Scientific Report 2014

Transcription

GLADSTONE INSTITUTES FINDINGS 2014
Gladstone Scientific Advisory Board Members
Cardiovascular Disease
Virology and Immunology
Neurological Disease
Shaun R. Coughlin, MD, PhD
Cardiovascular Research Institute
University of California, San Francisco
Elizabeth H. Blackburn, PhD
Department of Biochemistry and Biophysics
George Q. Daley, MD, PhD
Children’s Hospital Boston
Harvard Medical School
Howard Hughes Medical Institute
Richard A. Flavell, PhD, FRS
The Anlyan Center for Medical Research
and Education
Yale University School of Medicine
Carol Barnes, PhD
Division of Neural Systems,
Memory, and Aging
University of Arizona
Leroy Hood, MD, PhD
Institute for Systems Biology, Seattle, WA
Andrew R. Marks, MD
Helen Wu Center for Molecular Cardiology
Columbia University College of
Physicians & Surgeons
Deborah Nickerson, PhD
University of Washington
School of Medicine
Eric N. Olson, PhD
Department of Molecular Biology
University of Texas Southwestern
Medical Center at Dallas
Janet Rossant, PhD
Developmental and
Stem Cell Biology Program
The Hospital for Sick Children, Toronto
University of Toronto
Christine Seidman, MD
Department of Genetics and Medicine
Jonathan Seidman, PhD
Department of Genetics
Irving L. Weissman, MD
Institute for Stem Cell Biology
and Regenerative Medicine
Stanford University School of Medicine
Robert C. Gallo, MD
Institute of Human Virology
University of Maryland at Baltimore
Ashley T. Haase, MD
Department of Microbiology
University of Minnesota
Mary E. Klotman, MD
School of Medicine
Department of Medicine
Duke University Medical Center
Richard M. Locksley, MD
Sandler Asthma Basic Research Center
Harmit S. Malik, PhD
Fred Hutchinson Cancer Research Center
Fred H. Gage, PhD
Laboratory of Genetics
Salk Institute for Biological Studies
Richard L. Huganir, PhD
Department of Neuroscience
Brain Science Institute
The Johns Hopkins University
School of Medicine Joseph B. Martin, MD, PhD
Department of Neurobiology
Harry T. Orr, PhD
Institute of Human Genetics
Department of Laboratory
Medicine and Pathology
University of Minnesota
Dennis J. Selkoe, MD
Center for Neurologic Disease
Brigham and Women’s Hospital
Harvard University
GLADSTONE INSTITUTES
FINDINGS 2014
02 President’s Report
Cardiovascular Disease Research
04
06
08
10
12
14
16
18
20
22
24
26
28
Director’s Report
Thomas P. Bersot, MD, PhD
Benoit G. Bruneau, PhD
Bruce R. Conklin, MD
Sheng Ding, PhD
Robert V. Farese, Jr., MD
Kathryn N. Ivey, PhD
Nevan J. Krogan, PhD
Robert W. Mahley, MD, PhD
Katherine S. Pollard, PhD
Deepak Srivastava, MD
Kiichiro Tomoda, PhD
Shinya Yamanaka, MD, PhD
Virology and Immunology Research
30
32
34
36
38
40
42
44
46
48
50
52
Director’s Report
Phillip W. Berman, PhD
Marielle Cavrois, PhD
Gilad Doitsh, PhD
Robert M. Grant, MD, MPH
Warner C. Greene, MD, PhD
JJ Miranda, PhD
Melanie Ott, MD, PhD
Shomyseh Sanjabi, PhD
Eric Verdin, MD
Leor S. Weinberger, PhD
Neurological Disease Research
54
56
58
60
62
64
66
68
70
72
Director’s Report
Katerina Akassoglou, PhD
Steve Finkbeiner, MD, PhD
Li Gan, PhD
Yadong Huang, MD, PhD
Anatol Kreitzer, PhD
Lennart Mucke, MD
Ken Nakamura, MD, PhD
Jorge J. Palop, PhD
Gladstone Trustees
Albert A. Dorman
Andrew S. Garb
Richard D. Jones
Gladstone Scientific Leadership
R. Sanders Williams, MD
President, Gladstone Institutes
Director, Cardiovascular Disease Research
Director, Virology and Immunology Research
Lennart Mucke, MD
Director, Neurological Disease Research
Scientific Report Staff
Michael L. Penn, Jr., MD, PhD Vice President, Strategy
Gary C. Howard, PhD Editor
Celeste R. Brennecka, PhD Assistant Editor
Crystal R. Herron, PhD Assistant Editor
Sylvia Richmond Copy Editor
John C.W. Carroll Art Director
Giovanni Maki Graphic Designer
Teresa R. Roberts Design Assistant
Chris Goodfellow Photographer
Stephen Gonzales Producer for Web
GLADSTONE INSTITUTES FINDINGS 2014
74 The Roddenberry Center for Stem Cell Biology and Medicine
76 Gladstone Center for Translational Research
78 Facilitating Research: Gladstone’s Core Laboratories
79 Gladstone Institutes Publications List
93 The Gladstone Index
The cover image is a detail from
a quantitative genetic interaction
mapping analysis of point mutants of
RNA polymerase II in budding yeast.
For more information, please see Dr.
Nevan Krogan’s report on page 18.
President’s Report
R. Sanders Williams, MD
CELL REPROGRAMMING
BY TRANSIENT EXPOSURE
to extracellular signals and small molecules replaces the use of retroviruses or chromosomal insertion of transgenes
GLADSTONE
FOUNDATION
is formed to support Gladstone’s position as a
world leader in biomedical research
PYROPTOSIS
DRIVES
DEPLETION
FDA APPROVES
a regimen that prevents HIV infection in individuals at risk of contracting virus
AVANT-GARDE,
PIONEER, AND
NEW INNOVATOR of HIV-infected CD4 cells
awards from the NIH
THE
GENETIC
CAUSE
provides bridge between
genome and function
for transcriptional
pausing
SINGLE-STEP
TRANSFORMATION
of human fibroblasts into cardiomyocytes, neural stem
cells, brown fat, Tregs, or pancreatic b cells
of a heritable diarrheal
disorder revealed
HIGH-RES
NETWORK
BIOLOGY
A NEW
SIGNAL
MYOCARDIAL
INFARCTION
REPAIRED
by direct cellular reprogramming in
mice and pigs in vivo
PROTEIN DEACETYLATION
regulates metabolic function in stress response
SCARLESS SCULPTING
of the genome in human iPS cells
METLIFE
FOUNDATION
AWARD
for advancing research in
Alzheimer’s disease
GLOBAL LANDSCAPE
of HIV-human protein complexes described
02 Gladstone Institutes | Findings 2014
LATENT VIRUSES
REACTIVATED
by small molecules
PROTEIN
ACETYLATION
functions in the pathogenesis of
Type I diabetes
DOUBLESTRAND
BREAKS
in DNA are a consequence of normal
neuronal activity
AUTOMATED VIDEO
IMAGING SYSTEM
detects biochemical and morphological signs of neurodegeneration in individual human neurons by the thousands
MONOCLONAL
ANTIBODY
blocks neuroinflammation
ALZHEIMER’S
BIOMARKER
holds potential to empower new therapeutics
MAPPING THE
EPIGENETIC
LANDSCAPE
reveals otherwise unknowable events
that guide cardiac development
LATENT
VIRUSES
REACTIVATED
by small molecules
ANTIVIRAL VIRUSES
target rapidly evolving pathogens via an attenuated virus
derived from interfering particles
THE
NOBEL
PRIZE
for discovery of induced
pluripotent stem cells
HUMAN
MICROBIOME
research reveals surprising diversity of species
EXTRAMURAL
FUNDING DOUBLES
for Gladstone’s promising research
The scientific breakthroughs featured to the left — a nd much
more — have illuminated the past 2 years at Gladstone.
Little wonder why I find my job as Gladstone President to
be so exhilarating. Every day we are fulfilling our mission to
unravel the basics of biology in order to understand, prevent,
treat, and cure human diseases. Our rallying cries resound
through the labs: “Basic Science with a Purpose,” “Science
Overcoming Disease,” and (my favorite) “Science that Rocks.”
Our core values—uncompromising commitment to excellence
in science, deep knowledge of disease, teamwork, entrepreneurial spirit, interdisciplinary thinking, and zeal to provide
our trainees with an unsurpassed learning environment—have
the clear ring of truth. Plus we do this with distinctive flair
that derives from our identity as a small, nimble, spirited, independent, and free-standing research institute. Finally, we have
committed ourselves to a high level of accountability to our
sponsors, and to ourselves, by universally adopting the standard of the US National Academy of Sciences by which each
investigator continuously monitors their five, and only five,
most important and influential papers. You will find these
included on the bottom right-hand margin of each lab report,
below selected publications from the past 2 years. The goal,
of course, is for continuous improvement in that record of
achievement for each individual.
We welcome the distinguished members of our 2014
Scientific Advisory Boards, for whom this summary of
research at Gladstone has been prepared, and we invite all
others with an interest in solving big problems in medicine
to enjoy the rich feast of discovery and technological progress
you will find herein.
12 PARTNERSHIPS
with biotech and pharma companies
BAYBIO AWARD
—R. Sanders Williams
for scientific achievment
Findings 2014 | Gladstone Institutes 03
Director’s Report
“In our laboratories, basic
scientists work side by side with
physician scientists who, like me,
bring the patient perspective to
our work. Together, we spend
our days examining things on a
microscopic level — so that we can
have an impact on a global level.”
Cardiovascular Disease Research
Heart disease remains the leading cause of death in adults
and children. In addition to the severe mortality associated
with cardiovascular disorders, morbidity is significant in the
five million adults with heart failure and over one million survivors of congenital heart malformations in the United States.
