anti–rat/mouse LAMP-2A, anti-HA to detect HA-hLAMP-2A, anti-myc, and MemCode protein stain.
(C) Hsc70 interacts with phosphomimetic unexpanded polyQ Httex1p in vitro. Purified 25QP-H4 or 46QP-H4 wt and mutant proteins radiolabeled with
35
S were incubated with isolated GST-Hsc70 or GST protein bound to glutathione-agarose beads. Where indicated, 5 mM ATP or ADP was added to the
reaction. Bound proteins were washed, subjected to SDS-PAGE, and detected by phosphoimager autoradiography. (D) Hsc70 reduces Htt-mediated toxicity
in Hdh111/111 cells. Hdh7/7 or Hdh111/111 cells were nucleofected with vector, Hsp70, or Hsc70 together with GFP. 47 h later, XTT cell viability assays were
performed, and the percentage of relative survival was calculated correcting for transfection efficiency of the GFP control ± SEM from triplicates.
1092
on Htt-mediated toxicity in this model. The phosphomimetic
97QP-DD displayed significantly reduced toxicity compared
with either control or 97QP-AA (Fig. 7 A), suggesting that phos
phorylation of Httex1p may reduce neurotoxicity. Because
we showed that the phosphomimetic 97QP-DD Httex1p is
more nuclear localized (Fig. 2 E), but is less toxic than 97QP
in the slice cultures, the data present a potential contradiction
to the extensive studies showing that nuclear accumulation of
mutant Htt significantly enhances neurodegeneration (Saudou
et al., 1998; Schilling et al., 1999; Cornett et al., 2005). These
results suggest that while nuclear accumulation of mutant Htt
Figure 7. Phosphorylation may reduce toxicity but
increase insolubility of Htt. (A) Mimicking 97QP
Httex1p phosphorylation reduces its toxicity in acutely
transfected rat cortico-striatal slice explants. Plasmids
encoding YFP and 97QP-CFP, 97QP DD-CFP, 97QP
AA-CFP, or CFP control were biolistically cotransfected
into rat cortico-striatal brain slices. The number of
healthy medium spiny neurons in the striatal region of
each slice was scored 5 d after transfection. n = 9 for
each condition. Error bars represent SEM. Asterisk =
difference from 97QP at P < 0.0001. (B) Immuno
precipitated phosphorylated/acetylated wt Htt purified
from mouse brain runs in an insoluble fraction in the
SDS-PAGE stacking gel. 12 independent wt control
(W) or R6/2 (R) mouse brains were collected at 4, 8,
and 12 wk of age, snap frozen, and lysed. 500 µg
of lysate was subjected to immunoprecipitation with
PW0595 anti-Htt antibody or zero antibody control
(lysate from lanes 12 and 13 are identical). Western
analysis was performed with a series of antibodies
listed to the right of the panel. Immunoprecipitated Htt
is present as an insoluble species in the stacking gel
and at the top of the separating gel, and as a soluble
form at the standard 350-kD size, marked with an
arrow. MAB2166 does not detect the insoluble species well.
is toxic, nuclear localization facilitated by phosphorylation
could be part of a normal process of protein degradation that
becomes impaired upon expansion of the polyQ repeat, thus
promoting the accumulation of nuclear, toxic Htt that is implicated in HD. Confirming a potential in vivo role for this modification, mutant Htt-mediated neurotoxicity was significantly
reduced when the phosphomimetic was expressed compared
with control or phosphoresistant mutant Htt in BACHD mice,
suggesting that Htt phosphorylation may slow the progression of HD in vivo and represents a valid therapeutic target
(Gu et al., 2009).
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Phosphorylated and acetylated Htt can be
detected in mouse brain
Discussion
Delineation of Htt clearance mechanisms is of great significance because an accumulation of mutant Htt is implicated in
HD pathogenesis. We demonstrate that the IKK complex phosphorylates Htt at S13 and may activate its degradation, similar
to IKK-mediated degradation of IB and FOXO3a (Karin and
Ben-Neriah, 2000; Karin et al., 2002; Hu et al., 2004). The proposed selective degradation of phosphorylated wt Htt, which
involves both the proteasome and lysosome, may include a
transient nuclear localization mediated by phosphorylation of
Htt, where it is subsequently acetylated, ubiquitinated, and
SUMOylated in an order that remains to be established. Proteins
involved in lysosomal degradation pathways, Hsc70, LAMP-2A,
and Atg7, appear to modulate the levels of these modified
forms of Htt in mammalian cells. Our data also suggest that
IKK-–mediated Htt S13 phosphorylation is more efficient for
wt than for expanded polyQ truncated Htt polypeptides (Fig. 1 D
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We next examined whether modified Htt species could be detected in an extensively studied HD mouse model, R6/2, which
contains two wt copies of the mouse HD gene and is transgenic
for exon 1 of the human huntingtin gene originally carrying
150 CAG repeats (Mangiarini et al., 1996). R6/2 mice have a
rapid phenotypic progression (are severely impaired by 14 wk)
and intranuclear inclusions throughout the brain. Htt was
immunoprecipitated from whole brain tissue from 4-, 8-, and
12-wk R6/2 versus wt control mice (Fig. 7 B; antibody PW0595,
Enzo Life Sciences, Inc.). S13- and S16-phosphorylated and
K9-acetylated endogenous mouse Htt showed reactivity to relatively insoluble Htt species in both R6/2 and wt control brain.
A major percentage of modified Htt remained in the stacking
gel and at the top of the separating gel with less migrating to the
size of the standard 350-kD full-length Htt band. No fragments
originating from the transgene were visualized using this
method with the modification-specific antibodies, suggesting
that the transgene was not efficiently phosphorylated and acetyl
ated, although it could be detected after immunoprecipititation
with the Enzo Life Sciences, Inc. antibody (unpublished data).
Wt Htt antibodies Ab1, MAB5490/1H6, and the Enzo Life Sciences, Inc. antibody, each raised against unique Htt species, recognized wt-soluble and the insoluble Htt species in the stacking
gel, whereas MAB2166 did not recognize the insoluble Htt,
consistent with our data above suggesting that MAB2166 does
not substantially detect the phosphorylated/acetylated Htt species (Fig. S4) or that the insoluble fraction consists primarily of
truncated N-terminal Htt species, which do not contain the
MAB2166 epitope. We also find that Htt phosphorylated on
threonine 3 (T3) is present in the insoluble fraction, consistent
with our recent observation that mimicking phosphorylation of
T3 increases Htt aggregation (Aiken et al., 2009). Overall, our
data suggest that the S13/S16-phosphorylated and K9-acetylated
forms of wt endogenous Htt are detectable in both wt and R6/2
mice, and that these modified forms represent relatively insoluble species.
and Fig. 3 C), which may result in reduced clearance of mutant
Htt by inhibiting this phosphorylation-driven mechanism, and
ultimately contribute to disease. Finally, modified species recognized by phospho- and acetyl-specific antibodies are present
in mouse brain.
Mimicking phosphorylation of Htt serines 13 and 16
increases soluble Httex1p-GFP nuclear localization (Fig. 2 E).
In previous studies, we showed that Htt interacts with the acetyl
transferase CBP (Steffan et al., 2000, 2001); therefore CBP, a
nuclear protein, is a candidate acetyltransferase for Htt lysine
9, as was demonstrated for lysine 444 (Jeong et al., 2009).
CBP/p300 contains ubiquitin ligase activity which regulates
protein degradation (Grossman et al., 2003); therefore, this E3
ligase activity of CBP could also be involved in regulating Htt
ubiquitination. A futher connection between the IKK-mediated
phosphorylation of Htt and CBP activites may exist, as CBP
interacts directly with IKK- in the nucleus (Verma et al.,
2004) and is a substrate for IKK- (Huang et al., 2007), suggesting that CBP and the IKK complex could function together
to modulate Htt stability.
Relevant to the potential role for S13 and S16 phosphorylation in nuclear localization, nuclear caspase-6 cleavage of
mutant Htt has been implicated in its pathogenic potential
(Graham et al., 2006; Warby et al., 2008). We find that IKK
activates phosphorylation of Htt fragments, one of which is
consistent with the predicted size of a wt Htt caspase-6 cleavage
product (Fig. 4 B) and the fragment recently shown to be generated in neurons with activation of IKK- (Khoshnan et al.,
2009). It is therefore an intriguing possibility that phosphory
lation by IKK ultimately promotes Htt nuclear localization, polySUMOylation, acetylation by CBP, and subsequent caspase-6
cleavage, which all facilitate a regulated form of clearance.
Phosphorylation of Htt by IKK appears to activate its degradation at least in part by the lysosome, dependent on LAMP-2A
levels (Fig. 6), the integral membrane receptor protein that
can directly import proteins across the lysosomal membrane
for CMA (Massey et al., 2006b). The CMA chaperone Hsc70
preferentially interacts with phosphomimetic wt Httex1p and
reduces Htt-mediated toxicity (Fig. 6, C and D). Combined, this
data may suggest that phosphorylated Htt is degraded in
a LAMP-2A–dependent mechanism through CMA. Because
phosphorylation can trigger nuclear localization and acetylation
of specific Htt species (Fig. 2, C–E) and overexpression of
Hsc70 increases levels of acetylated and phosphorylated endogenous full-length and fragmented Htt (Fig. S5), the findings
implicate Hsc70 in either mediating an interaction of Htt with
the IKK complex, or alternatively activating the IKK complex,
as has been demonstrated for the ubiquitin ligase parkin, which
is mutated in Parkinson’s disease (Henn et al., 2007).
CMA activity declines with age due to a gradual decrease
of LAMP-2A levels in lysosomes (Cuervo and Dice, 2000),
whereas artificially maintaining LAMP-2A levels in aging rat
liver similar to those in young animals can restore CMA activity
to youthful levels and improve organ function (Zhang and
Cuervo, 2008). Because HD is a neurodegenerative disease associated with aging and we have found clearance of phosphorylated
Htt dependent on LAMP-2A, a reduction in LAMP-2A levels
over time may be tied to HD pathogenesis. We propose a hypothetical model for the progression of HD at the molecular level
(Fig. 8), where IKK phosphorylates Htt and activates a cascade
of Htt post-translational modifications and caspase cleavage
(Khoshnan et al., 2009) associated with rapid Htt degradation
by the proteasome and the lysosome in unaffected neurons. In a
presymptomatic HD neuron, IKK could be induced by the presence of the mutant protein (Khoshnan et al., 2004), thus stimulating an IKK-mediated mechanism of Htt clearance, consistent
with the innate immune activation that occurs in premanifest
patients well before symptom onset (Björkqvist et al., 2008).
As long as LAMP-2A levels remain high, patients can degrade
mutant Htt before it can cause toxicity, despite progressively
inhibited proteasome activity and reduced efficiency of mutant
Htt phosphorylation. As aging and mutant Htt together progressively impair proteasome and overall lysosomal activity, and as
LAMP-2A levels decline with age, modified Htt may accumulate, enhancing HD pathogenesis.
From this model, it follows that increasing the efficiency
of Htt clearance by the lysosome, or increasing levels or mobility (Kaushik et al., 2006) of functional LAMP-2A in the lysosomal membrane early in the disease process, could delay HD
onset and serve as a therapeutic strategy. Treatment choice may
vary depending on the stage of HD. In mammals, early treatment
to increase Htt phosphorylation and acetylation may be useful
when levels of LAMP-2A are adequate, as suggested by the
reduced toxicity in acutely transfected rat slice cultures (Fig. 7 A)
and complete lack of neurotoxicity in BACHD mice when Htt is
mutated to mimic the phosphorylated form (Gu et al., 2009).
However, when LAMP-2A levels are low or its function is impaired, drugs that activate the formation of the post-translationally
modified Htt species normally targeted for degradation by the
Materials and methods
Plasmid constructs
pcDNA3.1-based plasmids (Invitrogen) containing the Htt exon 1 DNA
between the HindIII and BamHI sites were used as described previously
(Steffan et al., 2004). These plasmids contained alternating CAG/CAA
repeats, coding for either a normal range (25) or expanded (46 or 97)
polyglutamine tracts followed by the proline-rich region of Htt ending with
the amino acids HRP to create Httex1p. The plasmids were opened at
BamHI and XbaI and DNA encoding various tags was inserted in frame
with Httex1p to create C-terminal tagged Httex1p. The following tags were
used: GS*, VL*, HBH (a gift from Peter Kaiser and Christian Tagwerker,
University of California, Irvine, Irvine, CA [Tagwerker et al., 2006]), EGFP,
and H4 (HIS-HA-HA-HIS: GSHHHHHHMGYPYDVPDYAEFYPYDVPDYAVHHHHHH*) where a stop codon is denoted by the asterisk. The H4 tag was
created by a two-step double-stranded oligonucleotide ligation.
Mutations in Httex1p were created using double-stranded oligo
nucleotides containing HindIII-compatible ends encoding the first 17 amino
acids of Huntingtin, which were ligated between the HindIII site of
pcDNA3.1 in the polylinker and the HindIII site in exon I, immediately 5
to the CAG repeat. K6R K9R K15R (3R) was used as described previously
(Steffan et al., 2004). To create S16 mutations, the HindIII site was shifted
using oligonucleotides containing BspMI (BfuAI) sites, then double-stranded
oligonucleotides were again used to create S13 together with S16 mutants. pHsc70 was constructed by ligating the BamHI fragment from pGSTHsc70 encoding Hsc70 into the BamHI site of pcDNA3.1. For plasmids
used in the acutely transfected striatal slice culture assay, 97QP Httex1p wt
and mutants were cloned into pGWIZ (Gene Therapy Systems) in frame
with CFP using the PstI site.
The following plasmids were obtained collaboratively or as gifts:
pHis-SUMO-1 (A. Dejean, Institut Pasteur, Paris, France); pHis-Ubiquitin
(D. Bohmann, University of Rochester, Rochester, NY); p-human-HA-LAMP-2A
(pAMC1) and pGST-Hsc70 (A.M. Cuervo, Albert Einstein College of Medicine, Bronx, NY; Cuervo and Dice, 1996); pCMV-Hsp70 (P. Muchowski
[University of California, San Francisco, San Francisco, CA] and H. Kampinga [University of Groningen, Groningen, Netherlands; Michels et al.,
1997); pMyc-Actin (H. Rommelaere, Ghent University, Ghent, Belgium);
pFLAG-IKK, pFLAG-IKK, and pHA-IKK (A. Khoshnan and P. Patterson,
California Institute of Technology, Pasadena, CA); pYFP (L. Kaltenbach and
D. Lo; Duke University, Durham, NC); 586aa Htt constructs 15Q (pCINeoHtttt586-15) and 128Q (pCINeoHtt 586–128; M. Hayden, University
of British Columbia, Vancouver, Canada); pcDNA3.1-CBP (A. Kazantsev
Figure 8. Proposed molecular mechanism for the development of HD.