Gladstone’s Strategy
In the Gladstone Institute of Cardiovascular Disease (GICD), we
have developed scientific areas of enormous potential, involving
developmental and stem cell biology, adding important chemical, systems, and computational biology approaches to the
study of human disease. Understanding disease processes at
a more refined and mechanistic level, ultimately leading to
intervention, requires interdisciplinary efforts that incorporate
these diverse approaches. We have strategically fostered an environment that intellectually and geographically focuses diverse
“state-of-the-art” talent in a coordinated fashion to tackle problems that lead to human suffering. Our investments in using cellular reprogramming technology, including induced pluripotent
stem (iPS) cells to model human diseases and novel approaches
of direct reprogramming, together with the broader Gladstone
stem cell program, serve as the foundation for the Roddenberry
Center for Stem Cell Biology and Medicine at Gladstone. (See
page 74.) The Stem Cell Center was inaugurated with a successful International Symposium on Cellular Reprogramming
at Gladstone in 2012. Strategies employed in this Center and
in GICD are beginning to reveal the genomic and epigenomic
bases for cardiac disease and are leading to novel approaches
to cardiac regeneration. Additional efforts aimed at metabolic
syndrome and obesity address an enormous risk factor for cardiovascular disease and a looming worldwide health liability.
To achieve our mission, we have effectively recruited and
integrated renowned experts in cardiac, stem cell, chemical,
computational, and systems biology. Convergence of such
diverse approaches and technology on unsolved biomedical
problems has allowed deep interrogation of human cell fates,
disease states, and the mechanisms underlying disease. This collaborative approach has been recognized and facilitated by multiple center grants involving four to five principal investigators,
including three from the National Institutes of Health (NIH)
and two from the California Institute for Regenerative Medicine
(CIRM), and often incorporates expertise from collaborators at
Stanford University, University of California, Berkeley, and the
Institute for Systems Biology in Seattle. Over the last 2 years,
we have developed or incorporated new technology to create
large numbers of cardiomyocytes, endothelial cells, and smooth
muscle cells from pluripotent stem cells to explore the progression of cell-fate decisions and mechanisms underlying abnormal
cell biology associated with genetically defined human disease.
For these purposes, we have rapidly harnessed the power of
genome engineering with TALEN and Cas9-based (CRISPR)
approaches to manipulate the human genome. Our investigators
have made many key observations, highlighted below, and have
been recognized with numerous awards, including the 2012
Nobel Prize in Physiology or Medicine to Shinya Yamanaka,
whose laboratory at Gladstone has made many fundamental
insights into the regulation of pluripotency.
GICD Research Highlights
Thomas Bersot, associate investigator, continued to train
medical professionals in managing lipid disorders. In addition,
he studied the effects of blood cholesterol and lipoprotein composition in people taking an inhibitor of cholesterol absorption.
Benoit Bruneau, associate director and senior investigator,
demonstrated the role of chromatin remodeling in transcriptional pathways governing cardiac differentiation and mapped
the epigenetic changes that drive early cardiac cell fate decisions at the genome-wide level.
Bruce Conklin, senior investigator, established iPS cells
from patients with genetically defined cardiac arrhythmias and
cardiomyopathies and made major investments in emerging
genome engineering technologies to interrogate consequences
of human disease-causing mutations.
Sheng Ding, senior investigator, used chemical biology,
growth factors, and microRNAs (miRNAs) to help guide cell
fate changes in vitro and pioneered the approach of partial
reprogramming toward pluripotency, followed by directed differentiation into specific cell types, including cardiomyocytes,
neurons, hepatocytes, and insulin-producing cells.
Robert Farese, Jr., senior investigator, further characterized
enzymes that regulate fat synthesis and their relationships to
diabetes and obesity. His laboratory has pioneered the deep
study of lipid droplets, a cellular organelle that stores lipids for
energy and membrane synthesis, and is revealing novel targets
for treating obesity and its metabolic consequences.
Kathryn Ivey, staff research investigator, revealed a novel mechanism by which the activity of co-transcribed muscle miRNAs are
differentially regulated through specific miRNA-protein interactions that may be relevant for myopathic conditions.
Nevan Krogan, senior investigator, has taken a systems
approach to dissect interactomes in cells, using analyses of
protein complexes on a large scale. He used this method to
understand the function of the HIV genome and has now
applied a similar approach to determine the protein-protein
interactome of the major transcription factors active in the
cardiovascular system.
Robert Mahley, senior investigator, continued large complex-trait genetic studies to identify loci linked to below-normal
levels of high-density lipoproteins in the Turkish population.
In addition, analysis of Turkish ancestry using whole-genome
sequencing data of multiple unique populations is revealing
novel contributions that may help explain their increased
genetic risk for heart disease.
Katherine Pollard, senior investigator, has continued to
investigate the most rapidly evolving areas of the genome,
largely representing enhancer elements, as they may be involved
in adaptive responses to many disease states. She has developed many new algorithms to analyze genome-wide epigenetic
and transcriptome data of normal and abnormal human cells,
as well as metagenomics data from the Human Microbiome
Project that enabled discovery of hundreds of new microbes
that may contribute to human disease.
Deepak Srivastava, senior investigator, investigated the
transcriptional and translational pathways controlling early
cardiac cell differentiation and utilized cellular reprogramming technologies to model genetically defined cardiovascular
disease. He also leveraged knowledge of cardiac developmental
biology to directly reprogram fibroblasts to cardiomyocyte-like
cells in vitro and in vivo and has translated this work to human
cells in vitro and to pig studies in vivo.
Kiichiro Tomoda, staff research investigator, showed that
by manipulating culture conditions, female fibroblasts could be
reprogrammed to pluripotent stem cells that reactivated both
X chromosomes, more similar to the true pluripotent state, and
that upon differentiation one X chromosome from these cells
became inactive as expected.
Shinya Yamanaka, senior investigator, continued to innovate and refine the iPS cell technology that his laboratory
discovered, which involves reprogramming adult cells into pluripotent cells similar to embryonic stem cells. Dr. Yamanaka’s
team at Gladstone described methods to make more fully
reprogrammed cells and revealed novel pathways regulating
the differentiated versus pluripotent state of cells involving networks of miRNAs and transcription factors.
The urgent need to address the etiology and risk factors of
cardiovascular disease, while building the scientific basis for
novel therapeutic development, is being aggressively pursued.
The promise of stem cell therapy and cellular reprogramming
for heart disease is tangible, but will require a focused and multi
disciplinary effort. The intersection of human genetic variation
with iPS cell technology and new genome engineering technology affords an unprecedented opportunity to characterize
disease in human cellular models. The ability to deeply investigate such human cells through emerging technologies, combined with the use of in vivo mouse models to study complex
cell-cell interactions and organ physiology, represents a powerful
approach for discovery. The outstanding trainees and scientists
that populate GICD are the drivers of innovation and creativity
and represent the future legacy of Gladstone. I look forward to
their many successes and breakthroughs in the coming years.
Back row from left to right:
Kiichiro Tomoda, Robert
Mahley, Bruce Conklin;
middle row: Robert Farese, Jr.,
Benoit Bruneau, Kathryn Ivey;
front row: Sheng Ding,
Deepak Srivastava, Shinya
Yamanaka. Not pictured:
Thomas Bersot, Nevan Krogan,
Katherine Pollard.
“The decline in CVD
mortality that began
in 1968 is expected
to reverse in the next
few years if young
children continue to
become overweight
and obese.”
Thomas P. Bersot, MD, PhD
Associate Investigator
labs.gladstone.ucsf.edu/bersot
HIGHLIGHTS
• Heart disease is the number-one
killer in America and the developed
world
• The Gladstone Lipid Disorders
Training Center educates healthcare
providers to better manage CVD
risk factors
• The Gladstone Lipid Clinic encourages healthy lifestyles, which reduce
the risk of CVD
LAB MEMBER
Gayatri Saldivar
The increasing epidemic of obesity and diabetes emphasizes the necessity to provide the best possible training
for managing the risk factors for heart disease.
Heart disease continues to be the number-one killer in America and the developed world. The good news is that annual
deaths caused by coronary heart disease
have dropped 60% since 1968. The notso-good news is that in 2010 cardiovascular disease (CVD) still caused one
in four deaths in the United States. If
stroke and heart failure are included,
the toll was about one in three deaths
(~725,000 deaths). While we now have
better treatments for CVD and have
improved how we manage CVD risk
factors, these healthy trends are being
offset by a population that is steadily
becoming more overweight and obese,
which is also increasing the prevalence
of diabetes. Consequently, obesity and
overweight have increased CVD deaths
in adults by nearly 20% since 1968. As of
2010, 69% of adults in the United States
were overweight or obese, as were 18%
of children 6–19 years old and 12% of
children 2–5 years old. Even worse, the
decline in CVD mortality that began in
1968 is expected to reverse in the next
few years if young children continue to
become overweight and obese.
Gladstone Lipid Disorders
Training Center
Cognizant of the threat posed by overweight and obesity, we established the
Gladstone Lipid Disorders Training
Center in 1990. Since then, we have
educated over 4200 healthcare providers
to help them better manage CVD risk
factors in their patients, including overweight and obesity. The training program offers two types of courses that
are offered several times per year. In
addition, we train healthcare providers
in the Community Health Network of
the San Francisco Department of Public
Health and at the San Francisco General
Hospital. Although most attendees are
physicians, more nurse practitioners,
clinical pharmacists, dietitians, and
exercise physiologists are taking the
courses. These non-physician providers
are assuming substantial responsibility
for managing the CVD risk factors of
primary care patients because there are
not enough physicians to deal with the
overwhelming number of patients with
weight issues and heightened CVD risk.
Training Courses
The basic 2½-day course covers the physiology and pathophysiology of plasma
lipid metabolism, hypertension, and
diabetes mellitus. We review evidence
supporting the use of risk assessment
tools, diagnostic procedures, and therapies. Extensive time is devoted to diet,
exercise, and weight management, which
are the cornerstones of CVD prevention.
We also review safe and appropriate use
of medications. In addition, attendees
take part in a 1-day demonstration clinic
where they see actual Gladstone Lipid
Clinic patients. This demonstration provides attendees with practical experience
in patient management.
The other course we offer is a 1-day
update course for previous students that
covers new diagnostic methods used to
assess the risk of sustaining a clinical vascular disease event, as well as the treatment implications of recently completed
clinical studies. In addition, we discuss
new drug therapies and significant new
developments in lifestyle management.
A Healthy Lifestyle
At the Gladstone Lipid Clinic, we
stress the value of a healthy lifestyle,
which can reduce the risk of vascular
disease by 50% or more and adds to
the benefits of drug therapy and invasive treatments, such as angioplasty,
stenting, and bypass surgery. Patient
compliance with lifestyle change recommendations is dif f icult, so our
teaching efforts are coordinated to
focus on this important issue.
Publications
SELECTED RECENT
1. Can AS et al. (2010) Optimal
waist:height ratio cut-off point
for cardiometabolic risk factors in
Turkish adults. Public Health Nutr.
13:488.
TOP FIVE OVERALL
1. Bersot TP et al. (1976) Interaction
of swine lipoproteins with the low
density lipoprotein receptor in
human fibroblasts. J. Biol. Chem.
251:2395.
2. Ling H et al. (2009) Genome-wide
linkage and association analyses
to identify genes influencing adiponectin levels: the GEMS Study.
Obesity (Silver Spring) 17:737.
3. Mahley RW et al. (1970) Identity of
very low density lipoprotein apoproteins of plasma and liver Golgi
apparatus. Science 168:380.
4.Mahley RW et al. (2000) Low
levels of high density lipoproteins in Turks, a population with
elevated hepatic lipase. High
density lipoprotein characterization and gender-specific effects
of apolipoprotein e genotype.
J. Lipid Res. 41:1290.
The training courses offered by the Gladstone Lipid Disorders Training Center are
endorsed by the American Heart Association. Since the founding of the Center in
1990, these courses have educated over 4200 healthcare providers on ways to help
patients reduce their risk of cardiovascular disease.
5. Rall SC Jr et al. (1989) Type III
hyperlipoproteinemia associated
with apolipoprotein E phenotype
E3/3. Structure and genetics of an
apolipoprotein E3 variant. J. Clin.
Invest. 83:1095.
Benoit G. Bruneau, PhD
Associate Director and Senior Investigator
labs.gladstone.ucsf.edu/bruneau
HIGHLIGHTS
• Mapped the epigenomic landscape
of cardiac differentiation
• Identified the first specified cardiac
precursor in the early mouse embryo
• Defined chromatin remodeling as an
important tissue-specific mode of
gene regulation
LAB MEMBERS
Jeffrey Alexander
SiangYun Ang
Claire Cutting
Paul Delgado
W. Patrick Devine
Matthew George
Daniel He
Swetansu Hota
Irfan Kathiriya
Luis Luna-Zurita
Dario Miguel-Perez
Elphège-Pierre Nora
Tatyana Sukonnik
John Wylie
Joshua Wythe
“We believe that
primary defects in
patterning in early
heart development
are at the root of
congenital heart
disease.”
Understanding how genes are regulated on a broad
scale is helping us discover the hidden mysteries surrounding the fate of cardiac cells.
The main focus of our laboratory is to
understand how a heart becomes a heart:
what cell lineage decisions take place to
direct cardiac differentiation and what
morphogenetic and patterning processes
occur to assemble all of the components
of the heart into a functional organ? We
are primarily interested in the regulation
of these processes by transcriptional regulatory mechanisms that include DNAbinding transcription factors, chromatin
remodeling complexes, and histone modifications. We have used this knowledge to
understand disease mechanisms, but also
to devise strategies for cardiac regeneration.
Why study heart development? We
believe that primary defects in patterning
in early heart development are at the root
of congenital heart disease, which affect
approximately 1% of live-born children.
We want to understand how these defects
occur, to perhaps be able to uncover new
and improved diagnostic or even therapeutic options. Also, by understanding
how cardiac lineage specification occurs,
we can better design stem cell-based
interventions of cardiac repair, based on
the knowledge of what drives an uncommitted cell toward a specific cardiac fate.
We have recently focused our efforts on
cardiac chromatin remodeling and modification factors, enzymes that unwind
DNA or modify histones to turn genes
on or off. We are particularly interested in
how these factors control cardiac cell lineage decisions. These chromatin remodeling factors may also be key to pushing a
stem cell into becoming a heart cell, perhaps opening up new avenues for cardiac
regenerative medicine.
Chromatin States
Over the last 2 years, we have taken
advantage of the ability to efficiently differentiate embryonic stem cells into cardiomyocytes on a large scale in order to
profile chromatin states during the progression of cardiac differentiation. This
has led to exciting new insights into how
epigenetic states change during differentiation. For example, we have discovered
that contractile protein genes are coregulated at the chromatin level, in a time
course that is unlike any other group of
genes. We are now disrupting key regulatory molecules to understand the
mechanism underlying these epigenomic
transitions. This approach is bearing fruit,
as we have discovered that a chromatin
remodeling factor, Brg1, is essential for the
epigenetic activation of enhancer elements
during early mesodermal fate specification.
Three-Dimensional Chromatin
We are now exploring the three-dimen
sional conformation of the genome
during differentiation and deploying
emerging genome engineering tools to
disrupt genomic elements rapidly and
broadly. These approaches are widely
applicable, and we are expanding our
studies to include human induced pluripotent stem cell models of disease, as
well as in vivo mouse models. With this
integrated approach we hope to be able
to decipher the functional nodes that
control the differentiation of cardiomyocytes. This will shed light on fundamental
questions in transcriptional regulation
and developmental biology, and also will
be directly relevant to human congenital
heart defects.
Publications
SELECTED RECENT
1. Delgado-Olguín P et al. (2012)
Epigenetic repression of cardiac
progenitor gene expression by Ezh2
is required for postnatal cardiac
homeostasis. Nat. Genet. 44:343.
2. Gaborit N et al. (2012) Cooperative
and antagonistic roles for Irx3 and
Irx5 in cardiac morphogenesis and
postnatal physiology. Development
139:4007.
3. Wamstad JA et al. (2012) Dynamic
and coordinated epigenetic regulation of developmental transitions in
the cardiac lineage. Cell 151:206.
4.Wythe JD et al. (2013) ETS factors
regulate Vegf-dependent arterial
specification. Dev. Cell 26:45.
TOP FIVE OVERALL
1. Delgado-Olguín P et al. (2012)
Epigenetic repression of cardiac
progenitor gene expression by Ezh2
is required for postnatal cardiac
homeostasis. Nat. Genet. 44:343.
2. Koshiba-Takeuchi KT et al. (2009)
Reptilian heart development and
the molecular basis of cardiac
chamber evolution. Nature 461:95.
3. Lickert H et al. (2004) Baf60c is
essential for function of BAF chromatin remodelling complexes in
heart development. Nature 432:107.
Lineage tracing of early cardiac progenitors in a fetal mouse heart. Twin clones,
labeled green and red, originated in early mesoderm. The green clone is in the right
atrium, while the red clone is in the left ventricle. This illustrates the early patterning of mesoderm into the different regions of the heart.
4.Takeuchi JK et al. (2009) Directed
transdifferentiation of mouse
mesoderm to heart tissue by
defined factors. Nature 459:708.
and coordinated epigenetic regulation of developmental transitions in
the cardiac lineage. Cell 151:206.
Bruce R. Conklin, MD
“Genome engineering
in human iPS cells
will allow us to
pinpoint many causes
of cardiac disease.”
Senior Investigator
labs.gladstone.ucsf.edu/conklin
HIGHLIGHTS
• Established an efficient method to
produce “isogenic” iPS cell lines
• Developed a series of assay tools
involving GPCRs
• Created models of sudden death
syndrome
LAB MEMBERS
Tilde Eskildsen
Ethan Hua
Miller Huang
Nathaniel Huebsch
Luke Judge
Allan Kuchinsky
Paweena Lizarraga
Mohammadali Mandegar
Yuichiro Miyaoka
Trieu Nguyen
Jason Park
Juan Perez-Bermejo
Caitlin Russell
Nathan Salomonis
Mark Scott
Marie Sears
Alice Sheehan
Po-Lin So
Matthew Spindler
An Truong
Jennie Yoo
Fumiaki Yumoto
We are focused on constructing isogenic iPS cell
disease models to study the molecular mechanisms
associated with cardiomyopathies.
Human Cardiac Disease Models
We use induced pluripotent stem (iPS)
cells to model human cardiac genetic
disease. We focus on genes associated
with heart failure from cardiomyopathy
or abnormal heart rhythm resulting in
“sudden death.” The heart provides an
ideal system to determine the molecular basis of human genetic findings.
Hundreds of gene loci have already been
associated with heart disease; yet, until
recently, modeling these gene variants
in human cardiac tissue has been difficult. Human iPS cells now allow us to
produce many cardiovascular tissues,
which have already been used to successfully uncover disease phenotypes.
Our first studies focused on iPS cells
from patients who have genetic diseases,
and more recently, my lab has focused
on engineering iPS cells to have specific mutations since they allow more
in-depth studies of disease mechanism
in a controlled background.