Normal neuronal function: IKK phosphorylates wt Htt activating its posttranslational modification, caspase cleavage, and clearance by the proteasome and lysosome. Presymptomatic HD neuronal function: With chronic
expression of mutant Htt, proteasome activity is reduced, and lysosomal
degradation of mutant Htt becomes essential. Mutant Htt triggers activation of the IKK complex; however, it is less efficiently phosphorylated than
wt Htt. With the clearance mechanism activated, mutant and wt Htt in the
presymptomatic cell are degraded by the lysosome. Symptomatic HD neuronal function: Lysosomal degradation of Htt is impaired through reduction
of LAMP-2A levels or other loss of lysosomal function caused by aging and
by mutant Htt expression. Uncleared mutant Htt and Htt fragments accumulate and take on toxic functions, enhancing HD pathogenesis.
lysosome might actually increase HD pathogenesis through
increased nuclear accumulation and aggregation of the mutant
protein. At this stage of disease, serine-modified Htt would
accumulate, leading to increased pathology as a result of the
intrinisic toxicity of the modified Htt. Consistent with this,
we find that mimicking Httex1p phosphorylation increases its
toxicity and aggregation in Drosophila photoreceptor neurons
where components of the mammalian machinery to degrade
phosphorylated Htt, specifically LAMP-2A, are not present
(unpublished data). Thus, during late-stage HD, it may be
harmful to increase pathways involved in IKK activation,
SUMOylation, and acetylation, whereas in presymptomatic
stages, these pathways may be protective.
The Htt protein itself may play an integral role in autophagic clearance of proteins. Conditional knockout of Htt in the
mouse central nervous system results in an accumulation of
neuropil protein aggregates containing ubiquitin and p62/
SQSTM1 (unpublished data). Htt has been shown to associate
with autophagosomes (Atwal and Truant, 2008) and lysosomes
(unpublished data) and may therefore play a functional and regulatory role in a selective protein clearance mechanism ultimately involved in its own processing. Extensive investigation
will be necessary to test this possibility and elucidate the precise mechanisms involved.
1095
and D. Housman, Massachusetts Institute of Technology, Cambridge, MA;
Kazantsev et al., 1999); pcDNA3.1-mRFP (S. Finkbeiner, University of
California, San Francisco, San Francisco, CA); and pcDNA3-CHIP and
pcDNA-CHIPUbox (C. Patterson, University of North Carolina at Chapel
Hill, Chapel Hill, NC; Jiang et al., 2001).
shRNA for rat Atg7 5-GAAGTACCACTTCTACTAC-3was cloned
into pSUPER vector (Appllied Biosystems). shRNA for rat LAMP-2A,
5-GACTGCAGTGCAGATGAAG-3 in pSUPER was previously constructed
(Massey et al., 2006a). IKK pool 1 contained the following shRNAs
in pLKO.1 (Addgene): 5-CCGGGCACTGGGAAAGTATCTGAAACTCGAGT
TTCAGATACTTTCCCAGTGCTTTTT-3, 5-CCGGCCAGCCAAGAAGAGTG
AAGAACTCGAGTTCTTCACTCTTCTTGGCTGGTTTTT-3, 5-CCGGCTTAC
CTGAATCAGACAAGAACTCGAGTTCTTGTCTGATTCAGGTAAGTTTTT-3,
5-CCGGGCATCTAGTAGAGCGGATGATCTCGAGATCATCCGCTCTACT
AGATGCTTTTT-3, 5-CCGGCGTTGTTAGTGAAGACTTGAACTCGAGTTC
AAGTCTTCACTAACAACGTTTTT-3. IKK pool 2 shRNAs in pLKO.1 were:
5-CCGGGCATCATAAGGAGTTGGTGTACTCGAGTACACCAACTCCT
TATGATGCTTTTT-3, 5-CCGGCCAGATTATGAAGAAGTTGAACTCGAGT
TCAACTTCTTCATAATCTGGTTTTT-3, 5-CCGGCCAGCCTCTCAATGT
GTTCTACTCGAGTAGAACACATTGAGAGGCTGGTTTTT-3, 5-CCGGGC
AAATGAGGAACAGGGCAATCTCGAGATTGCCCTGTTCCTCATTTGC
TTTTT-3, 5-CCGGGCGTGCCATTGATCTATATAACTCGAGTTATATAGATC
AATGGCACGCTTTTT-3.
Primary antibodies
Three affinity-purified rabbit polyclonal antibodies were generated
(New England Peptide) against post-translationally modified 1–17aa
Htt peptides. Antibodies were generated against the following peptides:
anti-S16-P: H2N-CMATLEKLMKAFESLK(pS)F-amide; anti-S13-P: H2NCMATLEKLMKAFE(pS)LKSF-amide; anti-K9-Ac: Ac-CLEKLM(Ac-K)AFE(pS)LK
(pS)F-amide. Two rabbits were immunized with each peptide; antisera were
pooled and run over an unmodified Htt 1–17aa peptide column (affinity
matrix 20401; Thermo Fisher Scientific) to remove antibodies recognizing
unmodified Htt species. The flow-through from each was then run over the
modified peptide column for each respective project. The elutions from
these columns were used as the modification-specific antibodies for this
study, and tested for specificity using a peptide dot blot (Fig. S1). These
three antibodies were also tested on Westerns of lysates from Hdh7/7 and
Hdh111/111 cells nucleofected with IKK or vector +/ siRNA for Htt (a gift
from R. Friedlander, Brigham and Women’s Hospital, Boston, MA); levels
of antigenic species were reduced with Htt siRNA in both cell lines, demonstrating modified Htt specificity. JG1 is another rabbit polyclonal anti-Htt
1–17aa antibody generated by our laboratory.
We also used the following antibodies for this work: CAG53b (a gift
from E. Wanker, Max Delbrueck Center for Molecular Medicine, Berlin,
Germany); anti-Htt PW0595 (Enzo Life Sciences, Inc.); anti-Myc 9E10
(Millipore); anti-FLAG (Sigma-Aldrich), anti-HA 16B12 (Covance); anti–
-tubulin clone B-5-1-2 (Sigma-Aldrich); anti-Htt Ab1 (M. DiFiglia, Harvard
University, Cambridge, MA); anti-Htt EM48 MAB5374 (Millipore); anti-Htt
1H6 (Abnova; Fig. S4); anti-Htt MAB2166 (Millipore); anti-Htt MAB5490
(Millipore; Fig. 7); anti-Htt 3B5H10 (S. Finkbeiner, Univeristy of California,
San Francisco); anti–rat LAMP-2A Igp96 (Invitrogen); anti-nestin (Millipore);
anti-Atg7 (Abcam); anti–SUMO-1 (PW9460; Enzo Life Sciences, Inc.),
anti-ubiquitin (13–1600, Invitrogen; and sc8017, Santa Cruz Biotechnology, Inc.); anti-IB clone IB-245 (Invitrogen); anti–phospho-IB Ser 32
14D4 (Cell Signaling Technology); and anti-phosphorylated Htt threonine 3
[anti-T3-P; Aiken et al. [2009]). Secondary antibodies used for Western
analysis were goat anti–mouse HRP (The Jackson Laboratory) and goat
anti–rabbit HRP (Thermo Fisher Scientific).
Immunofluorescence analysis
Cells were transfected (Lipofectamine 2000) or nucleofected (Lonza) with
IKK- (1/2 IKK- and 1/2 pcDNA) and 24 h later fixed at room temperature with 4% PFA, permeabilized, and blocked with 5% BSA and 4% donkey serum. Primary antibodies were diluted at 1:1,000. Secondary
antibodies were anti–rabbit conjugated with Cy3 (Jackson Immuno
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Protein purification and Western analysis
His-tagged proteins were purified under denaturing conditions using
magnetic Ni-NTA nickel beads (QIAGEN) as described previously (Steffan
et al., 2004) for Fig. 1 D, Fig. 2, A and B, and Fig. S2, A and B. Western
blots were processed with SuperSignal West Pico and Dura reagents
(Thermo Fisher Scientific). Quantitative densiometric analyses (Fig. 6 A
and Fig. S4) were performed on digitalized images of immunoblots using
Scion Image 4.0 software (Scion Corporation) and SEM calculated from
densiometric levels of Western signal from triplicate preparations of protein
extracts. Densitometric levels of phosphorylated or acetylated Htt protein
were normalized to levels of -tubulin loading control. Filter retardation assays were performed as follows: Cell debris pellets were taken after centrifugation for 10 min at 16,000 g. Pellets were resuspended in 100 µl of Tris
buffer (20 mM Tris and 15 mM MgCl2 at pH 8.0) and 100 µl of 4% SDS100 mM DTT in PBS was added. These samples were boiled for 5 min and
then filtered through nitrocellulose membrane via a dot-blot apparatus. The
membranes were then dried at room temperature for 30 min, stained with
MemCode reversible protein stain (Thermo Fisher Scientific), blocked, and
primary antibodies were added for Western analysis. Native buffer A,
used for lysis of cells in Figs. 3, 4, 6, S3, S4, and S5: 10 mM Tris-HCl,
pH 7.5, 10% glycerol, 400 mM NaCl, 1 mM EDTA, 1 mM PMSF, 0.5%
NP-40, 20 mM N-ethylmaleimide, 1 mM PMSF, phosphatase inhibitors 1 and 2
(Sigma-Aldrich), complete mini protease inhibitor pellet (Roche), 10 ng/ml
aprotenin, 10 ng/ml leupeptin, 5 mM nicotinamide and 5 mM butyrate,
pH 7.5. Native buffer B, used for purification in Fig. 2 D: 50 mM NaH2PO4,
pH 8.0, 150 mM NaCl, 0.1% Tween 20, 1 mM DTT, 5 mM ADP, 10 mM
Imidazole, 1 mM PMSF, 10 ng/ml aprotenin, 10 ng/ml leupeptin, complete mini protease inhibitor pellet (Roche), and phosphatase inhibitors
1 and 2 (Sigma-Aldrich). All St14A cells used in Westerns showing phosphorylated Htt were treated with phosphatase inhibitor Calyculin A (Enzo
Life Sciences, Inc.) 10–30 min (20 nM) before lysis.
In vitro kinase assay
The in vitro kinase assay was performed as described previously (Liu et al.,
2007) using 75 ng recombinant IKK- or IKK- protein (Millipore) and
25QP-H4 or 46QP-H4 purified from St14A cells using magnetic Ni-NTA
nickel beads (QIAGEN) under native conditions with the following buffers.
Lysis buffer: 50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 10 mM imidazole,
0.05% Tween 20, 1 mM PMSF, 10 ng/ml aprotenin, 10 ng/ml leupeptin,
and phosphatase inhibitors 1 and 2 (Sigma-Aldrich). Wash buffer: 50 mM
NaH2PO4, pH 8.0, 300 mM NaCl, 20 mM imidazole, 0.05% Tween 20,
and phosphatase inhibitors 1 and 2 (Sigma-Aldrich). Recombinant IKK was
diluted in enzyme dilution buffer: 20 mM MOPS/NaOH, pH 7.0, 1 mM
EDTA, 5% glycerol, 0.01% Brij35, 0.1% -mercaptoethanol, and 1 mg/ml
BSA. The kinase assay was performed in 5X kinase buffer: 40 mM MOPS/
NaOH, pH 7.0, and 1 mM EDTA. Before the assay, purified Htt bound to
Ni-NTA beads was incubated at 95°C for 3 min, and then placed on ice
for 5 min. The assay was performed at 30°C with light agitation for 10 min
under the following conditions: 2.5 µl recombinant IKK subunit, 5 µl 5X
kinase buffer, 2.5 µl 1 mM ATP, 2.5 µl 0.1 M MgAc, and 12.5 µl purified
Htt in water. The assay was stopped with addition of Western sample loading buffer, boiled 10 min, run on 12% SDS-PAGE, and Western analysis
was performed.
Mass spectrometry analysis
ST14A cells were transiently transfected with Htt25QP-HBH + IKK-
(Fig. 1 C) or Htt25QP-HBH + CBP + IKK- (Fig. 2 C); 48 h after transfection,
the cells reached confluency. Cells were then treated with fresh media containing 10 nM Calyculin A (EMD) for 30 min, or 20 nM Calyculin A (Enzo Life
Sciences, Inc.) for 10 min. Cells were washed with cold 1x PBS, then harvested and lysed in 1 ml lysis buffer each (50 mM Tris-HCl, pH 8.0, 8 M
urea, 500 mM NaCl, 50 mM NaH2PO4, 10 mM imidazole, 0.5% Triton
X-100, and complete mini protease inhibitor [Roche]). The DNA was sheared
and the cells further lysed by passing through a 20-guage needle 20 times,
and cellular debris was removed by centrifugation. Clarified lysates were
then incubated with 25 µl Ni-Sepharose 6 Fast Flow (GE Healthcare) or
Ni-NTA magnetic nickel (QIAGEN) bead slurry for 3 h or overnight at
Cell culture and transfections
The Hela, St12.7, ST14A, and N548mu and the wild-type STHdhQ7/HdhQ7
and homozygous mutant STHdhQ111/HdhQ111 cell lines were propagated
as described previously (Steffan et al., 2004; Apostol et al., 2008). NIH3T3 cells were grown in DMEM and 10% newborn calf serum (Hyclone).
All cells were transfected with Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen) except STHdhQ7/HdhQ7, which were nucleofected. rhTNF- and rmIl-1 (R&D Systems) were used to pharmacologically
activate IKK.
Research Laboratories, Inc.) and anti–mouse conjugated with FITC (Jackson
ImmunoResearch Laboratories, Inc.), and they were used at 1:1,000.