Genome and Tissue Engineering
The late Richard Feynman once said,
“What I cannot create, I do not understand.” Although this is well known
in the field of engineering, we are just
beginning to apply the principle to
human biolog y. Up until recently,
human genetics was primarily observational, but newly developed genome
engineering tools now allow us to
directly test the cellular consequences of
discrete genetic changes. We have developed efficient methods to edit one residue at a time in living human iPS cells,
resulting in “isogenic” iPS cell lines that
are identical except for a single alteration.
These isogenic iPS cell disease models are
now yielding phenotypes that are helping
to explain the molecular basis of several
human diseases. In addition, we are constructing collections of isogenic disease
cell lines that carry a range of disease
mutations, from the most severe (rare)
to the moderate (common) forms of cardiomyopathy. We are currently focused
on the most severe cardiomyopathies to
develop cell-based assays. We are hopeful
that these severe cardiomyopathies will
allow us to understand the molecular
basis of the more common cardiomyopathies as well.
The heart is a complex tissue that is
tightly integrated via chemical and electrical coupling. We are working with
tissue engineers to recapitulate these
complex tissues so that we can better
model cardiac disease. The disease cell
lines we are making help to provide a
“yard stick” to measure robustness of
engineered tissues. The cardiac tissues
that best reflect human cardiac contraction and electrical coupling are most
likely to provide insights into specific
human disease genes.
Targeting Better Therapies
We are hopeful that human iPS cellbased disease models will provide a path
to develop safer, more effective drug therapies. Personalized medicine can benefit
from experimental testing of gene variants to prove (or disprove) hypothetical
associations with drug responses. The
gene variants that we are testing could
help avoid unwanted cardiac toxicity,
while also pointing to new therapeutic
opportunities. Human iPS cell disease
models could provide the drug development platform of the future. Our prior
work with G protein-coupled receptors
gives us a strong background in drug discovery. We previously developed a novel
series of assay tools (G protein chimeras)
that are used by 80% of major pharmaceutical companies and has contributed
to the development of several approved
drugs. In the future, we imagine that
safer and more effective drugs will be
developed using genetically defined iPS
cell-derived disease models.
Publications
SELECTED RECENT
1. Conklin BR (2013) Sculpting
genomes with a hammer and
chisel. Nat. Methods 10:839.
2. Kreitzer FR et al. (2013) A robust
method to derive functional neural
crest cells from human pluripotent
stem cells. Am. J. Stem Cells 2:119.
3. Ma Z et al. (2014) Three dimensional filamentous human diseased
cardiac tissue model. Biomaterials
35:1367.
4.Miyaoka Y et al. Isolation of single-base genome-edited human iPS
cells without antibiotic selection.
Nat. Methods (in press).
5. Spindler MJ et al. (2013) AKAP13
Rho-GEF and PKD-binding domain
deficient mice develop normally
but have an abnormal response
to β-adrenergic-induced cardiac
hypertrophy. PLoS One 8:e62705.
TOP FIVE OVERALL
1. Conklin BR et al. (2008) Engineering GPCR signaling pathways
with RASSLs. Nat. Methods 5:673.
2. Conklin BR et al. (1993) Substitution of three amino acids switches
receptor specificity of Gqα to that
of Giα. Nature 363:274.
3. Dahlquist KD et al. (2002) GenMAPP, a new tool for viewing and
analyzing microarray data on biological pathways. Nat. Genet. 31:19.
4.Redfern CH et al. (1999) Conditional expression and signaling of
a specifically designed Gi-coupled
receptor in transgenic mice. Nat.
Biotechnol. 17:165.
5. Tingley WG et al. (2007) Genetrapped mouse embryonic stem
cell-derived cardiac myocytes and
human genetics implicate AKAP10
in heart rhythm regulation. Proc.
Natl. Acad. Sci. USA 104:8461.
Three iPS cell-derived cardiomyocytes stained for proteins involved in cardiomyopathy
(red = troponin T, green = BCL2-associated athanogene 3, yellow = overlap).
Sheng Ding, PhD
Senior Investigator
labs.gladstone.ucsf.edu/ding
HIGHLIGHTS
• Developed conditions to reprogram
fibroblasts into definitive endoderm
lineages
• Discovered a small molecule and its
mechanism of reprogramming white
fat cells to brown fat cells
• Identified the mechanism of a new
small molecule to reprogram Th17
cells to iTreg cells
LAB MEMBERS
Nan Cao
Ke Li
Changsheng Lin
Kai Liu
Peng Liu
Tianhua Ma
Itedale Namro Redwan
Baoming Nie
Shibing Tang
Haixia Wang
Xiaojing Wang
Min Xie
Shaohua Xu
Tao Xu
Chen Yu
Mingliang Zhang
Yu Zhang
Saiyong Zhu
“We hope our studies
will ultimately
facilitate therapeutic
applications that
stimulate the body’s
own regenerative
capabilities.”
Our current work focuses on screening chemical
libraries to identify and further characterize small
molecules that control cell fate and/or function.
We have been interested in developing
new approaches to study stem cell
biology and regeneration. Our current
work focuses on screening chemical
libraries to identify and further characterize small molecules that control cell
fate and/or function in various systems.
These functions include maintenance of
tissue-specific stem cells, directed differentiation of pluripotent stem cells
toward new cell lineages, reprogramming of lineage-restricted somatic cells
to alternative cell fates (e.g., toward
induced pluripotent stem (iPS) cells
or transdifferentiation), programming
naive T cells, and regulation of cancer
stem cells.
Characterizing Small Molecules
for Reprogramming
In our laboratory, we identified small
molecules and generated cells, which we
further characterized in vitro and in vivo.
For example, our recent efforts identified
novel small molecules that functionally
replace reprogramming transcription
factors as well as significantly improve
efficiency and speed of iPSC reprogramming. In addition, mechanistic studies
of these small molecules have revealed
new insights underlying fundamental
processes in reprogramming.
A New Paradigm
Our efforts led to a new paradigm in
transdifferentiation. We used the CellActivation and Signaling-Directed
(CASD) lineage-conversion strategy,
which employs transient overexpression
of iPSC transcription factors (TFs) (cell
activation) in conjunction with lineage
specific soluble signals (signal-directed) to
reprogram somatic cells into diverse lineage-specific cell types without entering
the pluripotent state. Using this novel
concept and strategy, we directly converted
fibroblasts to cardiac, neural, endothelial,
or definitive endoderm precursor cells.
Advantages of CASD-Based
Transdifferentiation
In conventional transdifferentiation,
different cell specifications are determined by ectopic expression of different
sets of lineage-specific TFs. However,
such CASD-based transdifferentiation
might be advantageous in that a single
set of TFs is used for all cell types, and
such transient gene expression might
be more easily replaced with safer and
more convenient methods without the
risk of genetic modifications. In addition, generating lineage-specific precursor cells using such approaches would
allow for the isolation, expansion, and
characterization of reprogrammed cells
for various applications. Furthermore,
it is conceivable that existing and new
small molecules identified for controlling iPS cell reprogramming, as well
as stem cell self-renewal and differentiation, may be valuable in this transdifferentiation paradigm. For example,
combinations of small molecules that
induce or enhance iPSC reprogramming
were also found to enable CASD-based
transdifferentiation with a reduced
number of genetic factors.
We hope our continued studies will
ultimately facilitate therapeutic applications of stem cells and the development
of small-molecule drugs to stimulate the
body’s own regenerative capabilities by
promoting survival, migration/homing,
proliferation, differentiation, and reprogramming of endogenous stem/progenitor cells or more differentiated cells.
Transient
Treatment with
Molecules
Fibroblast
Publications
SELECTED RECENT
1. Li H et al. (2012) Versatile pathway-centric approach based on
high-throughput sequencing to
anticancer drug discovery. Proc.
2. Li K et al. Small molecules facilitate
the reprogramming of mouse fibroblasts into pancreatic lineages. Cell
Stem Cell (in press).
3. Li W et al. (2013) Chemical
approaches to stem cell biology
and therapeutics. Cell Stem Cell
13:270.
4.Wang H et al. Small molecules
enable cardiac reprogramming of
mouse fibroblasts with a single
factor, Oct4. Cell Report (in press).
5. Zhu S et al. Mouse liver repopulation with hepatocytes generated
from human fibroblasts. Nature
(in press).
Epigenetically
“Activated”
Cell Population
Neuron
and
Glial Cell
Small Molecule
Inhibitor of
Pluripotency
Neural
Induction
Medium
Neural
Precursor
Cell
Hepatocyte
Definitive
Endoderm
Induction
Medium
Cardiac
Induction
Medium
iPS Cell
Medium and
Extended
Expression
of iPS Cell
TFs
Pancreatic
Cell
TOP FIVE OVERALL
1. Chen S et al. (2004) Dedifferentiation of lineage-committed cells by
a small molecule. J. Am. Chem. Soc.
126:410.
2. Efe JA et al. (2011) Conversion of
mouse fibroblasts into cardiomyocytes using a direct reprogramming
strategy. Nat. Cell Biol. 13:215.
Definitive
Endoderm
Precursor
Cell
3. Li H et al. (2012) Versatile pathway-centric approach based on
high-throughput sequencing to
anticancer drug discovery. Proc.
Cardiomyocyte
4.Li W et al. (2011) Rapid induction
and long-term self-renewal of primitive neural precursors from human
embryonic stem cells by small molecule inhibitors. Proc. Natl. Acad.
Sci. USA 108:8299.
Cardiac
Precursor
Cell
iPS Cell
5. Shi Y et al. (2008) A combined
chemical and genetic approach for
the generation of induced pluripotent stem cells. Cell Stem Cell 2:525.
A novel path to transdifferentiation. Temporally restricted ectopic overexpression
of reprogramming factors in fibroblasts leads to the rapid generation of epigenetically “activated” cells, which can then be coaxed to “relax” back into various
differentiated states, ultimately giving rise to somatic cells entirely distinct from the
starting population. TF, transcription factor.