Slides were stained with DAPI to detect nuclei. ProLong Gold Antifade
(Invitrogen) imaging medium was used. Images were collected at room
temperature with an inverted microscope (Observer.Z1; Carl Zeiss, Inc.)
with a Plan-Apochromat 63X objective, NA 1.40. AxioVision AxioVs40
v 4.7.1.0 software (Carl Zeiss, Inc.) was used to generate 3D deconvoluted
images. The camera used was an AxioCam MRm (Carl Zeiss, Inc.).
room temperature. The beads were then washed twice in the lysis buffer,
and four times with wash buffer (50 mM Tris-HCl, pH 6.3, 8 M urea, 500 mM
NaCl, 50 mM NaH2PO4, 20 mM imidazole, and 0.5% Triton X-100). The
beads of the same condition were pooled and the urea buffer was
replaced with 50 mM NH4CO3 before digestion.
Chymotryptic digestion (2% by weight) of 25QP-HBH was performed on the Ni beads used to purify the protein to maximize peptide recovery. The digestion occurred overnight at 37°C. Resulting peptides were
extracted from the beads with 25% acetonitrile, 0.1% formic acid three
times. The extracts were pooled, concentrated using a SpeedVac, and
acidified by 0.1% formic acid before mass spectrometric analysis. Resultant peptides were then separated and analyzed by reverse-phase liquid
chromatography coupled to tandem mass spectrometry (LC MS/MS) on
a quadropole-orthogonoal-time-of-flight tandem (Guerrero et al., 2008)
(QSTAR XL; Applied Biosystems/PE Sciex) or an ultra-high performance
Thermo Electron linear trap quadropole (LTQ)-Orbitrap hybrid (Fang et al.,
2008) (Thermo Fisher Scientific) mass spectrometer.
Extraction of the monoisotopc masses (m/z) of parent ions, their
charge states, and their corresponding fragment ions was performed automatically using Analyst software (Applied Biosystems) for QSTAR data or
using extract MSn (Matrix Science) for LTQ-Orbitrap data. These data were
then submitted for automated database searching for protein identification
using the Protein Prospector (University of California, San Francisco) search
engine. Post-translational modifications were confirmed by manual inspection of the MS/MS spectra.
Toxicity assays
For XTT cell viability assays, STHdhQ7 and STHdhQ111 and cell lines were
plated in 24-well plates (0.75 × 105 cells per well) in complete media as
described previously (Apostol et al., 2008). The next day, cells were shifted
to nonpermissive conditions (i.e., 39°C and low serum media) for 48 h
for STHdhQ111 and STHdhQ7 lines followed by incubation for 4 h with XTT
and phenazine methosulfate (0.2 mg/ml and 0.2 µg/ml, respectively;
Sigma-Aldrich) for 4 h, and plates were read at 450 nM.
Rat cortico-striatal brain slice neurodegeneration assay
All animal experiments were performed in accordance with the Institutional Animal Care and Use Committee and Duke University Medical Center Animal Guidelines. Brain slice preparation and biolistic transfection
were performed as described previously (Lo et al., 1994; Southwell et al.,
2008) with some modifications. In brief, brain tissue was dissected from
postnatal day 10 CD Sprague Dawley rats (Charles River Laboratory) and
placed in ice-cold Neurobasal A culture medium containing 10% heatinactivated pig serum, 5% heat-inactivated rat serum (Lampire), 10 mM KCl,
10 mM Hepes (Sigma-Aldrich), 1 mM sodium pyruvate (Sigma-Aldrich),
100 U/ml penicillin/streptomycin, and 1 mM l-glutamine. All media
reagents were obtained from Invitrogen unless otherwise noted. Brain
tissue was cut into 250-µm-thick coronal slices using a Vibratome and incubated for 30 min at 32°C/5% CO2 before biolistic transfection. Gold
particles (1.6 µm; Bio-Rad Laboratories) were coated with the indicated
plasmids, loaded into Tefzel tubing (McMaster-Carr), and transfected with
the Helios Gene Gun (Bio-Rad Laboratories) at 95 psi. Brain slices were
incubated at 32°C/5%CO2 until analysis at 5 d post-transfection. Medium
spiny neurons were visualized by fluorescence microscopy of YFP and
scored by neuron morphology. Medium spiny neurons were considered
healthy if they were of uniform size and shape and contained visible dendrites. Data were analyzed with Prism software (GraphPad Software, Inc.)
and significance was determined by unpaired Student’s t test.
Calculation of the nucleus/cytoplasm ratio for 97QP-GFP versus 97QP-DD-GFP
Cell culture and transfection. Primary cultures of rat cortical neurons were
prepared from embryonic rats (E 19–20) and transfected using calcium
GST pull-down assay
For GST pull-down assays, 35S-labeled His-tagged Httex1p-H4 proteins
were synthesized in a TNT coupled reticulocyte lysate system (Promega),
purified, and eluted under native conditions using magnetic Ni-NTA nickel
beads (QIAGEN) in the buffers suggested by the manufacturer. The eluted
proteins were dialyzed using SlideALyzer 3.5K Dialysis Casettes (Thermo
Fisher Scientific) against 20 mM MOPS, pH 7.3/0.25 M sucrose buffer.
GST-Hsc70 was purified from Escherichia coli using glutathione-agarose
beads, and incubated with purified radioactive Httex1p-H4 proteins in
20 mM Tris-HCl, pH 7.2, 150 mM NaCl, 0.1% Tween 20, and 1 mM DTT
supplemented with ATP or ADP corresponding to the incubation conditions,
washed five times, and subjected to SDS-PAGE and autoradiography.
phosphate with plasmids at 5 d in vitro, as described previously (Xia et al.,
1996; Finkbeiner et al., 1997). Specifically, neurons were cotransfected
with pcDNA3.1-mRFP (Arrasate et al., 2004) and 97QP-GFP or 97QP-PPGFP in a 1:1 molar ratio using a total of 3 µg of DNA in each well of a
24-well plate. After transfection, neurons were maintained in serum-free
medium with Forskolin (10 µM; Sigma-Aldrich) and IBMX (100 µM; SigmaAldrich). Neurons were fixed with 4% paraformaldehyde in PBS (15 min)
20 h after transfection, permeabilized with 0.1% Triton X-100 in PBS
(30 min), and incubated with 1 M glycine in PBS (20 min). Finally, neurons
were washed twice for 5 min each with 2.5 µg/ml of Hoechst 33258 in
PBS dye to stain the nuclei.
Robotic microscope imaging system. The microscope imaging system
has been described previously (Arrasate et al., 2004; Arrasate and
Finkbeiner, 2005). Basically, the system is based on an inverted microscope
(TE300 Quantum; Nikon). Xenon lamp (175 W) illumination was supplied
by a liquid light guide. The Nikon working distance objective 20X (NA
0.45) was used. Fluorescence excitation and emission filters were moved
into or out of the optical path by two ten position filter wheels (Sutter Instrument Co.) under computer control. Images were collected with a 12/14 bit
digital cooled CCD camera (Orca II; Hamamatsu Photonics) and digitized
with MetaMorph software (Universal Imaging). Before image acquisition,
care was taken to adjust the gain and offset of the camera and the analogueto-digital converter to ensure that the intensities of all the pixels of each image were within the detection range of the instrument, and that the settings
were the same across the samples that were examined. The whole system
is mounted on a vibration isolation table.
Image analysis. Measurements of htt expression were extracted from
images generated with the microscope imaging system described above.
The validity of estimating htt expression levels in live cells from images of
the fluorescence of the GFP fusion tag to which it is fused has been demonstrated previously by directly comparing this approach to other methods
that are highly quantitative but unsuitable for live-cell imaging (Arrasate
et al., 2004). The expression of 97QP-GFP or 97QP-DD-GFP was estimated
by measuring GFP fluorescence intensity within a region of interest from the
image that corresponded to a portion of the cytoplasm or the nucleus in
neurons that had not developed inclusion bodies. Hoechst staining was
used to localize the nucleus. Pixel intensities within a similar sized region
from an adjacent acellular portion of the image were collected as a measurement of background signal. Pixel values from these background
measurements were subtracted from the corresponding pixel intensity
measurements made from the nucleus and the cytoplasm. These calculations produced background-corrected measurements of htt 97QP-GFP or
97P-DD-GFP from the cytoplasm and nucleus.
Statistical analysis. The ratio of GFP intensity in the nucleus over
the cytoplasm was calculated for individual neurons by dividing the
background-corrected pixel intensities from the region of interest within the
nucleus by the background-corrected pixel intensities from the region of interest within the cytoplasm of the same neuron. Differences in the mean of
these ratio measurements were compared by t test with commercially available software (Prism 3.0; GraphPad Software, Inc.).
Htt immunoprecipitation from mouse brain
Age-matched R6/2 and wt control brains were collected and snap frozen.
Whole brain tissue was dounced 20 times on ice in T-Per lysis buffer
(Thermo Fisher Scientific) containing an EDTA-free mini protease inhibitor
pellet (Roche), a Phos-Stop pellet (Roche), 5 mM sodium fluoride, and
1 mM sodium orthovanadate. Lysates were microfuged at 16,000 g at 4°C
for 15 min, and the supernatant saved. Htt was immunoprecipitated from
the 500 µg of supernatant using Protein G-Plus Agarose (Santa Cruz
Biotechnology, Inc.) with 1 l PW0595 antibody (Enzo Life Sciences, Inc.)
or zero antibody control, and run on 8% SDS-PAGE and blotted to nitro
cellulose for standard Western analysis.
Online supplemental material
Fig. S1 shows the specificity of the modification-specific antibodies. Fig. S2
shows the role of IKK in the regulation of mutant Httex1p ubiquitination and
SUMOylation. Fig. S3 shows cellular localization of phosphorylated Htt.
Fig. S4 shows loss of an epitope for popular anti-Htt antibody MAB2166
with post-translational modification of Htt. Fig. S5 shows the role of Hsc70
and the ubiquitin ligase CHIP in the regulation of levels of phosphorylated
and acetylated Htt. Online supplemental material is available at http://
www.jcb.org/cgi/content/full/jcb.200909067/DC1.
1097
This paper is dedicated to the memory of Dr. Charles H. Sawyer, whose example was its constant inspiration.
We thank Drs. David Housman, Matthew Blurton-Jones, Masashi Kitazawa,
Peter Kaiser, Alex Osmand, Christian Landes, Bin Liu, and Daniel Keys for
insightful discussion; Denise Dunn and Emily Mitchell for technical assistance;
and Anne Dejean, Dirk Bohmann, Paul Muchowski, Harm Kampinga, Peter
Kaiser, Christian Tagwerker, Cam Patterson, Heidi Rommelaere, Alex Kazantsev,
Robert Friedlander, Erich Wanker, and Marian DiFiglia for their generous gifts
of reagents for these experiments.
This work was supported by the Hereditary Disease Foundation (J.S.
Steffan, L.M. Thompson, J.L. Marsh, D.C. Lo, and P.H. Patterson); the Fox Family
Foundation (J.S. Steffan and L.M. Thompson); the High Q Foundation (J.S. Steffan,
L.M. Thompson, J.L. Marsh, and D.C. Lo); the Huntington’s Disease Society of
America Coalition for the Cure (L.M. Thompson); the Taube-Koret Center for
Huntington’s Disease Research (S. Finkbeiner); and National Institutes of Health
awards NS52789 (L.M. Thompson and J.L. Marsh), HD36081 (J.L. Marsh),
NS045283 (J.L. Marsh and L.M. Thompson), NS043466 (S.O. Zeitlin),
GM74830 (L. Huang), 2R01NS039074 (S. Finkbeiner), 2R01NS045191 (S.
Finkbeiner), 2P01AG022074 (S. Finkbeiner), P01AG031782 (A.M. Cuervo)
and NS055298 (P.H. Patterson), and T32GM0731130 (C.T. Aiken).
Submitted: 10 September 2009
Accepted: 20 November 2009
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1099
[Autophagy 5:5, 1-3; 1 July 2009]; ©2009 Landes Bioscience
Autophagic Punctum
Protein turnover differences between neurons and other cells
This manuscript has been published online, prior to printing. Once the issue is complete and page numbers have been assigned, the citation will change accordingly.
Andrey S. Tsvetkov,1-3 Siddhartha Mitra1,4 and Steven Finkbeiner1-4,*
1Gladstone Institute of Neurological Disease; San Francisco, CA USA; 2Taube-Koret Center for Huntington’s Disease Research; San Francisco, CA USA; 3Neuroscience Program;
Departments of Neurology and Physiology; 4Biomedical Sciences Program and Medical Scientist Training Program; University of California; San Francisco, CA USA
Key words: huntington disease, autophagy, neurodegeneration, rapamycin, everolimus, LC3
In a recent study, we investigated the relationship between
formation of an inclusion body (IB) and activity of the ubiquitin-proteasome system (UPS) in a primary neuron model of
Huntington disease. We applied single-cell longitudinal acquisition and analysis to simultaneously monitor mutant huntingtin,
which causes Huntington disease, IB formation, UPS function
and neuronal toxicity. We found that proteasome inhibition is
toxic to striatal neurons in a dose-dependent fashion. The UPS
is more impaired in neurons that go on to form IBs than in those
that do not; however, after IBs form, UPS function improves.
Our findings suggest that IBs are a protective cellular response to
mutant protein that also improves intracellular protein degradation The study also revealed some surprising differences in the
ways that neurons regulate protein turnover compared with nonneuronal cells, which we discuss further in this article.
To determine if concurrent changes in autophagy affected our
measurement of UPS activity, we examined the activity of the
autophagic pathway after treatment with the UPS inhibitor epoxomicin. We found that levels of LC3-II, which indicate the extent of
autophagy, were unchanged in primary striatal neurons, suggesting
no upregulation of autophagy. However, consistent with previous
reports, proteasome inhibition in HEK293 cells led to LC3-II
accumulation. We concluded that autophagy regulation in neurons
might be different than in other cell types.
Although implicated in neurodegeneration, autophagy has been
characterized mostly in yeast and mammalian non-neuronal cells,
and the few studies in neurons reach different conclusions. We
sought to determine if common autophagy enhancers would stimulate autophagy in cultured primary striatal and cortical neurons.