“Diseases of lipid
excess, such as
obesity, diabetes, and
atherosclerosis, are
major global health
problems.”
Robert V. Farese, Jr., MD
Senior Investigator
labs.gladstone.ucsf.edu/farese
HIGHLIGHTS
• Lipid droplets exist as two populations: small and expanding
• Lipid droplets expand by synthesizing lipids through key enzymes to
the lipid droplet surface
• Loss of DGAT1 results in congenital
diarrhea syndrome in humans
Although diseases of lipid excess (e.g., obesity and diabetes) are epidemic, we actually know very little about
how fats are made and stored at the molecular level.
Lipids are central to all aspects of life,
most prominently as constituents of biological membranes and as major energy
reservoirs. Diseases of lipid excess, such
as obesity, diabetes, and atherosclerosis,
are major global health problems.
Lipid Homeostasis
LAB MEMBERS
Mohan Chitraju
Erin Currie
Grisell Diaz-Ramirez
Delphine Eberle
Joel Haas
Laura Mitic
Andrew Nguyen
Thi Nguyen
Manuele Piccolis
Ivana Semova
Marina Vayner
We study cellular lipid homeostasis.
Our recent focus is on the mechanisms
of lipid synthesis and storage in lipid
droplets. When lipids are in excess in
cells, they are esterified to neutral lipid
forms for storage. For example, excess
levels of sterols or fatty acids are stored
as sterol esters or triglycerides (TGs),
respectively. The storage forms of lipids
are the products of enzymes in the
endoplasmic reticulum (ER), which
are then stored in cytosolic organelles
called lipid droplets. Our work focuses
on the biochemistry and regulation of
lipid synthesis enzymes, the formation
and growth of lipid droplets, and the
utilization of lipids from lipid droplets
for energy and cell membranes. Our
studies range from biophysical and biochemical approaches to cell biology to
physiology in whole organisms. Our
work is performed in close partnership with the laboratory of Dr. Tobias
Walther (Yale University).
Mechanisms of Lipid Expansion
In recent studies, we identified key mechanisms involved in lipid droplet expansion. Nearly all cells have two populations
of lipid droplets: small and expanding.
To expand, smaller lipid droplets require
increases in both volume (neutral lipids,
such as TG) and surface (phospholipids).
We identified that some lipid droplets
acquire enzymes of TG synthesis, which
can locally synthesize the droplet core
lipids. These expanding lipid droplets also
require local phospholipid synthesis to
maintain surfactants at the lipid droplet
surface. We identified a mechanism for
upregulating phosphatidylcholine synthesis at lipid droplet surfaces through
the activation of phosphocholine cytidylyltransferase 1 (CCT1), the rate-limiting
enzyme. Most recently, we have begun to
investigate the mechanism by which key
enzymes of TG synthesis and hydrolysis
(lipases) gain access to the lipid droplet
surface. We have identified that lipid
droplets connect to the ER via membrane
bridges that allow enzymes to migrate to
the monolayer surfaces of lipid droplets.
DGAT1
We previously identified one of the
enzymes of TG synthesis, diacylglycerol
O-acyltransferase 1 (DGAT1), as a drug
target for metabolic diseases, such as obesity, hypertriglyceridemia, and hepatic
steatosis, and several DGAT1 inhibitors
are being tested in clinical trials. Most of
the information on DGAT1 inhibition
comes from studies in mice. Last year,
together with collaborators at Harvard,
we identified null mutations of DGAT1
in humans. Homozygous DGAT1 deficiency leads to a syndrome of congenital
diarrhea. Our work showed that humans
lack DGAT2 in the intestine, likely
making them more susceptible to the
effects of DGAT1 deficiency. We continue to explore the mechanisms underlying intestinal dysfunction associated
with human DGAT1 deficiency.
Frontotemporal Dementia
Publications
Our laboratory also researches the basic
SELECTED RECENT
mechanisms of frontotemporal dementia
(FTD), the most common cause of
dementia in people under age 65. As part
of the Consortium for FTD Research, we
use a variety of model systems to study
the pathogenesis of FTD and search for
cures. Our work is focused on understanding the biochemistry and functions
of progranulin, a secreted protein that plays
key roles in neuronal health and in modulating inflammation. We study progranulin function in the nervous system and
2. Haas JT et al. (2012) DGAT1 mutation in a family with a congenital
diarrheal disorder. J. Clin. Invest.
122:4680.
3. Martens LH et al. (2012) Progranulin deficiency promotes neuroinflammation and neuronal loss
following toxin-induced injury.
J. Clin. Invest. 122:3955.
4.Nguyen AD et al. (2013) Secreted
progranulin is a homodimer and is
not a component of high density
lipoproteins (HDL). J. Biol. Chem.
288:8627.
metabolic diseases.
Enzyme That
Catalyzes PC
Synthesis
(Surface)
Small
LDs
1. Armakola M et al. (2012) Inhibition
of RNA lariat debranching enzyme
suppresses TDP-43 toxicity in
ALS disease models. Nat. Genet.
44:1302.
5. Wilfling F et al. (2013) Triacylglycerol synthesis enzymes mediate
lipid droplet growth by relocalizing
from the ER to lipid droplets. Dev.
Cell 24:384.
Enzyme That
Catalyzes TG
Synthesis
(Core)
TOP FIVE OVERALL
Expanding
LDs
Cytosol
ER Lumen
Fat droplets form at the ER. Our studies suggest that enzymes in the ER synthesize
fats, such as TGs, and give rise to small lipid droplets (LDs) in the cytoplasm. Some
of these droplets subsequently reconnect with the ER, which allows fat synthesis
enzymes to migrate to their surfaces. Here these TG-synthesis enzymes make fat and
expand the LD core. Other enzymes bind to the surface of expanding LDs and catalyze
phospholipid synthesis to coat the surfaces. Most cells appear to have the capacity to
make both small and expanding LDs. Understanding how these processes are regulated
should provide molecular targets for treating diseases of lipid excess.
1. Cases S et al. (1998) Identification
of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key
enzyme in triacylglycerol synthesis.
Proc. Natl. Acad. Sci. USA 95:13018.
2. Guo Y et al. (2008) Functional
genomic screen reveals genes
involved in lipid droplet formation
and utilization. Nature 453:657.
3. Krahmer N et al. (2011) Phosphatidylcholine synthesis for lipid
droplet expansion is mediated by
localized activation of CTP:phosphocholine cytidylyltransferase.
Cell Metab. 14:504.
4.Smith SJ et al. (2000) Obesity
resistance and multiple mechanisms of triglyceride synthesis in
mice lacking DGAT. Nat. Genet.
25:87.
5. Wilfling F et al. (2013) Triacylglycerol synthesis enzymes mediate
lipid droplet growth by relocalizing
from the ER to lipid droplets. Dev.
Cell 24:384.
“We are just
beginning to learn
how the biogenesis,
stability, and even
activity of miRNAs
are controlled.”
Kathryn N. Ivey, PhD
Staff Research Investigator
labs.gladstone.ucsf.edu/ivey
HIGHLIGHTS
• Muscle-enriched miRNAs, miR-1
and miR-133, direct cardiac differentiation from pluripotent cells
• The activities of miR-1 and miR-133
are regulated by differential protein
interactions
• In mice, miR-1 loss causes postnatal
heart failure and gene dysregulation
LAB MEMBERS
Abhishek Kumar
Donaldo Salas
The heart develops and functions through the precise
actions of myriad factors at specific moments and in
response to signaling and transcriptional networks.
MicroRNAs (miRNAs), a type of small,
non-coding RNA, regulate the dosage
of myriad factors and are thus critical
contributors to cardiac development and
function. The expression of miRNAs,
like that of protein-coding genes, is
spatially and temporally regulated, and
some miRNAs are transcribed only
in the heart or vasculature. However,
post-tra nscriptiona l processing is
required to generate active, mature, cytoplasmic miRNAs from primary miRNA-encoding transcripts.
We understand the basic molecular
events mediating these processing steps.
However, we are just beginning to learn
how the biogenesis, stability, and even
activity of miRNAs are controlled in
a context-dependent manner through
interactions with specific factors. This
level of gene regulation is a novel area for
investigation that may be manipulated to
control the cellular activity of miRNAs.
miR-1 Expressed in Muscle
miR-1 is enriched in cardiac and skeletal muscle. The targeted deletion of
miR-1 in mice perturbs cardiac development and alters adult heart function.
In contrast, deletion of other individual
miRNAs often results in no overt phenotype. miR-1 is produced from bicistronic transcripts that also generate
miR-133, such that the two miRNAs
are initially transcribed in a 1:1 ratio.
Our previous work showed that, despite
their co-expression, miR-1 and miR-133
have both cooperative and opposing
functions, with miR-1 promoting differentiation, while miR-133 maintains
cardiac and skeletal muscle in the progenitor state. Interestingly, sequencing
has revealed that miR-1 makes up ~40%
of mature miRNA in cardiac cells, even
though hundreds of unique miRNAs are
expressed in the heart. This finding suggests that miR-1 is stabilized post-transcriptionally, while miR-133 is more
rapidly turned over. Finally, unlike
most miRNAs, mature miR-1 accumulates in the nucleus, where its function
is entirely unknown.
Post-Transcriptional miRNA
Regulation
My laboratory is investigating the unique
characteristics of miR-1 with a goal of
understanding the unexplored potential
of post-transcriptional miRNA regulation, specifically during development
and in diseases of cardiac and skeletal
muscle. As part of that effort, we identified a unique interaction between miR-1
and an RNA-binding protein, mutations
of which are associated with a musclewasting disease. We found that this
300
200
miR-1
C
Adult
miR-133
AAAA
miR-133a
Muscle
Progenitor
SELECTED RECENT
1. Hassel D et al. (2012)
MicroRNA-10 regulates the angiogenic behavior of zebrafish and
human endothelial cells by promoting vascular endothelial growth
factor signaling. Circ. Res. 111:1421.
2. Heidersbach A et al. (2013)
microRNA-1 regulates sarcomere
formation and suppresses smooth
muscle gene expression in the
mammalian heart. eLife 2:e01323.
3. Ivey KN et al. (2013) MicroRNAs
as developmental regulators. In
Mammalian Development: Networks,
Switches, and Morphogenetic Processes (Tam PPL, et al. eds.) pp 33.
4.Limphong P et al. (2013) Modeling
human protein aggregation cardiomyopathy using murine induced
pluripotent stem cells. Stem Cells
Transl. Med. 2:161.
TOP FIVE OVERALL
E14.5
12
10
8
6
4
2
0
Publications
5. White MP et al. (2013) Limited
gene expression variation in human
embryonic stem cell and induced
pluripotent stem cell-derived endothelial cells. Stem Cells 31:92.
E9.5
B
Molecules per 10ng
Total RNA (x 10 6)
A
Adult
We are also investigating the role of the
aforementioned protein interaction in
the unique nuclear localization of miR-1
and other miRNAs. In addition to their
well-defined role in post-transcriptional
regulation of gene expression, miRNAs
may also act in the nucleus to direct or
E14.5
Protein Interactions
suppress transcription of specific genes.
Our current studies seek to fully describe
the temporal dynamics of nuclear
miRNA localization in cardiac and skeletal muscle, identify the chromosomal
locations with which miR-1 interacts, and
determine the effects of that interaction
on gene expression in the heart. We anticipate that nuclear muscle miRNAs may
regulate gene expression directly and that
disruption of this process contributes to
diseases of the heart and skeletal muscle.
In combination, increased understanding
of miRNA regulation and elucidation of
unique miRNA functions could provide
the framework for the development of
new therapies to promote normal heart
development and maintain healthy cardiac and skeletal muscle in adults.
E9.5
interaction limits the normal activity of
miR-1, and, we have elucidated the basic
mechanism underlying this functional
blockade. Our future studies will investigate the effects of this protein interaction on miR-1 activity in normal and
diseased heart and skeletal muscle using
induced pluripotent stem cell technology
to create these cell types from healthy
and diseased individuals.
miR-1
Mature
Muscle
The non-coding RNA miR-1 is critical for heart development and homeostasis. A) Unlike
other miRNAs, miR-1 accumulates in the nucleus of mature cardiac and skeletal muscle.
B) miR-1 is also present at exceedingly high levels, far in excess of the co-transcribed
miRNA, miR-133. C) miR-1 and miR-133 have partially opposing functions.
1. Cordes KR et al. (2009) miR-145
and miR-143 regulate smooth
muscle cell fate and plasticity.
Nature 460:705.
3. Ivey KN et al. (2008) MicroRNA
regulation of cell lineages in mouse
and human embryonic stem cells.
Cell Stem Cell 2:229.
4.Ivey KN et al. (2008) Transcriptional regulation during development of the ductus arteriosus.
Circ. Res. 103:388.
5. Sheehy N et al. (2010) The neural
crest-enriched microRNA miR-452
regulates epithelial-mesenchymal
signaling in the first pharyngeal
arch. Development 137:4307.
“In our systems-tomechanism approach,
we are quantitatively
characterizing
biological processes.”
Senior Investigator
labs.gladstone.ucsf.edu/krogan
HIGHLIGHTS
• Evolutionary comparison of genetic
interactomes reveals modular
conservation
• Functional prioritization of
post-translational modifications
using protein structural and conservation analysis
• Point mutant genetic analysis
reveals new insights into structure-function relationships
LAB MEMBERS
Stefan Bohn
Hannes Braberg
Si-Han Chen
Manon Eckhardt
Kathy Franks-Skiba
David Gordon
Ruth Huettenhain
Jeffrey Johnson
Tasha Johnson
Joshua Kane
Billy Newton
Assen Roguev
Priya Shah
Erik Verschueren
Ariane Watson
Jason Wojcechowskyj
Jiewei Xu
Systems biology provides powerful, holistic insights into
functional and evolutionary relationships, but it makes
uncovering novel mechanistic insights challenging.
We develop experimental and computational methods to bridge the gap between
the relationships and mechanisms determined through systems biology. Creative
approaches are essential to generate and
analyze large-scale biological datasets
from different organisms and to extract
detailed molecular insights into the functions of individual pathways, complexes,
and proteins. In our systems-to-mechanism approach, we are quantitatively
characterizing protein-protein and
genetic interactions as well as post-translational modifications (PTMs) in various
biological processes, including DNA
repair, transcriptional regulation, and
RNA processing.
Evolution of Genetic Interactomes
In an effort to study the evolution of
genetic interaction networks, we have
been creating and comparing genetic
interaction maps from two distinct
eukaryotic species, Saccharomyces cerevisiae and Schizosaccaromyces pombe, work
that has led to the notion of “modular
conservation,” a finding that genetic interactions between genes whose proteins are
co-complexed are highly conserved. This
work has uncovered a hierarchical model
for the evolution of genetic interactions,
with conservation highest within protein complexes, lower within biological
processes, and lowest between distinct
biological processes. However, despite
the large evolutionary distance and
extensive rewiring of individual interactions, remarkably, both networks retain
conserved features and display similar
levels of functional cross-talk between
biological processes, suggesting general
design principles of genetic interactomes.
Thus, information collected from model
systems about connections between individual genes may not be as useful as inferences derived from functional module
definitions and the level of cross-talk
between different processes.
To extend this evolutionary analysis
as well as to functionally probe specific
biological processes in more complex
organisms, we have developed a genetic
interaction mapping approach in mammalian cells using RNA interference
(RNAi) technology. Initial studies have
confirmed that modular conservation is
also observed in higher organisms, and
we are now using this platform to aid in
understanding the underlying biology
behind different disease and infection
states.
Sub-Protein Genetic Analysis of
Molecular Machines
To date, virtually all genetic interaction data collected have used complete
deletions or knockdowns of genes, perturbations that affect all functions associated with a given protein. Furthermore,
human sequencing efforts are identifying
many mutated genes that are correlatively linked to different disease states,
and much work is required to understand the underlying biology that results
from these mutations. To address both
of these issues, we are now extending
our genetic analysis to the next level by
genetically characterizing specific amino
acid changes on multifunctional, structurally defined machines (initially in
budding yeast). Furthermore, we have
been heavily involved in the identification and characterization of PTMs,
including phosphorylation and ubiquitination, using mass spectrometry, and
have been using structural information
and conservation to help functionally
prioritize these PTMs, many of which
are perturbed in different disease states.
Genetically analyzing specific mutants
of amino acids that are modified will be
essential to understanding the functional
significance of these different PTMs.
In our proof-of-principle project, we
identified 53 different point mutants
in budding yeast RNA polymerase II
(RNAPII) and subjected them to genetic
interaction profiling by crossing them to
a panel of >1000 mutants. The resulting
genetic interaction data allowed us to
assign specific functions to individual
residues of this molecular machine,
uncover novel transcription factors,
and reveal a remarkable coordination
between transcription initiation, elongation rate, and mRNA splicing. Finally,
we discovered that our genetic data can
be used for the structural elucidation of
the protein-protein interaction interfaces
within RNAPII.
We are now extending this analysis
to genetically characterize other molecular machines, including the nucleosome
and several chaperones. Ultimately, we
believe that the analysis of genetic
interactions in this fashion will provide unprecedented insight into structure-function relationships involving
macromolecular complexes and will be
highly relevant when trying to understand the role that individual mutations
play in specific disease states in human
cells using RNAi-based genetic interaction methodologies.
E-MAP Analysis
Genetic Interaction Map
RNA Polymerase II
Point Mutations
Direct
Analysis
Growth
Phenotypes
Test
Predictions
Fitness
Transcription
Rates
Predict Functional Relationships
Chromosome
Stability
Start Site
Selection
Splicing
Efficiency
Using quantitative genetic interaction mapping analysis (E-MAP), 53 different point
mutants of RNAPII in budding yeast were analyzed, which led to linking unique residues
to different processes, including splicing and chromosome segregation. Additionally,
the genetic interaction information revealed a remarkable coordination between
transcription initiation, transcription rate, and mRNA splicing. Finally, the genetic data
can inform structural analysis of multifunctional molecular machines.
Publications
SELECTED RECENT
1. Beltrao P et al. (2012) Systematic
functional prioritization of protein
post-translational modifications.
Cell 150:413.
2. Braberg H et al. (2013) From structure to systems: high-resolution,
quantitative genetic analysis of
RNA polymerase II. Cell 154:775.
3. Haber JE et al. (2013) Systematic
triple mutant analysis uncovers
functional connectivity between
pathways involved in chromosome
regulation. Cell Rep. 3:2168.
4.Ryan CJ et al. (2012) Hierarchical
modularity and the evolution of
genetic interactomes across
species. Mol. Cell 46:691.
5. Ryan CJ et al. (2013) High-resolution network biology: connecting
sequence with function. Nat. Rev.
Genet. 14:865.
TOP FIVE OVERALL
1. Bandyopadhyay S et al. (2010)
Rewiring of genetic networks in
response to DNA damage. Science
330:1385.
2. Fiedler D et al. (2009) Functional
organization of the S. cerevisiae
phosphorylation network. Cell
136:952.
3. Keogh MC et al. (2006) A phosphatase complex that dephosphorylates gH2AX regulates DNA
damage checkpoint recovery.
Nature 439:497.
4.Roguev A et al. (2008) Conservation and rewiring of functional
modules revealed by an epistasis map in fission yeast. Science
322:405.