First, we treated primary neurons with bafilomycin A1 to block the
*Correspondence to: Steven Finkbeiner; Gladstone Institute of Neurological
Disease; 1650 Owens Street; San Francisco, California 945158 USA; Tel.:
415.734.2000; Fax: 415.355.0824; Email: [email protected]
Submitted: 03/18/09; Revised: 04/03/09; Accepted: 04/09/09
Previously published online as an Autophagy E-publication:
http://www.landesbioscience.com/journals/autophagy/article/8705
Punctum to: Mitra S, Tsvetkov A S, Finkbeiner S. Single Neuron UbiquitinProteasome Dynamics Accompanying Inclusion Body Formation in Huntington
Disease. J Biol Chem 2009; 284:4398-403. Epub 2008 Dec 10.
1
fusion of autophagosomes with lysosomes. LC3-II levels increased
in primary rat neurons and HeLa cells, indicating autophagosome
accumulation (Fig. 1A). These results suggest that autophagy is
constitutively active in neurons and that fusion of autophagosomes
to lysosomes is similar in all cells.
We then determined if autophagy is induced similarly in
neurons and non-neuronal cells. Others showed that GFP-LC3
transgenic mice exhibited no autophagy in the brain after starvation. But was this because the brain is protected from starvation?
To ensure that neurons were deprived of nutrients, we eliminated
contributions from glial cells (our cultures are 95% neurons) and
homeostatic mechanisms outside the central nervous system. The
pathways that mediate starvation-induced autophagy in neurons
evidently differ from those in non-neuronal cells; starvation in
Hank’s balanced salt solution increased LC3-II levels in HeLa cells,
but not in neurons (Fig. 1B).
We also assessed the effects of rapamycin. In neurons, rapamycin
pretreatment blocked BDNF-induced phosphorylation of p70S6K
(Fig. 1C), indicating mTOR inhibition. Next we determined
if rapamycin or everolimus induced autophagy. As expected,
rapamycin potently increased LC3-II levels in HeLa cells. However,
neither chemical increased LC3-II levels in primary neurons (Fig.
1D). While surprising, this result is consistent with our observations in nutrient-deprived neurons. Even in non-neuronal
cells, rapamycin effects are complex. Nanomolar concentrations
completely inhibit mTOR activity, but only vast excesses induce
autophagy in non-neuronal cells, suggesting rapamycin may act
on additional cellular targets. In fly and mouse HD models,
it attenuates mutant htt toxicity and promotes htt clearance.
However, more experiments are needed to show that the benefits
of rapamycin are due only to autophagy. In autophagy-deficient
fibroblasts, for example, rapamycin apparently inhibits huntingtin
aggregation by reducing protein synthesis.
Lithium chloride induces autophagy in non-neuronal cells by
inhibiting inositol monophosphatase—a mechanism that is independent of mTOR. We found that lithium chloride effectively
induced autophagy in control cells, but LC3-II did not accumulate
in primary neurons (Fig. 1E), suggesting that neurons may differ
somewhat from other cells in both mTOR-dependent and -independent mechanisms of autophagy.
Western blots show steady-state LC3-II levels but not the flux
through the autophagic pathway. If autophagy basal rates are high
Autophagy
Figure 1. Common inducers of autophagy in non-neuronal cells fail to stimulate autophagy in primary neurons. Relative intensities of LC3-I and LC3-II
bands reflect levels of autophagy. (A) Bafilomycin A1 (bafA; 4 h, 1 nM) induced LC3-II accumulation in striatal and cortical neurons and in HeLa cells.
(cont), control untreated cells. (B) Striatal and cortical neurons were incubated in Hanks’ solution. Starvation (2 h in Hanks’ solution) induced autophagy
in HeLa cells but not neurons. Longer incubations gave similar results. (C) Pretreatment with rapamycin (2 µM) blocked BDNF-induced phosphorylation
of p70S6K in striatal neurons. (D) Striatal and cortical neurons incubated in medium with the mTOR inhibitors 2 µM rapamycin (rap) or everolimus
(everol) for 48 h. Shorter or longer incubations and higher and lower concentrations (2 nM to 20 µM) gave similar results (not shown). Rapamycin (2
µM, 24 h) induced autophagy in HeLa cells. (E) Lithium chloride (LiCl, 10 mM) induced autophagy in COS-7 cells but not neurons. (F) Autophagy inducers did not increase the flux through the autophagic pathway. In striatal neurons, inhibition of lysosomal degradation with bafilomycin A1 (overnight)
led to LC3-II accumulation (compare lanes 1 and 2). Starvation of these treated cells did not increase LC3-II levels (compare lanes 2–4). Incubation of
these treated cells with 2 µM rapamycin (compare 2, 5, 6), 2 µM everolimus (compare 2, 7, 8), or 10 mM LiCl (compare 2, 9, 10) did not increase
LC3-II levels. (G) Striatal and cortical neurons were incubated in medium with niguldipine (nig, 4 µM), trifluoperazine (3F, 8 µM), or loperamide (lop,
5 µM) (overnight). Note LC3-II accumulation. (H) Starvation (starv, Hanks’ solution, 8 h), rapamycin (rap, 2 µM, 24 h), and lithium chloride (LiCl, 10
mM) induced autophagy in astrocytes.
in neurons, steady-state levels of autophagosomes and LC3-II
might not change in neurons under autophagy-inducing conditions. To determine if flux through autophagy changes with drugs,
we examined LC3-II levels with these drugs and bafilomycin
A1, which blocks fusion of autophagosomes and lysosomes. We
incubated primary neurons with bafilomycin A1, under starvation
conditions, or with rapamycin, everolimus, or lithium chloride
(Fig. 1F). Simultaneous application of bafilomycin A1 and drugs
that induce autophagy in non-neuronal cells did not increase
LC3-II accumulation in neurons over conditions in which only
fusion is inhibited. Thus, starvation, rapamycin, everolimus and
lithium chloride do not significantly increase autophagic flux in
primary neurons.
Since at least one study reports LC3-II accumulation in
rapamycin-treated neurons, we thought perhaps our western
blots might not detect LC3-II in neurons undergoing autophagy.
www.landesbioscience.com
We tested other small molecules that induce autophagy in nonneuronal cells. Niguldipine, trifluoroperazine and loperamide
robustly induced autophagy in primary neurons (Fig. 1G).
Therefore, we conclude that the upregulation of autophagy, in
principle, can be induced and detected in our system.
Is there another way to reconcile the different results? They
might be affected by contaminating non-neuronal cells in the
mixed primary cultures. To test this, we prepared pure primary
cultures of astrocytes and starved them or treated them with
rapamycin or lithium chloride. Like the cell lines, astrocytes
responded by inducing autophagy (Fig. 1H).
These results raise the intriguing possibility that autophagy in
neurons is regulated by mechanisms that differ, at least in part,
from those in non-neuronal cells. Our results underscore the
potential importance of using primary neurons to study the role
of autophagy in neurodegeneration and the consideration of these
Autophagy
2
potential differences in any efforts to target this pathway therapeutically.
Acknowledgements
This work was supported by R01 2NS039746 and 2R01
NS045191 from the National Institute of Neurological Disease
and Stroke, P01 2AG022074 from the National Institute on
Aging, the Taube-Koret Center for Huntington’s Disease Research,
and the J. David Gladstone Institutes (S.F.); a Milton Wexler Award
and a fellowship from the Hereditary Disease Foundation (A.T.);
NIH-NIGHMS UCSF Medical Scientist Training Program and a
fellowship from the UC-wide adaptive biotechnology (GREAT)
program (S.M.); and RR018928 from the National Center for
Research Resources. We thank Dr. Walter Schuler (Novartis) for
everolimus, helpful discussions, and bringing to our attention the
difference in the doses of rapamycin that inhibit mTOR and those
that are typically used to induce autophagy. We also thank Jayanta
Debnath for LC3 antibodies and helpful discussions, Dr. Ana
Maria Cuervo for helpful advice, and members of the Finkbeiner
laboratory for helpful discussions. Kelley Nelson provided administrative assistance, and Gary C. Howard edited the manuscript.
3
Autophagy
9104 • The Journal of Neuroscience, July 15, 2009 • 29(28):9104 –9114
Neurobiology of Disease
Loss of Hsp70 Exacerbates Pathogenesis But Not Levels of
Fibrillar Aggregates in a Mouse Model of Huntington’s Disease
Jennifer L. Wacker,1 Shao-Yi Huang,2 Andrew D. Steele,6 Rebecca Aron,2 Gregor P. Lotz,2 QuangVu Nguyen,1
Flaviano Giorgini,1 Erik D. Roberson,2 Susan Lindquist,6 Eliezer Masliah,7 and Paul J. Muchowski1,2,3,4,5
1Department of Pharmacology, University of Washington, Seattle, Washington 98195, 2Gladstone Institute of Neurological Disease, 3The Taube-Koret
Center for Huntington’s Disease Research, and Departments of 4Biochemistry and Biophysics and 5Neurology, University of California, San Francisco, San
Francisco, California 94158, 6Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, and
7Department of Neurosciences, University of California, San Diego, La Jolla, California 92093
Endogenous protein quality control machinery has long been suspected of influencing the onset and progression of neurodegenerative
diseases characterized by accumulation of misfolded proteins. Huntington’s disease (HD) is a fatal neurodegenerative disorder caused by
an expansion of a polyglutamine (polyQ) tract in the protein huntingtin (htt), which leads to its aggregation and accumulation in
inclusion bodies. Here, we demonstrate in a mouse model of HD that deletion of the molecular chaperones Hsp70.1 and Hsp70.3 significantly exacerbated numerous physical, behavioral and neuropathological outcome measures, including survival, body weight, tremor,
limb clasping and open field activities. Deletion of Hsp70.1 and Hsp70.3 significantly increased the size of inclusion bodies formed by
mutant htt exon 1, but surprisingly did not affect the levels of fibrillar aggregates. Moreover, the lack of Hsp70s significantly decreased
levels of the calcium regulated protein c-Fos, a marker for neuronal activity. In contrast, deletion of Hsp70s did not accelerate disease in
a mouse model of infectious prion-mediated neurodegeneration, ruling out the possibility that the Hsp70.1/70.3 mice are nonspecifically
sensitized to all protein misfolding disorders. Thus, endogenous Hsp70s are a critical component of the cellular defense against the toxic
effects of misfolded htt protein in neurons, but buffer toxicity by mechanisms independent of the deposition of fibrillar aggregates.
Introduction
Many neurodegenerative diseases, including Alzheimer’s disease
(AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis
(ALS), prion disease and HD, are characterized by conformational
changes in disease-causing proteins that result in misfolding and aggregation and have collectively been termed protein-conformational
disorders. In contrast to AD, PD, and ALS, in which the vast
majority of cases are idiopathic, HD is one of a number of inherited neurodegenerative disorders, collectively termed polyQ
diseases, which are caused by an expansion of CAG repeats, coding for glutamine, in their respective disease proteins. The deposition of aggregation-prone proteins that contain expanded
polyQ repeats in inclusion bodies is a neuropathological hallmark of the majority of these disorders.
The accumulation of misfolded proteins in cells triggers a
protective stress response that includes the upregulation of heat
Received May 13, 2009; accepted June 11, 2009.
Support for this study was provided by National Institute of Neurological Disease Grants NS47237 and NS054753
(P.J.M.) and National Institute of Aging Grant AG022074 (P.J.M., E.M.). We thank S. Ordway and G. Howard for
editorial assistance and Artur Topolszki for assistance with mouse colony management for PrP studies. S.L. is an
investigator of the Howard Hughes Medical Institute.
Correspondence should be addressed to Paul J. Muchowski, Gladstone Institute of Neurological Disease,
University of California, San Francisco, 1650 Owens Street, San Francisco, CA 94158. E-mail:
[email protected].
F. Giorgini’s current address: Department of Genetics, University of Leicester, Leicester LE1 7RH, UK.
E. D. Roberson’s current address: Department of Neurology, University of Alabama at Birmingham, Birmingham,
AL 35294.
DOI:10.1523/JNEUROSCI.2250-09.2009
Copyright © 2009 Society for Neuroscience 0270-6474/09/299104-11$15.00/0
shock proteins (Hsps) that function as molecular chaperones to
help to restore cellular homeostasis (Lindquist, 1986). Postmitotic neurons, unable to dilute misfolded and/or aggregated
proteins through cell division, are particularly vulnerable to the
deleterious effects of misfolded proteins (Muchowski and
Wacker, 2005). Accordingly, the endogenous protein quality control
system is speculated to be critical in controlling the onset and severity
of protein-conformational diseases that affect the brain.
The 70 kDa Hsps (Hsp70s) are abundantly expressed molecular chaperones that participate in a variety of fundamental cellular processes. Hsp70s promote the renaturation of misfolded
and/or aggregated proteins through ATP-dependent cycles of
binding and release and are likely to provide a first line of defense
against aggregation-prone disease proteins in vivo (Hartl and
Hayer-Hartl, 2002). Indeed, genetic screens and directed studies
have shown that Hsp70 and its partners potently modulate the
aggregation and/or suppresses the toxicity of mutant polyQ proteins in cell-, yeast-, worm- and fly-based models of polyQ aggregation and disease (Warrick et al., 1998; Chai et al., 1999; Warrick
et al., 1999; Jana et al., 2000; Krobitsch and Lindquist, 2000; Muchowski et al., 2000; Kobayashi and Sobue, 2001; Zhou et al., 2001;
Gunawardena et al., 2003; Nollen et al., 2004). Hsp70 overexpression
also conferred a dose-dependent improvement in behavioral phenotypes of transgenic mouse models of Spinocerebellar ataxia-1
(SCA1) and Spinal and bulbar muscular atrophy (SBMA) (Cummings et al., 2001; Adachi et al., 2003). Conversely, overexpression of
Hsp70 in the R6/2 mouse model of HD had only a marginal effect on
Wacker et al. • Hsp70 and Huntington’s Disease
weight loss and no effect on other behavioral and neuropathological
features (Hansson et al., 2003; Hay et al., 2004).
The goal of this study was to determine whether endogenous
Hsp70s can modulate the onset, progression and/or severity of
pathogenesis in a mouse model of HD. We used the well characterized R6/2 transgenic model of HD, in which expression of htt
exon 1 with ⬃150 CAG repeats causes a progressive HD-like
behavioral phenotype, including a robust decline in motor performance, alterations in activity level, weight loss and premature
death (Mangiarini et al., 1996). R6/2 mice also accumulate mutant htt exon 1 in intranuclear and cytoplasmic inclusion bodies
(Davies et al., 1997), a feature of HD brains (DiFiglia et al., 1997).