5. Schuldiner M et al. (2005) Exploration of the function and organization of the yeast early secretory
pathway through an epistatic mini
array profile. Cell 123:507.
“Despite the
importance of Turkey
and the Levant in
analyzing human
genetic diversity,
this area represents
a major gap in
population genetics.”
President Emeritus and Senior Investigator
labs.gladstone.ucsf.edu/mahley
HIGHLIGHTS
• Identified genetic association
between low levels of HDL-C and
coronary disease in Turks
• Turkish population structure and
genetic ancestry revealed unique
genetic relatedness
• Expansion of genetic data will provide insights into ancient Turkish
history and disease gene mapping
LAB MEMBERS
Uğur Hodoğlugil
K. Erhan Palaoğlu
Studies of human ancestry provide an opportunity to
map the history of the relationship between genetic
variation and disease risk.
The Anatolian peninsula (present-day
Turkey) connects the Middle East,
Europe, and Asia and, thus, has been
subject to major population movements, including ancient migrations
out of Africa. Despite the importance
of Turkey and the Levant in analyzing
human genetic diversity, this area of the
world represents a major gap in population genetics. Biodata and DNA samples
from the Turkish Heart Study (THS)
provide insight into the genealogical
relationships important for developing
tools for disease gene mapping.
Mapping Heart Disease
One unique disease area that has been
studied in the THS is coronary artery
disease and lipid metabolism. The
Turkish population is known for a high
prevalence of heart disease, resembling
the highest incidence seen in some
Eastern European populations. We
determined that one of the major risk
factors is a low level of high density lipoprotein cholesterol (HDL-C), which
was associated with candidate genes
identified in a genome-wide scan. These
include polymorphisms in the cholesterol ester transfer protein, ATP-binding
cassette transporter A1, apolipoprotein
A-V, and hepatic lipase. Most recently,
a u nique gene — g lucu ronic acid
epimerase — was shown to be associated
with both HDL-C and triglyceride levels
in Turks. Interestingly, the single nucleotide polymorphism (SNP) frequency
pattern across the locus for this gene
resembles an Asian pattern, whereas the
SNP frequency surrounding this locus
on chromosome 15q21-23 was similar
to a European pattern. Thus, we have
used genetic mapping to analyze genetic
diversity, human history, and disease
genes in the Turkish population.
SNP Genotyping
Our initial studies used principal component (PC) analysis to understand global
ancestry and demonstrate long-distance historical migration patterns.
STRUCTURE software was used for
model-based clustering and for predicting genetic relatedness and population structure. Samples from three regions
of Anatolia were analyzed using over
500,000 SNP genotypes and compared
with Human Genome Diversity Project
(HGDP) data. To obtain a more representative sampling from Central Asia,
we genotyped samples from a Kyrgyz
population in Bishkek, Kyrgyzstan. PC
analysis revealed a significant overlap
between Turks and Middle Easterners
and a relationship with Europeans and
South and Central Asians; however, the
Publications
SELECTED RECENT
1. Hodoğlugil U et al. (2012) Turkish
population structure and genetic
ancestry reveal relatedness among
Eurasian populations. Ann. Hum.
Genet. 76:128.
2. Mahley RW et al. (2012) Apolipoprotein E sets the stage: response
to injury triggers neuropathology.
Neuron 76:871.
3. Mahley RW et al. (2012) Smallmolecule structure correctors
target abnormal protein structure and function: the structure
corrector rescue of apolipoprotein
E‑associated neuropathology.
J. Med. Chem. 55:8997.
4.Mahley RW (2013) Shinya
Yamanaka, MD, PhD—2012 Nobel
Prize Laureate: how his dream of a
research career provides vision for
the next generation of young scientists. Anadolu Kardiyol. Derg. 13:204.
America
Oceania
East Asia
Turkey
Africa
Middle
East
Europe
A
Caucasus
Methodologic advances in analyzing
human history allow testing of possible
Central
Asia
SNP Genotyping and Gene Mapping
ancestry and migration between population groups. Work from the laboratory
of Dr. David Reich at the Broad Institute
of Harvard and the Massachusetts
Institute of Technology advances this
new approach using a software package,
A DMIXTOOLS, to study population history [Patterson N et al. (2012)
Genetics 192:1065]. We collaborated with
Dr. Reich to apply his SNP genotyping
array, consisting of 629,443 SNPs, to
analyze genetic diversity, human history,
and pattern variation related to disease
gene mapping. Samples from six regions
of Turkey and Bishkek, Kyrgyzstan will
be added to the collection, and data from
the THS will become part of the larger,
worldwide data set to address a major gap
in genetic diversity knowledge. These
data will provide a better understanding
of ancient Turkish history and improve
tools for disease gene mapping.
South Asia
(Pakistan)
Turkish genetic structure was unique.
STRUCTUR E analyses supported
the PC analysis and defined parental
ancestry components. For example, where
K colored segments (K) = 4, the genetic
ancestry of Turks was 38% European,
35% Middle Eastern, 18% South Asian,
and 9% Central Asian (see Figure). Data
from the Turkish samples in all three
regions of Anatolia did not show subpopulation structure and suggested Turkish
population homogeneity (see Figure). In
some regards, these data support historical
population migration patterns. However,
the significant genetic relatedness of the
Turks with the South Asian (Pakistani)
samples was surprising and may reflect
earlier migratory and admixture events.
Ancestry Coefficient (%)
100
80
TOP FIVE OVERALL
60
40
20
Turkey
Aydin
Istanbul
Kayseri
European
0.04
0.02
PC1
Adygei
0
Middle East
Palestinian
Druze
Turkish
Europe
Russian
Orcadian
Basque
French
Sardinian
Italian
Tuscan
Middle
Eastern
–0.02
–0.04
–0.04
–0.02
Caucasus
Adygei
0
PC2
0.02
Kyrgyz
Central
Asian
Central
Asia
Hazara
Uygur
Kyrgyz
0
Turkish
–0.02
South
Asian
–0.04
–0.02
Karitiana
N. Han
Turkey
Aydin
Istanbul
Kayseri
0.04
0.02
Papuan
Yakut
Japanese
Kyrgyz
Mongola
Hazara
Uygur
Pathan
Balochi
C
PC1
B
Turkish
Adygei
Italian
French
Palestinian
Bedouin
Mozabite
Yoruba
Mandenka
0
0
PC2
0.02
0.04
South
Asia
(Pakistan)
Burusho
Brahui
Balochi
Makrani
Pathan
Sindhi
A) Estimated individual ancestry and population structure (FRAPPE analysis). Each
individual is represented by a thin vertical line, which is partitioned into K colored
segments (K = 7). Colors represent the inferred ancestry from parental populations.
B, C) PC analysis demonstrating genetic relatedness across major geographic regions.
Each symbol represents one individual. B) PC analysis of populations from the HDGP,
Turkish, and Kyrgyz samples. C) PC analysis focusing on selected Eurasian populations.
1. Hodoğlugil U et al. (2010) Polymorphisms in the hepatic lipase
gene affect plasma HDL-cholesterol levels in a Turkish population.
J. Lipid Res. 51:422.
2. Mahley RW (1988) Apolipoprotein
E: cholesterol transport protein
with expanding role in cell biology.
Science 240:622.
3. Mahley RW et al. (1995) Turkish
Heart Study: lipids, lipoproteins,
and apolipoproteins. J. Lipid Res.
36:839.
4.Mahley RW et al. (2000) Low
levels of high density lipoproteins
in Turks, a population with elevated hepatic lipase: high density
lipoprotein characterization and
gender-specific effects of apolipoprotein E genotype. J. Lipid Res.
41:1290.
5. Yu Y et al. (2005) Multiple QTLs
influencing triglyceride and HDL
and total cholesterol levels identified in families with atherogenic
dyslipidemia. J. Lipid Res. 46:2202.
Katherine S. Pollard, PhD
Senior Investigator
labs.gladstone.ucsf.edu/pollard
HIGHLIGHTS
• Gene regulatory enhancers are the
fastest evolving sequences in the
human genome
• Many uniquely human enhancers
are active during embryonic
development
• Several developmental enhancers
differ between humans and
primates
LAB MEMBERS
Aram Avila-Herrera
Patrick Bradley
Genevieve Erwin
Lucia Franchini
Tara Friedrich
Joshua Ladau
Molong Li
Stephen Nayfach-Battilana
Ann Ryu
Sean Whalen
“Understanding
the genetic basis of
the human-specific
aspects of our
biology and health
is fundamentally
important.”
My laboratory pioneered the statistical phylogenetic
approach that identified human accelerated regions,
the DNA sequences most unique to humans.
Only a few of the fast-evolving human
accelerated regions (H ARs) of our
genome encode genes. Human proteins
are nearly identical to chimpanzees and
other animals. What makes us human is
how we use genes. Work in my laboratory
is revealing that, since we diverged from
chimpanzees and ancient hominids, the
evolutionary rewiring of our genome is
concentrated in a relatively small number
of mutations inside developmental
enhancers, the DNA sequences that control gene expression in the embryo.
Machine Learning Algorithms
We developed machine learning algorithms that integrate hundreds of diverse
datasets, including evolutionary patterns, transcription factor binding measurements, epigenetics, gene expression,
and sequence motifs, to predict if a DNA
sequence functions as a gene regulatory
enhancer. We applied this algorithm to
the 721 HARs that we found and predicted that one third of them function
as enhancers active during embryonic
development in heart, brain, limb, and
other tissues.
In parallel, we also analyzed the
sequence of each HAR across 50 vertebrate genomes to determine which evolutionary forces might have driven the
rapid evolution of HARs in humans.
These analyses allowed us to distinguish
positive selection from loss of function
mutations and a non-adaptive, recombination-associated process known as
biased gene conversion. We were also able
to predict which human-specific mutations in HARs were most likely to affect
their function, for example, by creating or
destroying binding sites for transcription
factors. These computational approaches
enabled us to rank HARs based on
evidence that they might function as
human-specific gene regulatory elements.