To determine whether inducible Hsp70s play a protective role in
the R6/2 model of HD, we crossed transgenic R6/2 mice with
knock-out mice that lack both Hsp70.1 and Hsp70.3.
Materials and Methods
Animals and breeding strategy. The University of Washington Animal
Care and Use Committee, the University of California San Francisco
IACUC Committee, or the Massachusetts Institute of Technology (MIT)
Committee on Animal Care approved all experiments and procedures
involving mice. Mice were maintained and bred in accordance with National Institutes of Health guidelines. Hemizygous transgenic R6/2 tg/⫺
male founder mice were kindly provided by Dr. James Olson (Fred
Hutchinson Cancer Research Center, Seattle, WA). The R6/2 tg/⫺ male
mice were backcrossed five times to C57BL/6 female mice to generate a
colony of R6/2 tg/⫺ mice. The Hsp70.1/3 knock-out mice were originally
generated by simultaneously targeting the Hsp70.1 and Hsp70.3 genes so
that homologous recombination with the targeting construct resulted in
a 12 kb deletion of both Hsp70.1 and Hsp70.3 coding regions as well as
insertion of a neomycin-resistance gene (Hampton et al., 2003). A breeding pair of double knock-out Hsp70.1/3 ⫺/⫺ mice (herein referred to as
Hsp70 ⫺/⫺ mice) were obtained with the permission of Dr. David Dix
from Dr. Philip Mirke (University of Washington, Seattle, WA) and used
to establish a colony of Hsp70 ⫺/⫺ mice that was maintained on a
C57BL/6 background for R6/2 studies and on 129Sv/Ev for prion studies.
Hsp70 ⫺/⫺ females were mated with R6/2 tg/⫺ males. Resulting R6/2 tg/⫺;
Hsp70 ⫺/⫹ males were mated with Hsp70 ⫺/⫺ females to yield four genotypes: R6/2 ⫺/⫺;Hsp70 ⫺/⫹, R6/2 tg/⫺;Hsp70 ⫺/⫹, R6/2 ⫺/⫺;Hsp70 ⫺/⫺,
and R6/2 tg/⫺;Hsp70 ⫺/⫺. Female mice of these four genotypes were analyzed alongside female R6/2 tg/⫺;Hsp70 ⫹/⫹and R6/2 ⫺/⫺;Hsp70 ⫹/⫹
mice for a total of six genotypes. The number of mice in each cohort that
was analyzed in the behavioral paradigms was as follows: R6/2 tg/⫺;
Hsp70 ⫹/⫹ (n ⫽ 21), R6/2 tg/⫺;Hsp70 ⫹/⫹ (n ⫽ 18), R6/2 ⫺/⫺;Hsp70 ⫺/⫹
(n ⫽ 27), R6/2 tg/⫺;Hsp70 ⫺/⫹ (n ⫽ 22), R6/2 ⫺/⫺;Hsp70 ⫺/⫺(n ⫽ 18),
and R6/2 tg/⫺;Hsp70 ⫺/⫺ (n ⫽ 18). The experimenter was blind to the
genotype during all testing paradigms. At 4 weeks of age the mice were
weaned and housed randomly in groups of five. Mice were allowed access
to water and food ad libitum and maintained on a 12 h light-dark cycle. At
10 weeks of age, mice were given powdered chow mixed with water
(mash) to provide adequate nutrition and hydration.
Genotyping. Mouse tail DNA was analyzed by PCR to determine the
genotype. The R6/2 transgene was identified as described using the
following primer sequences to identify the R6/2 transgene: forwardCGCAGGCTAGGGCTGTCAATCATGCT and reverse-TCATCAGCTTTTCCAGGGTCGCCAT (Hockly et al., 2003). Hsp70 ⫺/⫺ and
Hsp70 ⫺/⫹ mice were genotyped using a protocol established by the mutant mouse regional resource center at UC Davis (http://www.
mmrrc.org/strains/372/0372.html). The primer sequences used to identify the targeted knock-out Hsp70.1/3-neo were: forward-GAACGGAGGATAAAGTTAGG and reverse-AGTACACAGTGCCAAGACG. The primer
sequences used to identify the wild-type (WT) Hsp70.3 allele were: forwardGTACACTTTAAACTCCCTCC and reverse-CTGCTTCTCTTGTCTTCG.
We used GeneMapper techniques to determine the CAG repeat number by measuring the size of fluorescently labeled PCR products that
cover the CAG repeat region in the exon 1 of HD gene. GeneMapper
results showed that the R6/2 tg/⫺;Hsp70 ⫹/⫹ mice used in the behavioral
J. Neurosci., July 15, 2009 • 29(28):9104 –9114 • 9105
assays of this study had a CAG repeat length of ⬃185. Genetic deletion of
Hsp70.1/3 did not have a dramatic effect on CAG repeat length, which
was ⬃181 in the R6/2 tg/⫺;Hsp70 ⫺/⫺ mice used in the behavioral assays.
R6/2 tg/⫺;Hsp70 ⫹/⫹ mice used in the neuropathological assays of this
study had a CAG repeat length of 115.
Prion studies. Hsp70.1/3 knock-out mice (Hsp70 ⫺/⫺) used for the
prion studies were the same mice used for the R6/2 study, other than
being maintained on a 129Sv/Ev pure background (Hampton et al.,
2003). The Hsp70 overexpressing transgenic mouse was maintained on a
hybrid C57BL/6-SJL background and expresses the rat inducible Hsp70
gene of a rat under a ␤-actin promoter (Marber et al., 1995). Hsp70 ⫺/⫺
(n ⫽ 19) and Hsp70 ⫹/⫹ (n ⫽ 12) mice were injected intracranially with
30 ␮l of the Rocky Mountain Laboratory (“RML”) strain of murine
prions, corresponding to a dose of ⬃3.5 log LD50/30 ␮l. Hsp70 Tg ⫹/⫺
(n ⫽ 14) and Hsp70 Tg ⫺/⫺ (n ⫽ 10) mice were injected intracranially
with 5.5 log LD50/30 ␮l. Hsp70 ⫺/⫺(n ⫽ 19) and Hsp70 ⫹/⫹ (n ⫽ 11)
mice were also injected with “22L” strain of murine prions at a dose of
⬃3.5 log LD50/30 ␮l. Hsp70 Tg ⫹/⫺ (n ⫽ 12) and Hsp70 Tg ⫺/⫺ (n ⫽ 11)
mice were injected intraperitoneally with 100 ␮l of 4.5 log LC50 RML.
Mice were monitored daily for typical prion symptoms, such as imbalance, priapism (males), and weight loss.
Survival. For the R6/2 study (performed at the University of Washington) mice were observed twice daily, in the early morning and late afternoon. Survival was evaluated as the time to which the mice either died
spontaneously, or exceeded a defined endpoint criterion. Motor performance, neurobehavioral and physical symptoms, weight, and ability to
feed were closely monitored. Mice were killed when they had lost ⬎20%
of their maximal weight, and were no longer actively eating or drinking.
For the prion studies (performed at MIT), mice were closely monitored
and killed when they were unable to reach the food bin or water spout or
regain posture after being placed on their side.
Rotarod experiments. A Rotamex rotarod (Rotamex 4/8, Columbus
Instruments International) was programmed to accelerate from 4 to 40
rpm over a period of 10 min and measure the latency to fall. Testing was
performed every 2 weeks, starting at week 4. During the first week of
testing (week 4) the mice performed three trials per day on four consecutive days. Data from day 1 of week 4 was excluded from the analysis as
the mice were learning the task. During the subsequent weeks of testing
(6, 8, 10, 12, and 14 weeks), the mice were tested on three consecutive
days for three trials per day and all of the trial data were included in the
analysis. For each week, the trials were pooled and used to calculate the
average latency to fall for each mouse.
Weight loss. Starting at 28 d, the mice were weighed twice weekly, at the
same time of day, to the nearest 0.1 g.
Neurobehavioral and physical phenotype assessment. Beginning week 6,
mice were evaluated once a week, as described (Ditzler et al., 2003) to
extensively characterize their neurobehavioral and physical phenotype.
Each mouse was removed from its home cage and placed into a new,
sterile cage where it was observed for 2 min. Briefly, to assess the neurobehavioral phenotype mice were scored for grooming, spontaneous
activity, and locomotor activity. During the same 2 min period the physical phenotype of each mouse was scored for palpebral closure, coat
appearance, body position and tail position. The scoring protocol for the
neurobehavioral and physical assessment is detailed in supplemental Table 1, available at www.jneurosci.org as supplemental material. At the
end of the 2 min period the mouse was removed and suspended by the tail
⬃10 cm above the cage for 30 s to analyze pathogenic clasping behavior.
Paw clasping behavior was scored from 0 to 2 points as described in
supplemental Table 1, available at www.jneurosci.org as supplemental
material.
Statistics. All data are expressed at the mean ⫾ SEM. For each outcome
measure a two-way ANOVA was performed to determine whether there
was a significant interaction between the R6/2 transgene and the
Hsp70.1/3 genes. Specifically, the Mixed Models ANOVA in SPSS 13 was
used with week as a repeated variable, mouse as a subject variable and the
R6/2 transgene or Hsp70 deletion as factors. An unstructured repeated
covariance was used to analyze weight, rotarod, clasping, tremor, body
postion, tail position, grooming, locomotor activity, and spontaneous
activity outcome measures. A compound asymmetry repeated covari-
9106 • J. Neurosci., July 15, 2009 • 29(28):9104 –9114
ance was used to evaluate eye closure and fur
phenotypes. In cases in which differences between the various genotypes were examined at
a single time-point, a one-way ANOVA in conjunction with the Bonferroni post hoc test was
performed in GraphPad Prism. The Kaplan–
Meier method was used to evaluate survival,
followed by the log rank test to identify significant changes in GraphPad Prism.
Biochemical experiments. At 14 weeks of age
mice were killed with CO2. The brains were
removed and homogenized with 5 ␮l/mg tissue
RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM
NaCl, 1% NP40, 0.5% sodium deoxycholate,
0.1% SDS, 1 mM ␤-mercaptoethanol, 1 mM Figure 1. Deletion of Hsp70.1 and Hsp70.3 decreases survival in the R6/2 mouse, but not in mice infected with prions. A,
PMSF, and a protease inhibitor cocktail (Roche Kaplan–Meier survival curve for the indicated genotypes [R6/2 ⫺/⫺;Hsp70 ⫹/⫹ (n ⫽ 21), R6/2 tg/⫺;Hsp70 ⫹/⫹ (n ⫽ 18),
Diagnostics) and centrifuged at 10,000 ⫻ g for R6/2 ⫺/⫺;Hsp70 ⫺/⫹ (n ⫽ 27), R6/2 tg/⫺;Hsp70 ⫺/⫹ (n ⫽ 22), R6/2 ⫺/⫺;Hsp70 ⫺/⫺(n ⫽ 18), and R6/2 tg/⫺;Hsp70 ⫺/⫺
90 min at 4°C. A Bradford assay was used to (n ⫽ 18)] demonstrates that the absence of Hsp70.1/3 significantly decreased survival of R6/2 mice (log rank: p ⫽ 0.033). No
determine protein concentration of the super- nontransgenic, Hsp70 heterozygous knock-out, or Hsp70 homozygous knock-out mice died during the 14 week time course. B, C,
natant fraction. For Western blots, 3⫻ SDS Kaplan–Meier survival curves for Hsp70 ⫹/⫹ (n ⫽ 11, and 12, respectively) and Hsp70 ⫺/⫺ (n ⫽ 19) mice inoculated intracrasample buffer was added, and the samples were nially with 3.5 LC50 22L prion or 3.5 LC50 RML prion indicate that deletion of Hsp70.1/3 did not affect survival (log rank: p ⫽ 0.207
heated at 95°C for 5 min. Equal amounts of and 0.495, respectively).
protein (25 ␮g) were loaded in each well, separated by 4 –20% SDS/PAGE, transferred to niated secondary antibody, avidin coupled to horseradish peroxidase and
trocellulose membranes, blocked for 30 min at room temperature in 5%
reacted with DAB. Sections were analyzed and the numbers of Iba-1milk/TBST. After overnight incubation with primary antibody (made in
positive microglia were averaged and expressed as total number per 0.1
5% milk/TBST, blots were rinsed three times in TBST, incubated with
mm 3. Ten digital images per field were obtained and analyzed with
secondary antibodies for 2 h at room temperature, rinsed three times in
Image-Pro Plus (MediaCybernetics) to determine the number of microTBST and detected with enhanced chemiluminescence (GE Healthcare).
glia per unit area. Similar immunohistochemical methods were perAntibodies and concentrations were as follows: EM48 (1:500, a kind gift
formed to quantify astrocyte activation with a mouse monoclonal
from Dr. Xiao-Jiang Li, Emory University), GAPDH (1:200, Millipore
antibody against GFAP (1:1000, Abcam), c-Fos with a rabbit polyBioscience Research Reagents), secondary antibodies (1:5000, Jackson
clonal antibody (1:500, Abcam) and synaptophysin with a mouse
ImmunoResearch). All chaperone antibodies were from Stressgen Biomonoclonal antibody (1:200, Sigma). From each animal at least three
technologies, and dilutions were as follows: Hsp70 (1:1000), Hsp40 (1:
blind-coded random sections were analyzed, and the results were
10,000), Hsp27 (1:1000), Hsp25 (1:5000), Hsc70 (1:1000), Hsp90 (1:
averaged and expressed as mean value. Two sets of mice were used for
5000). To detect formic acid-sensitive monomer/oligomers, 100 ␮g of
the pathology experiments. For the first analysis, the mice that remained
total protein lysate was incubated with 100 ␮l of formic acid at room
alive after the 14 week behavioral study were perfused and the brains were
temperature for 1 h. Treating the lysate with formic acid releases mutant
harvested. For the second analysis, mice were bred specifically for the
htt species that migrate at the approximated molecular weight of a tribiochemical and pathology experiments so that a more comprehensive
mer/tetramer, although it is possible that this species is an aberrantly
analysis could be performed with a larger number of mice/genotype.
migrating monomer. Formic acid was removed in a speed vacuum and 30
Shown here are the results of the second analysis, but results were similar
␮l of SDS loading buffer was added. The samples were neutralized with 2
in both groups of animals.
␮l of 5 M NaOH and heated at 95°C for 5 min. For the filter assay, 30 ␮l of
1⫻ SDS sample buffer (4% SDS) were incubated with 100 ␮g of total
Results
protein lysate at 95°C for 5 min and then filtered onto a cellulose acetate
membrane with a slot blot manifold. For densitometry films were
Deletion of Hsp70.1 and Hsp70.3 decreases survival in the
scanned using ArcSoft PhotoStudio 5.5, and analyzed with ImageQuant
R6/2 mouse, but not in prion-infected mice
V2005 (GE Healthcare).
To determine whether endogenous Hsp70s play an important
Neuropathology. At 14 weeks of age, mice were deeply anesthetized
role in combating the toxic effects induced by a mutant htt
with halothane and perfused with 100 ml of phosphate buffer, followed
fragment, we crossed the R6/2 mouse model of HD to knockby 100 ml of 4% paraformaldehyde in phosphate buffer, pH 7.4. Brains
out mice lacking the inducible Hsp70.1 and Hsp70.3 genes
were removed, cryoprotected overnight in 30% sucrose and frozen in
(herein
referred to as Hsp70.1/3). We subsequently analyzed a
cooled isopentane. To investigate the effects of Hsp70.1/Hsp70.3 deletion
number
of physical and behavioral outcome measures in six
on levels of mutant htt immunoreactivity in R6/2 mutant mice, the secgenotypes of mice: R6/2 ⫺/⫺;Hsp70 ⫹/⫹, R6/2 ⫺/⫺;Hsp70 ⫺/⫹,
tions were immunolabeled overnight with a rabbit polyclonal antibody
R6/2 ⫺/⫺;Hsp70 ⫺/⫺, R6/2 tg/⫺;Hsp70 ⫹/⫹, R6/2 tg/⫺;Hsp70 ⫺/⫹,
(EM48, Millipore Bioscience Research Reagents) against a glutathione
and R6/2 tg/⫺;Hsp70 ⫺/⫺.
S-transferase fusion protein containing the first 256 aa of htt lacking the
polyQ and polyproline stretches. Sections were washed in PBS and then
Kaplan–Meier survival analysis demonstrated that deletion of
placed in biotinylated secondary antibody (1:100) (Vector Laboratories)
one copy of Hsp70.1/3 did not alter the lifespan of the R6/2 mouse
for 2 h. Sections were placed in 20% diaminobenzidine (DAB) (Vector
(details of all statistical analyses used in this study can be found in
Laboratories), mounted, dried, and coverslipped with Entillin (Fisher).
Materials and Methods). The survival curves of the R6/2 tg/⫺;
Three immunostained sections per mouse were imaged with an Olympus
Hsp70 ⫹/⫹ and the R6/2 tg/⫺;Hsp70 ⫺/⫹ mice were indistinguishdigital microscope. A total of 10 digital images per section and region of
able, as were the endpoint survival rates of 83% and 82%, respecinterest were analyzed with Image-Pro Plus (MediaCybernetics) to detively (Fig. 1 A). Strikingly, deletion of both copies of Hsp70.1/3
termine the optical density per field and the mean diameter and number
profoundly affected R6/2 survival: only 50% of the R6/2 tg/⫺;
of intranuclear inclusions. Individual values were averaged and exHsp70 ⫺/⫺ mice were alive at the study endpoint. Survival analypressed as mean value. To quantify microglial activation, microtome
sis demonstrated a significant decrease in the lifespan of the R6/2 tg/⫺;
sections from R6/2 mice were immunostained with a mouse monoclonal
antibody against Iba-1 (1:1000, DakoCytomation) followed by biotinylHsp70 ⫺/⫺ mice relative to the R6/2 tg/⫺;Hsp70 ⫹/⫹ ( p ⫽ 0.033,
J. Neurosci., July 15, 2009 • 29(28):9104 –9114 • 9107
performances of wild-type and R6/2 mice
were well matched. In contrast, the R6/
2 tg/⫺;Hsp70 ⫺/⫺ mice were already significantly impaired at 4 weeks ( p ⬍ 0.001),
demonstrating that the absence of inducible Hsp70s decreases the age of onset of
the R6/2 motor phenotype. The intermediate motor phenotype of the R6/2 tg/⫺;
Hsp70 ⫺/⫹ mice, when compared with the
R6/2 tg/⫺;Hsp70 ⫹/⫹ and the R6/2 tg/⫺;
Hsp70 ⫺/⫺ mice, suggests that the relative
expression levels of inducible Hsp70s
modulate both the progression and severity of motor abnormalities in R6/2 mice.
Deletion of Hsp70.1 and Hsp70.3
exacerbates neurobehavioral
phenotypes in R6/2 mice
To characterize the neurobehavioral
and physical decline of our mice we used
a modified SHIRPA assessment (Rogers
et al., 1997). This behavioral protocol
was recently refined to provide a rapid,
reproducible and quantitative means of
examining numerous outcome meaFigure 2. Deletion of Hsp70.1/3 worsens motor deficits in R6/2 mice. A–D, Deletion of Hsp70.1/3 decreases the latency to fall sures that clearly distinguish R6/2 transof R6/2 mice (two-way ANOVA: p ⬍ 0.05) (A), and increases severity of clasping (two-way ANOVA: p ⬍ 0.001) (B), tremor genic mice from their wild-type litter(two-way ANOVA: p ⬍ 0.001) (C), and grooming (two-way ANOVA: p ⬍ 0.03) (D). E, F, Deletion of Hsp70.1/3 decreases R6/2 mates (Ditzler et al., 2003). The
spontaneous activity (two-way ANOVA: p ⬍ 0.001) but has only a moderate effect on locomotor activity. Error bars indicate SEM. protocol includes a number of neurobeNote that in the absence of the R6/2 transgene, the loss of one or both copies of Hsp70.1/3 does not influence any of the presented havioral (clasping, tremor, grooming,
outcome measures.
spontaneous and locomotor activities)
and physical (weight, palpebral closure,
log rank test) and the R6/2 tg/⫺;Hsp70 ⫺/⫹ mice ( p ⫽ 0.026). An
coat appearance, body and tail position) outcome measures
intact endogenous Hsp70 response, thus, appears to be critical for
(supplemental Table 1, available at www.jneurosci.org as supsurvival in the R6/2 mice.
plemental material).
Hsp70s are presumed to be broadly protective against the
Progressive clasping of the front and hind limbs that is trigtoxic effects of misfolded protein in the CNS. Therefore, we exgered by a tail suspension test is a conserved motor abnormality
amined the effect of deleting endogenous Hsp70s on survival in
observed in numerous mouse models of neurological disease and
two mouse models of prion disease. A dose corresponding to
is widely used as a marker of neuronal dysfunction (Mangiarini et
⬃3.5 log LD50 of the 22L strain of murine prions was injected
al., 1996; Carter et al., 1999; Stack et al., 2005). We analyzed
intracranially into Hsp70 ⫹/⫹ and Hsp70 ⫺/⫺ mice. Surprisclasping behavior once a week by suspending each mouse above
ingly, the absence of endogenous Hsp70s had no effect on lifesits cage for 30 s and scoring 0 for no clasp, 1 for a mild clasp in
pan (Fig. 1 B). The survival curves of the Hsp70 ⫹/⫹ and
which only the fore or hind-limbs press into the stomach, and 2
Hsp70 ⫺/⫺ mice were indistinguishable, and the median survival
for a severe clasp in which both fore and hind-limbs touch and
times of the Hsp70 ⫹/⫹ and Hsp70 ⫺/⫺ mice injected with the 22L
press into the stomach. Deletion of Hsp70.1/3 significantly worsprion strain were 25.0 and 24.1 weeks, respectively. Similarly, the
ened ( p ⬍ 0.001) the average clasping score of the R6/2 mice (Fig.
median survival times of the Hsp70 ⫹/⫹ and Hsp70 ⫺/⫺ mice in2 B). In contrast to the R6/2 tg/⫺;Hsp70 ⫹/⫹mice, the R6/2 tg/⫺;
jected with the RML prion strain were identical at 26.4 weeks
Hsp70 ⫺/⫹ and the R6/2 tg/⫺;Hsp70 ⫺/⫺ mice already displayed
significant clasping by 6 weeks ( p ⬍ 0.01). Moreover, the R6/2 tg/⫺;
post-prion inoculation, and the two survival curves were indisHsp70 ⫺/⫺ mice consistently exhibited the most severe clasping
tinguishable (Fig. 1C). Moreover, transgenic overexpression of
score, followed by the R6/2 tg/⫺;Hsp70 ⫺/⫹ mice and finally the
Hsp70 did not prolong survival of prion-infected mice (data not
R6/2 tg/⫺;Hsp70 ⫹/⫹ mice, suggesting a gene dose-dependent efshown).
fect on the onset, progression and severity of this R6/2 phenotype
(Fig. 2 B).
Deletion of Hsp70.1/3 worsens motor deficits in R6/2 mice
R6/2 mice develop a progressive, resting tremor in the limbs,
We used a panel of diverse outcome measures to systematically
trunk and head, which was scored as 0 (no tremor,) 1 (mild
characterize the effect of deleting endogenous Hsp70s on the phetremor), or 2 (severe tremor) (Mangiarini et al., 1996; Ditzler et
notypes of R6/2 mice. We first evaluated the effects of Hsp70
al., 2003). Tremor analysis showed that deletion of Hsp70.1/3
deletion on motor performance, as measured by rotarod analysis,
significantly increased ( p ⬍ 0.001) the score of the R6/2 mice
which is widely used to characterize the progressive decline in
(Fig. 2C). At 6 weeks, the R6/2 tg/⫺;Hsp70 ⫹/⫹ mice had a negligimotor performance of R6/2 mice (Carter et al., 1999; Hockly et
ble tremor score, whereas the R6/2 tg/⫺;Hsp70 ⫺/⫹ and the R6/2 tg/⫺;
al., 2002). We found that deletion of Hsp70.1/3 significantly enHsp70 ⫺/⫺ mice exhibited a significantly higher average score of
hanced ( p ⬍ 0.05) the severity of rotarod deficits in R6/2 mice
0.5 ( p ⬍ 0.001), demonstrating that deletion of one or both
(Fig. 2A). As expected, at the early time point of 4 weeks the
9108 • J. Neurosci., July 15, 2009 • 29(28):9104 –9114
alleles of Hsp70.1/3 decreased the age of tremor onset. The consistently intermediate score of the R6/2 tg/⫺;Hsp70 ⫺/⫹ mice relative to the R6/2 tg/⫺;Hsp70 ⫹/⫹ and R6/2 tg/⫺;Hsp70 ⫺/⫺ mice
suggests a gene dose-dependent effect of Hsp70.1/3 on the R6/2
tremor phenotype.
As R6/2 mice become symptomatic, either a complete lack of
grooming or a stereotypic, repetitive grooming behavior is often
observed (Mangiarini et al., 1996; Carter et al., 1999). Repetitive
hindlimb grooming is thought to mimic the choreiform movements displayed by HD patients (Mangiarini et al., 1996). Mice
received a score of 1 for normal grooming and a score of 2 for
abnormal grooming. Analysis of cumulative grooming scores revealed that the deletion of Hsp70.1/3 genes significantly worsened
( p ⬍ 0.03) the abnormal grooming behavior of the R6/2 at later
time points (Fig. 2 D). In this case, however, the loss of both alleles
of Hsp70.1/3 was required to enhance the progression and endpoint severity of the R6/2 grooming phenotype.
The progressive development of abnormalities in the activity
level of R6/2 HD mice has been well characterized (Dunnett et al.,
1998; Bolivar et al., 2003; Stack et al., 2005), and our modified
SHIRPA protocol included two measures of activity. We first
measured spontaneous activity by scoring the coverage of four
delineated cage quadrants by each mouse during a 2 min testing
period. A score of 1 denoted movement into all four quadrants, 2
denoted slow movement in three or less quadrants, 3 denoted no
movement or stereotypic darting/circling movements. We found
that deletion of Hsp70.1/3 significantly exacerbated ( p ⬍ 0.001)
the spontaneous activity phenotype of the R6/2 mouse, most noticeably after 8 weeks of age (Fig. 2 E). The absence of both alleles
of Hsp70.1/3 had a marked effect on the onset, progression and
endpoint severity. Despite the fact that deletion of one allele of
Hsp70.1/3 also had a more moderate effect, there was still a trend
toward gene dose dependence for this outcome measure. We also
performed the locomotor test as a second measure of activity by
scoring the number of times that each mouse touched the side of
the cage during a 2 min observation period. The locomotor activity test did not reveal a significant effect of the inducible
Hsp70s on the R6/2 phenotype, although there was a trend toward a gene dose-dependent effect of Hsp70.1/3 deletion to enhance motor abnormalities (Fig. 2 F). Thus, two distinct outcome
measures showed that Hsp70.1/3 affects the development, progression and severity of activity deficits in the R6/2 mouse.
Deletion of Hsp70.1/3 exacerbates the physical phenotypes of
R6/2 mice
To characterize decline in the physical phenotypes of the R6/2
mice, we measured weight and scored for coat appearance, body
position, tail position and palpebral closure. Female R6/2 mice
show a characteristic weight loss pattern: weight plateaus around
week 8 and declines significantly at 12 weeks (Hockly et al., 2003).
The weights of the R6/2 ⫺/⫺;Hsp70 ⫹/⫹, R6/2 ⫺/⫺;Hsp70 ⫺/⫹, and
the R6/2 ⫺/⫺;Hsp70 ⫺/⫺ mice were indistinguishable, demonstrating that the absence of inducible Hsp70s alone does not influence body weight (Fig. 3A). Analysis of the weight of the
R6/2 tg/⫺;Hsp70 ⫹/⫹ and R6/2 tg/⫺;Hsp70 ⫺/⫺ mice showed a significant interaction ( p ⬍ 0.05) on this phenotype and a
Hsp70.1/3 gene dose-dependent trend on weight loss was observed, suggesting that the inducible Hsp70s may modulate the
onset, progression and endpoint severity of the R6/2 weight loss
phenotype.
The coat appearance of the R6/2 mice declines as the disease
state progresses and is characterized by a score of 1 for a shiny,
well groomed coat and a score of 2 for a scruffy and/or piloerected
Figure 3. Deletion of Hsp70.1/3 exacerbates the physical phenotypes of R6/2 mice. The
absence of Hsp70.1/3 significantly exacerbates the weight loss phenotype (two-way ANOVA:
p ⬍ 0.05) (A), and worsens the coat appearance (two-way ANOVA: p ⬍ 0.001) (B), body
position (two-way ANOVA: p ⬍ 0.02) (C), and tail position (two-way ANOVA p ⬍ 0.001) (D) of
R6/2 mice. Error bars indicate SEM. Note that in the absence of the R6/2 transgene, the loss of
one or both copies of Hsp70.1/3 does not influence any of the presented outcome measures.
coat. Deletion of Hsp70.1/3 significantly worsened ( p ⬍ 0.001)
the R6/2 coat appearance phenotype (Fig. 3B). A decrease in the
age of onset, enhanced progression and increase in endpoint severity were observed with a trend toward Hsp70.1/3 gene dose
dependence. We also scored body position and tail position to
further evaluate the effect of Hsp70.1/3 on the decline of the R6/2
physical phenotype. The R6/2 body position phenotype was
scored as 1 for normal, and 2 for a hunched and rounded stature.
Tail position was scored as 1 for normal or horizontally extended,
and 2 for dragging/straub. Deletion of Hsp70.1/3 significantly
enhanced the severity of the body position outcome measure
( p ⬍ 0.02, Fig. 3C) and the tail position outcome measure ( p ⬍
0.001, Fig. 3D). In both cases a trend toward an Hsp70.1/3 genedose dependent enhancement of phenotypic severity was observed. The only component of the physical phenotype test that
was unaffected by the deletion of Hsp70.1/3 was palpebral closure
(data not shown). Importantly, all outcome measures included in
the neurobehavioral and physical phenotype assessment showed
that there were no significant differences between the wild-type
nontransgenic mice and the Hsp70.1/3 heterozygous or homozygous knock-out mice.
Deletion of Hsp70.1/3 increases the size of inclusion bodies in
R6/2 mice
To determine whether the exacerbated behavioral and physical
phenotypes observed in R6/2 mice that lacked inducible Hsp70s
correlated with changes in the density or size of inclusion bodies
formed by mutant htt exon 1, we examined serial sections from
the neocortex of 14-week-old mice with immunohistochemistry
and the EM48 anti-htt antibody. As expected, the R6/2 ⫺/⫺;
J. Neurosci., July 15, 2009 • 29(28):9104 –9114 • 9109
Western blots probed with the EM48
antibody on total brain homogenates
from R6/2 tg/⫺;Hsp70 ⫹/⫹ mice showed
that all of the reactivity corresponded to
large SDS-insoluble aggregates that were
retained in the stacking portion of the gel
(Fig. 5A) as described (Davies et al., 1997).
An identical pattern of reactivity is observed on Western blots that contain purified mutant htt exon 1 with 53Q that has
been aggregated into fibrillar protein assemblies (Wacker et al., 2004). Surprisingly, analysis of the pixel intensities relative to the GAPDH loading controls
showed that the average EM48 reactivities
for the R6/2 tg/⫺;Hsp70 ⫹/⫹ and R6/2 tg/⫺;
Hsp70 ⫺/⫺ mice were indistinguishable
(Fig. 5A, B). Previous studies showed that
HD brain homogenates treated with formic acid liberate a SDS-resistant oligomer
Figure 4. Deletion of Hsp70.1/3 increases the size of inclusion bodies in R6/2 mice. A, Representative images (600⫻) of as analyzed by Western immunoblots (Iuinclusion bodies in the neocortex of R6/2 mice as detected with the anti-htt antibody EM48. B, Quantification of inclusion body chi et al., 2003; Hoffner et al., 2005). Simnumber in the neocortex shows that R6/2 tg/⫺;Hsp70 ⫺/⫺ mice have an increase in the density of inclusion bodies compared with ilar to the results observed in HD brain
R6/2 tg/⫺;Hsp70 ⫹/⫹ mice, although this difference only showed a trend toward statistical significance ( p ⫽ 0.086). C, Repre- homogenates, we found that treatment of
sentative images (1000⫻) illustrating the size of inclusion bodies in the neocortex of R6/2 mice as detected with the anti-htt
total brain homogenates from R6/2 tg/⫺;
antibody EM48. D, Quantification of inclusion body size shows that the average size of the inclusion bodies was significantly larger
Hsp70 ⫹/⫹ mice with formic acid released
( p ⬍ 0.001) in R6/2 tg/⫺;Hsp70 ⫺/⫺ mice compared with R6/2 tg/⫺;Hsp70 ⫹/⫹ mice. Statistical comparisons were performed by
two
bands that reacted with EM48 which
one-way ANOVA (n ⫽ 6 –11 mice per group).
migrated at an apparent molecular mass
of 70 – 85 kDa, while concomitantly leading
Hsp70 ⫹/⫹, R6/2 ⫺/⫺;Hsp70 ⫺/⫺ and R6/2 ⫺/⫺;Hsp70 ⫺/⫹ mice
to nearly a complete loss of reactivity in the stacking gel (Fig. 5C).
did not display EM48-positive inclusion bodies (data not
Based on their apparent molecular mass, these bands may reflect
shown). Immunohistochemical analyses on cortical brain seca low molecular mass SDS-resistant oligomer or aberrantly mitions with EM48 suggested R6/2 tg/⫺;Hsp70 ⫺/⫺ had an increased
grating monomers of mutant htt exon 1. The levels of formic
density of inclusion bodies compared with R6/2 tg/⫺;Hsp70 ⫹/⫹
acid-sensitive monomers/oligomers (normalized to GAPDH remice (Fig. 4 A). However, quantification of the number of incluactivity) appeared to increase in the absence of Hsp70.1/3, but did
sion bodies in a defined brain volume indicated this difference
not reach statistical significance (Student’s t test p ⫽ 0.15). (Fig.
only showed a trend toward statistical significance ( p ⫽ 0.086)
5D). Identical findings were observed using the 3B5H10 antibody
(Fig. 4 B).
generated by the Finkbeiner laboratory (data not shown).
Analysis of average inclusion body diameter demonstrated that the
We next used filter-trap assays as an independent method to
R6/2 tg/⫺;Hsp70 ⫹/⫹ (4.22 ⫾ 0.55 ␮m) and R6/2 tg/⫺;Hsp70 ⫺/⫹ (3.81 ⫾
evaluate total SDS-insoluble material formed by mutant htt exon
0.34 ␮m) mice were indistinguishable ( p ⬎ 0.05, Fig. 4 D). In
1 in R6/2 brains in the presence and absence of inducible Hsp70s.
comparison, the R6/2 tg/⫺;Hsp70 ⫺/⫺ inclusion bodies (7.68 ⫾
In this assay, total brain homogenates were boiled in SDS and
0.44 ␮m) stained with EM48 were dramatically and significantly
filtered through a cellulose acetate membrane that contains 0.2
tg/⫺
⫹/⫹
larger ( p ⬍ 0.001) than in R6/2
;Hsp70
mice (Fig. 4C, D).
␮m pores. Previous studies with brains from R6/2 mice showed
The pixel intensity of EM48 staining in R6/2 mice lacking both
that this method traps large (⬎0.2 ␮m) SDS-insoluble aggregates
alleles of Hsp70.1/3 also appeared greater than in R6/2 mice alone.
of fibrillar material (Scherzinger et al., 1997). Consistent with the
In summary, these results indicate that the complete absence of
Western immunoblots, we found that the levels of SDS-insoluble
inducible Hsp70s increased the size of inclusion bodies formed by
material detected by EM48 in filter-trap assays were not signifimutant htt exon 1 in R6/2 mice, consistent with in vitro data
cantly different between R6/2 tg/⫺;Hsp70 ⫹/⫹ and R6/2 tg/⫺;
indicating Hsp70 can directly modulate the misfolding and agHsp70 ⫺/⫺ mice (Fig. 5E, F ). Interestingly, total brain homogegregation of mutant htt (Muchowski et al., 2000; Wacker et al.,
nates treated with formic acid and SDS were still detected by
2004).
EM48 in filter assays (Fig. 5G), suggesting that this treatment
releases oligomeric species larger than 0.2 ␮m in size. However, as
Deletion of Hsp70.1/3 does not modulate levels of
with the other assays, no significant differences were observed in
SDS-insoluble fibrillar protein aggregates formed by mutant
brain homogenates analyzed in this manner between R6/2 tg/⫺;
Htt exon 1 in R6/2 mice
Hsp70 ⫹/⫹ and R6/2 tg/⫺;Hsp70 ⫺/⫺ mice (Fig. 5G, H ). Similar
We next sought to determine whether the increased size of incluresults were also obtained when agarose native gels were used to
sion bodies in the absence of inducible Hsp70s in R6/2 mice was
detect oligomeric species in total brain homogenates from R6/2 tg/⫺;
attributed to increased levels of aggregates formed by mutant htt
Hsp70 ⫹/⫹ and R6/2 tg/⫺;Hsp70 ⫺/⫺ mice (Fig. 5I ). Thus, three
exon 1. We used Western immunoblots, filter-trap assays and
independent approaches used to evaluate mutant htt exon 1 agagarose gels to measure the relative levels of SDS-insoluble aggregregates in 14-week-old R6/2 brain homogenates showed no siggates and formic acid–sensitive htt species in the brains of 14nificant differences in aggregate levels in the presence or absence
week-old R6/2 tg/⫺;Hsp70 ⫹/⫹ and R6/2 tg/⫺;Hsp70 ⫺/⫺ mice.
9110 • J. Neurosci., July 15, 2009 • 29(28):9104 –9114
of inducible Hsp70s. Similar results were
obtained in 7-week-old R6/2 brain homogenates (data not shown).
Previous
studies
demonstrated
changes in the relative levels of chaperone
proteins in mouse models over the course
of polyQ disease (Cummings et al., 2001;
Hay et al., 2004). For example, expression
of mutant ataxin-1 in a mouse model of
SCA1 elicits an increase in Hsp70 expression (Cummings et al., 2001), whereas
levels of Hsp70 and other chaperones decreased progressively in R6/2 mice (Hay et
al., 2004). To test whether deletion of
Hsp70.1/3 caused compensatory changes
in the relative levels of other heat shock
proteins, we performed Western immunoblots on brain homogenates from R6/
2 tg/⫺;Hsp70 ⫹/⫹, R6/2 tg/⫺;Hsp70 ⫺/⫺, R6/
2 ⫺/⫺;Hsp70 ⫹/⫹ and R6/2 ⫺/⫺;Hsp70 ⫺/⫺
mice. At 14 weeks of age, no significant
changes were detected in the levels of
Hsp27 and Hsp90 (supplemental Fig. 1,
available at www.jneurosci.org as supplemental material), or Hsp25, Hsp40 and
Hsc70 (data not shown), relative to a
GAPHD loading control. Thus, the lack of
both Hsp70 alleles on a wild-type or R6/2
strain background did not appear to confer compensatory changes in levels of
other major heat shock proteins.
Deletion of Hsp70.1/3 exacerbates the
loss of c-Fos immunoreactivity and
other neuropathological deficits in
R6/2 mice
To determine the effect of Hsp70 deletion
on neuronal loss in R6/2 mice we used
immunohistochemistry and unbiased stereology with an antibody against the Figure 5. Deletion of Hsp70.1/3 does not modulate levels of SDS-insoluble fibrillar protein aggregates formed by mutant htt
neuronal-specific protein NeuN in brain exon 1 in R6/2 mice. A, B, Deletion of Hsp70.1/3 does not alter the levels of EM48 reactive SDS-insoluble aggregates (normalized to
sections from 14-week-old mice. These GAPDH reactivity) measured with Western immunoblots in 14-week-old R6/2 brain homogenates (Student’s t test p ⫽ 0.96). C, D,
analyses showed no significant change in Treatment of brain homogenates with formic acid liberates an SDS-resistant monomeric/oligomeric mutant huntingtin exon 1
NeuN immunoreactivity in the cortex or species. The levels of formic acid-sensitive monomers/oligomers (normalized to GAPDH reactivity) appeared to increase in the
striatum of R6/2 tg/⫺;Hsp70 ⫹/⫹ mice absence of Hsp70.1/3, but did not reach statistical significance (Student’s t test p ⫽ 0.15). E, F, The levels of SDS-insoluble
EM48-positive fibrillar aggregates in brain measured by a filter-trap assay do not change in the absence of Hsp70.1/3 (Student’s t
compared with WT animals, and no test p ⫽ 0.89). G, H, Formic acid-treated brain homogenates were subjected to the filter-trap assay, which showed no change in
significant difference in NeuN levels be- EM48 immunoreactivity in the absence of Hsp70.1/3 (Student’s t test p ⫽ 0.90). I, A native agarose gel used to examine EM48
tween R6/2 tg/⫺;Hsp70 ⫹/⫹ and R6/2 tg/⫺; immunoreactive oligomeric species in R6/2 brain homogenates shows no apparent change in the absence of Hsp70.1/3.
Hsp70 ⫺/⫺ mice (data not shown). In an
independent study, we recently found that
and this loss was further and significantly exacerbated ( p ⬍ 0.05)
immunoreactivity for the presynaptic protein synaptophysin and
in R6/2 tg/⫺;Hsp70 ⫺/⫺ mice (Fig. 6 A, B). Importantly, levels of
the calcium regulated immediate early gene product c-Fos, a surc-Fos were not significantly different between R6/2 ⫺/⫺;Hsp70 ⫹/⫹
rogate marker for neuronal activity, were decreased in R6/2 mice
and R6/2 ⫺/⫺;Hsp70 ⫺/⫺ mice. Levels of synaptophysin immunorelative to nontransgenic littermate controls, and that these
reactivity also appeared to be decreased in the cortex and striatum
changes were attenuated in R6/2 mice treated with a smallof R6/2 tg/⫺;Hsp70 ⫺/⫺ mice compared with R6/2 ⫺/⫺;Hsp70 ⫺/⫺
molecule inhibitor of kynurenine 3-monooxygenase in a manner
mice, although this decrease did not reach statistical significance
that correlated with survival (P. Guidetti, W. Kwan, S.-Y. Huang,
possibly explained by the lack of statistical power due to the small
J. Lee, C. Patrick, F. Giorgini, T. Möller, C. S. Cheah, T. Wu, K.
numbers of mice analyzed (n ⫽ 4 – 6 per group) (Fig. 6C, D). A
Scearce-Levie, J. M. Muchowski, E. Masliah, R. Schwarcz, and P.
recent study showed increased levels of immunoreactivity for the
J. Muchowski, unpublished observations). In the current study
tg/⫺
microglia-specific protein Iba1 in R6/2 mice (Simmons et al.,
;
immunohistochemical analysis of brain sections from R6/2
⫹/⫹
2007). We observed a significant ( p ⫽ 0.0297) increase in immumice showed a significant ( p ⬍ 0.05) decrease in c-Fos
Hsp70
noreactivity for Iba1, and a trend toward an increase in the
immunoreactivity relative to WT mice in the cortex and striatum,
J. Neurosci., July 15, 2009 • 29(28):9104 –9114 • 9111
ing, activity, weight, coat appearance, and
body position). The absence of both alleles of Hsp70.1/3 profoundly enhanced
the onset, severity and progression of
behavioral phenotypes in R6/2 mice, including a significant decrease in median
lifespan. R6/2 mice completely lacking inducible Hsp70s showed an increase in the
number and size of inclusion bodies, although these findings did not correlate
with a biochemical changes in the relative
levels of SDS-insoluble fibrillar aggregates
as measured by multiple independent approaches. Finally, we found that deletion
of Hsp70.1/3 exacerbated the loss of c-Fos,
a surrogate marker for neuronal activity,
in a highly significant manner. These findings indicate that the absence of inducible
Hsp70s increased neuronal sensitivity to
mutant htt exon 1 in the R6/2 mouse
model of HD, without affecting htt expression or its accumulation into SDSinsoluble aggregates.
Deletion of Hsp70.1/3 had no significant effect on lifespan in two mouse models of transmissible prion disease. This is
not simply because prion diseases are so
extreme that they can not be modified. Indeed, deletion of HSF1, a master regulator
of homeostatic stress responses, has a pronounced effect on the course of these
same prion models (Steele et al., 2008).
Thus, the striking effect of the absence of
inducible Hsp70s on R6/2 mice indicates
that a specific genetic interaction occurs
between the inducible Hsp70s and the
mutant htt fragment in vivo. Although the
inducible Hsp70s may play a pivotal role
in prion propagation in yeast (Tutar et al.,
Figure 6. Deletion of Hsp70.1/3 exacerbates the loss of c-Fos immunoreactivity and other neuropathological deficits in R6/2 2006; Loovers et al., 2007), our results
mice. A, B, Quantification of c-Fos immunohistochemistry in the neocortex from 14-week-old mice shows that R6/2 tg/⫺; suggest that the inducible Hsp70s do not
Hsp70 ⫺/⫺ mice have a significant decrease ( p ⬍ 0.05) in c-Fos levels compared with R6/2 tg/⫺;Hsp70 ⫹/⫹ mice. C, D, Quanti- influence toxicity in mice infected with esfication of synaptophysin immunohistochemistry in the neocortex from 14-week-old mice shows that R6/2 tg/⫺;Hsp70 ⫹/⫺ and ⫺/⫺ tablished strains of prions. Importantly,
mice have a significant decrease ( p ⬍ 0.05) in synaptophysin levels compared with R6/2 ⫺/⫺;Hsp70 ⫺/⫺ mice. E, F, Quantification of unlike HD, which is an autosomal domiIba1 and GFAP immunohistochemistry in the neocortex from 14-week-old mice shows that R6/2 tg/⫺;Hsp70 ⫹/⫺ and ⫺/⫺ mice have a nant inherited neurodegenerative disorsignificant increase ( p ⫽ 0.0297) in Iba1 levels, and a trend toward increased GFAP levels ( p ⫽ 0.1048), respectively, compared with
der, prion disease encompasses diverse
R6/2 ⫺/⫺;Hsp70 ⫺/⫺ mice.
etiologies in addition to acute infection
(Kingsbury et al., 1983). The inducible
Hsp70s may possibly play a role in supastrocyte-specific marker GFAP in the cortex of R6/2 tg/⫺;
pressing toxicity in other mouse models of spontaneous and/or
Hsp70 ⫺/⫺ mice compared with R6/2 ⫺/⫺;Hsp70 ⫹/⫺ and ⫺/⫺ mice
genetically derived prion disease.
(Fig. 6 E, F ). Insufficient brain material unfortunately precluded
The loss of one copy of Hsp70.1/3 did not decrease the lifespan
the analysis of other genotypes in these studies. Our results demof R6/2 mice, suggesting a potent activity of endogenous Hsp70
onstrate that endogenous Hsp70s protect against the loss of c-Fos
chaperones, even when present at half of their normal concentrain a highly significant manner, and suggest these chaperones are
tion, to mitigate pathogenic cascades and modulate disease onset,
critical regulators of neuronal activity and inflammatory reprogression and severity in vivo. However, the decrease in the age
sponses in R6/2 mice.
of onset observed for the majority of behavioral and physical testing
Discussion
parameters in both the R6/2 tg/⫺;Hsp70 ⫺/⫹ and the R6/2 tg/⫺;
Here we showed that endogenous Hsp70s critically regulate the
Hsp70 ⫺/⫺ mice demonstrates that an intact inducible Hsp70s retoxicity of a disease-causing misfolded protein in a mouse model
sponse is required to limit mutant htt toxicity at the earliest stages of
of HD. The absence of even one allele of the Hsp70.1/Hsp70.3
pathogenesis in R6/2 mice.
genes significantly exacerbated the severity of a number of outThe absence of both alleles of Hsp70.1/3 significantly income measures for the R6/2 mouse model of HD (rotarod, claspcreased the average size and appeared to increase the number of
9112 • J. Neurosci., July 15, 2009 • 29(28):9104 –9114
inclusion bodies in R6/2 brains, yet paradoxically did not alter the
total load of fibrillar aggregates detected biochemically. What
mutant htt species detected by EM48 in brain sections can account for the increased size and abundance of inclusion bodies?
Our biochemical studies excluded the possibility that the increased size and abundance of inclusion bodies were due to any
significant changes in fibrillar and/or large oligomeric species
that are insoluble in SDS. In addition, formic acid-treated R6/2
brain lysates had similar levels of mutant htt monomers and oligomers in the presence or absence of Hsp70s. Our previous in
vitro studies used atomic force microscopy and biochemical approaches to demonstrate that the cooperative activity of Hsp70
and Hsp40 stabilized a monomeric conformation of a mutant htt
fragment (HD53Q), while concomitantly suppressing the accumulation of annular and spherical oligomeric assemblies
(Wacker et al., 2004). However, a recent study indicated Hsp70
and Hsp40 can also partition onto SDS-soluble mutant htt oligomers in an ATP-dependent manner (G. P. Lotz, J. Legleiter, E.
Mitchell, S.-Y. Huang, C.-P. Ng, C. Glabe, L. M. Thompson, and
P. J. Muchowski, unpublished observations). Therefore we speculate that, in the absence of inducible Hsp70s in R6/2 mice, small,
SDS-soluble mutant htt exon 1 assemblies that accumulate may
account for the increase in inclusion body density and size in the
R6/2 tg/⫺;Hsp70 ⫺/⫺ mice. Consistent with this interpretation,
deletion of C terminus of Hsp70 interacting protein (CHIP) in a
mouse model of Spinocerebellar Ataxia Type 3 (SCA3) markedly
increased levels of ataxin-3 microaggregates in a manner that
correlated with exacerbated behavioral phenotypes in these mice
(Williams et al., 2009). We hypothesize that inducible Hsp70s
buffer toxicity by binding monomeric and/or low molecular mass
SDS-soluble oligomers that are likely off-pathway to fibril formation, but may be potentially pathogenic. However, based on the
multifunctional nature of Hsp70 it is very likely that this chaperone can also suppress protein misfolding toxicity by multiple
mechanisms independent of its direct effects on misfolded protein (see below).
Although larger inclusion bodies were observed in R6/2 mice
in the absence of Hsp70s, this does not necessarily suggest that
inclusion bodies are toxic entities. Indeed, in direct contrast to
the current study, we recently observed a strong positive correlation between survival and inclusion body size in mice treated
with an inhibitor of the mitochondrial enzyme kynurenine
3-monooxygenase (P. Guidetti, W. Kwan, S.-Y. Huang, J. Lee, C.
Patrick, F. Giorgini, T. Möller, C. S. Cheah, T. Wu, K. ScearceLevie, J. M. Muchowski, E. Masliah, R. Schwarcz, and P. J. Muchowski, unpublished observations). Furthermore, systematic
analysis of the effects of genetic enhancers (Willingham et al.,
2003) or suppressors (Giorgini et al., 2005) of mutant htt exon 1
toxicity in yeast showed no correlations between toxicity and
inclusion bodies. These experiments underscore the inherent
limitations of quantifying inclusion body size and abundance in
mouse brain sections using immunohistochemistry to draw
meaningful deductions of the role of these abnormal brain deposits on in vivo pathogenesis. We propose that the molecular
composition of SDS-soluble conformers that may exist in a diffuse fraction or in inclusion bodies, be they monomers or small
oligomers, will be the key to understanding which structures mediate pathogenesis. Thus, tools to identify and track such structures in situ, such as antibodies, will be required before unequivocal experiments can determine which are the toxic species of
mutant htt in mouse models of HD.
Although a primary function of Hsp70s in animal models of
polyQ disease may be to counteract the assembly process that
leads to the accumulation of toxic monomers/oligomers, the inducible Hsp70s may also buffer the toxicity of mutant htt monomers/oligomers by masking surfaces that promote pathogenic
interactions with essential cellular proteins. For example, in one
study, a mutant htt monomer underwent an intramolecular transition that facilitated an interaction with the Tata binding protein
(TBP) and ultimately resulted in the functional inactivation of
this important transcription factor (Schaffar et al., 2004). Addition of Hsp70 to the in vitro system prevented the conformational
rearrangement of mutant htt and thus inhibited the pathogenic
interaction with TBP, suggesting that the activity of Hsp70 to
bind and hold mutant htt monomers can prevent aberrant
protein-protein interactions that lead to neuronal dysfunction.
Mutant htt, in addition to causing transcriptional repression, has
also been shown to upregulate p53 associated transcriptional
events in neuronal cultures (Bae et al., 2005). p53 is a strong
suppressor of Hsp70 expression in specific neuronal subtypes
that are affected in HD (Tagawa et al., 2007), and, moreover,
genetic deletion of p53 ameliorates behavioral abnormalities in
the N171-82Q mouse model of HD (Schilling et al., 1999; Bae et
al., 2005). Thus, it is tempting to speculate that the effect of p53
on HD pathogenesis may be at least partially mediated by changes
in the expression of inducible Hsp70s.
The exacerbation of R6/2 phenotypes in mice lacking Hsp70s
may also be due to an overall disruption in the protein homeostasis network, as suggested from studies in Caenorhabditis elegans
by Morimoto and colleagues (Gidalevitz et al., 2006). Consistent
with this scenario, we observed that levels of the calcium regulated immediate early gene c-Fos and the presynaptic protein
synaptophysin were decreased in R6/2 mice lacking Hsp70s relative to controls, whereas levels of protein markers for inflammatory responses (Iba1 and GFAP) were increased. Although the
functional significance of these changes in R6/2 mice has not yet
been investigated, the levels of c-Fos, which is used a surrogate
marker for neuronal activity, are tightly linked to cognitive deficits in mouse models of AD (Palop et al., 2003). The apparent loss
of synaptophysin in R6/2 mice lacking Hsp70 is consistent with
previous studies in R6/2 mice (Cepeda et al., 2003) and, more
broadly, with studies that suggest synaptic loss may be important
for pathogenesis in HD (Li et al., 2003). Interestingly, as mutant
htt inhibits the acetyltransferase activity of CREB-binding protein (CBP) (Steffan et al., 2000; Steffan et al., 2001), which itself
controls c-Fos expression (Yuan et al., 2009), it is possible that
aberrant protein interactions between mutant htt and CBP, and
suppression of these interactions by Hsp70 (Schaffar et al., 2004),
mediate in part the effects we observed on c-Fos expression in
R6/2 mice. Our results also indicate endogenous Hsp70s may
influence inflammatory responses, consistent with previous reports (Van Molle et al., 2002; Hampton et al., 2003; Singleton and
Wischmeyer, 2006; Mycko et al., 2008). Collectively these studies
strongly suggest that Hsp70s may modulate pathogenesis of protein misfolding diseases in vivo by direct and indirect effects in
multiple cell types that may only be dissected by modulating
levels of these proteins and their interacting proteins in specific
cell types in vivo.
The majority of the behavioral outcome measures that we
examined in R6/2 mice showed a trend toward Hsp70.1/3 gene
dose dependence, demonstrating that the relative levels of inducible Hsp70s can dramatically alter pathogenesis in vivo. A recent
study used RNA interference (RNAi) to show that the expression
levels of Hsp70 dictate the susceptibility of primary neurons to
mutant htt toxicity (Tagawa et al., 2007). Thus, even a modest
increase in the levels of molecular chaperones may suffice to de-
crease the severity of protein-conformational disorders. Indeed,
treatment with arimoclomol, a compound that acts to amplify
the endogenous heat shock response to the accumulation of misfolded disease-causing proteins, significantly delayed disease
progression in a mouse model of ALS (Kieran et al., 2004) and is
being evaluated in a clinical trial in ALS patients. In addition,
geranylgeranylacetone, which acts to increase the levels of heat
shock proteins in vivo, significantly decreased the severity of the
neuromuscular phenotype in a mouse model of SBMA (Katsuno
et al., 2005). Pharmacological strategies aimed at enhancing the
production or activity of molecular chaperones, such as Hsp70,
may prove beneficial in the treatment of protein-conformational
disorders.
The demonstration in this study that the endogenous Hsp70.1
and Hsp70.3 chaperones are an integral component of the physiological response to an aggregation-prone disease protein in vivo
highlights the importance of investigating genetic modifiers of
disease pathogenesis as potential therapeutic targets. The multifunctional activity of Hsp70 in vivo contributes to its attraction as
a potential therapeutic target for diseases associated with protein
misfolding and aggregation. Modification of the levels and/or
activity of Hsp70 can potentially impact a number of important
cellular pathways that influence HD pathogenesis, and likely
plays similar role in modulating pathogenic cascades in diverse
protein-conformational disorders.
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