To validate our computational predictions, we compared the expression
patterns of HARs using a mouse transient transgenic reporter gene assay.
This approach enabled us to visualize
the expression pattern driven by the
human versus chimpanzee sequence of
a HAR in mouse embryos. We tested
our top 29 candidates and discovered
24 new developmental enhancers, five of
which showed differences in expression
patterns between the human and chimpanzee sequences.
Examining Ancient Humans
To study when HARs acquired their
human-specific mutations, we collaborated with Dr. Svante Pääbo at the Max
Planck Institute to array capture and
sequence the HARs in ancient DNA
samples from Neandertal and Denisovan
fossils. We discovered that about 10%
of mutations in HARs occurred after
modern humans split from these ancient
hominins, making them truly unique to
our species. We additionally analyzed
human genomes from diverse populations around the world and discovered
that more than 50 HARs have mutations
that arose very recently and are unique to
a single world population.
More New Tools
Another major project in our laboratory was the creation of SFams. This
protein family database encompasses
more diversity than existing databases,
and it processes new genome sequences
in an automated fashion as they rapidly
become available online. The motivation
for SFams is to extend our work on the
taxonomic diversity of microbial communities in the human body and natural
environments to encompass information
about the functions, pathways, and metabolic processes of these communities.
This effort will allow us to move from
describing “who is there” to understanding “what they are doing,” which is
a key step toward delineating the mechanisms through which microbes interact
with their hosts.
We also developed ProteinHistorian,
a software package for studying gene age.
Using this tool, we collaborated with the
Verdin laboratory at Gladstone to identify a novel sirtuin-substrate pair. We
also collaborated with the Ott laboratory, also at Gladstone, to characterize
post-translational modification of RNA
polymerase that appeared in the common
ancestor of multicellular organisms.
A Chimpanzee Sequence
SELECTED RECENT
1. Capra JA et al. (2013) How old is
my gene? Trends Genet. 29:659.
2. Capra JA et al. (2013) Many
human accelerated regions are
developmental enhancers. Philos.
Trans. R. Soc. Lond., B, Biol. Sci.
368:20130025.
3. The Human Microbiome Project
Consortium (2012) Structure,
function and diversity of the human
microbiome in an adult reference
population. Nature 486:207.
4.Ladau J et al. (2013) Global marine
bacterial diversity peaks at high latitudes in winter. ISME J. 7:1669.
5. Smith RP et al. (2013) A compact, in vivo screen of all 6-mers
reveals drivers of tissue-specific
expression and guides synthetic
regulatory element design. Genome
Biol. 14:R72.
1
1
2
1
Publications
2
2
TOP FIVE OVERALL
1. Pollard KS et al. (2002) Statistical inference for simultaneous
clustering of gene expression data.
Math. Biosci. 176:99.
B Human Sequence
1
2
2. Pollard KS et al. (2006) An RNA
gene expressed during cortical
development evolved rapidly in
humans. Nature 443:167.
1
1
2
2
3. Pollard KS et al. (2008) A genomewide approach to identifying
novel-imprinted genes. Hum. Genet.
122:625.
4.Pollard KS (2009, May) What
makes us human? Sci. Am. 300:44.
2xHAR.238 is a neurodevelopmental enhancer with uniquely human activity.
A) At embryonic day 11.5, transgenic mice carrying the chimpanzee sequence show
enhancer activity (blue) in the rostral dorsal pallium (arrowhead 1) and a caudal part
of the dorsal pallium (arrowhead 2), plus the hindbrain and spinal cord. B) Caudal
dorsal pallium activity is lost when using the human sequence. We predict that
2xHAR.238 regulates GLI2, a gene linked to abnormal cortex development.
5. Pollard KS et al. (2010) Detection
of non-neutral substitution rates on
mammalian phylogenies. Genome
Res. 20:110.
“We must understand
the molecular events
that occur during
cardiac differentiation
to develop regenerative
interventions for
cardiovascular disease.”
Director and Senior Investigator
labs.gladstone.ucsf.edu/srivastava
HIGHLIGHTS
• Demonstrated efficacy of in vivo
cardiac reprogramming for regen
erative purposes
• Discovered a combination of
transcription factors that can
reprogram human fibroblasts
into cardiomyocytes
• Found that miR-1 reinforces the
striated muscle phenotype by
regulating smooth muscle
gene expression
LAB MEMBERS
Yen Sin Ang
Emily Berry
Romit
Bhattacharya
Yen Bui
Karen
Carver-Moore
Paul Cheng
Amy Foley
Jidong Fu
Giselle Galang
Casey Gifford
Samantha Hastie
Amy
Heidersbach
Yu Huang
Isabelle King
Huey Jiin Liu
Lei Liu
Kimberly Cordes
Metzler
Tamer Mohamed
Li Qian
Ethan Radzinsky
Renee Rivas
Eva Samal
Christopher
Saxby
Neil Sheehy
Joseph Shieh
Nicole Stone
Christina
Theodoris
Charissa To
Vasanth
Vedantham
Mark White
Pengzhi Yu
Ping Zhou
Over the last several years, we have found that various
networks regulate early decisions about cardiac cell
fate, cellular differentiation, and cell behavior.
A common theme emerging from our
work is that complex net works — signaling, transcriptional, and translational networks — are intertwined in
positive and negative feedback loops
that precisely titrate critical pathways for
cardiac and vascular differentiation and
maintenance. The dosage of key genes is
important in these pathways. We, and
others, have found that heterozygous
mutations of many genes cause human
disease, and we are modeling different
gene dosages in induced pluripotent stem
(iPS) cells. Most recently, we have leveraged our knowledge of cardiac differentiation pathways to successfully reprogram
non-cardiac cells into new cardiomyocyte-like cells in vitro and in vivo.
Reprogramming Cells into
Cardiomyocytes
The combined knowledge of cardiac
differentiation pathways previously led
us to describe a combination of three
cardiac transcription factors — Gata4,
Mef2c, and Tbx5 — that was sufficient
to convert a cardiac fibroblast into a
cell containing globally altered gene
expression that was similar to a cardiomyocyte. We called this cell an induced
cardiomyocyte (iCM). In vitro, iCMs
developed sarcomeres and calcium transients, and in some cases, they could
even contract spontaneously. However,
introducing the reprogramming factors into the native environment of the
cells produced even more fully reprogrammed cardiac fibroblasts that contracted and electrically communicated
with neighboring myocytes. When we
examined models of coronary occlusion, in vivo reprogramming spurred
new myocyte generation and improved
cardiac function. Human fibroblasts
were more difficult to reprogram, but
after we screened for additional factors,
we found that, by adding two more
transcription factors — MESP1 and a
nuclear hormone receptor ESSRG — we
could induce cardiac reprogramming in
vitro, although incompletely. In an in
vivo pig model of myocardial infarction,
this combination of five human factors
appeared to reprogram cells more fully.
To understand the mechanism of
direct cardiac reprogramming, we are
performing temporal analyses of the transcriptome, DNA-binding, and epigenetic
changes that occur as early as 12 hours
and over the subsequent 10 weeks of
reprogramming. This approach will
reveal fundamental knowledge of how a
cell transitions its fate in the absence of
cell division. Together, our basic studies
and translational efforts, which leverage
years of cardiac development research,
may provide a roadmap for guiding cell
fate in vivo for regenerative purposes.
MicroRNAs Influence Signaling
and Transcription
In addition to investigating cellular
reprogramming to generate cardiomyocytes, we are also studying small noncoding RNAs of the microRNA family
(miRNAs). We have found that miRNAs
can influence major cellular events by
titrating signaling and transcriptional
events. MiRNAs generally function by
interacting with mRNAs and negatively
regulating their stability and/or translation. By studying the complete loss of
function of miR-1, the most abundant
miRNA in the heart, we found that
miR-1 is required for postnatal cardiac
function, and it reinforces the striated
muscle phenotype by regulating both
transcriptional and effector nodes of the
smooth muscle gene expression network.
Additionally, we found that endothelial
miRNAs promote signaling of vascular
endothelial growth factor (VEGF) by
repressing multiple negative regulators
of VEGF. Furthermore, endothelial
miRNAs are embedded in the major
transcriptional regulators of endothelial
cells. Finally, we reported that let-7, an
miRNA found in many differentiated
cell types, functions to promote multiple
differentiation pathways and thereby
serves as a barrier when reprogramming
cells to regain pluripotency.
Gata4/Mef2c/Tbx5
In Vitro iCMs
Post-natal
Cardiac Fibroblast
Publications
SELECTED RECENT
1. Fu J et al. (2013) Direct reprogramming of human fibroblasts toward
the cardiomyocyte lineage. Stem
Cell Reports 1:235.
2. Hassel D et al. (2012)
MicroRNA-10 regulates the angiogenic behavior of zebrafish and
human endothelial cells by promoting vascular endothelial growth
factor signaling. Circ. Res. 111:1421.
4.Qian L et al. (2012) In vivo reprogramming of murine cardiac
fibroblasts into induced cardiomyocytes. Nature 485:593.
5. Worringer KA et al. (2014) The
let-7/LIN-41 pathway regulates
reprogramming to human induced
pluripotent stem cells by controlling
expression of pro-differentiation
genes. Cell Stem Cell 14:40.
TOP FIVE OVERALL
1. Garg V et al. (2003) GATA4 mutations cause human congenital heart
defects and reveal an interaction
with TBX5. Nature 424:443.
Gata4/Mef2c/Tbx5
Fibroblasts in
Injured Heart
In Vitro iCMs
2. Ieda M et al. (2010) Direct reprogramming of fibroblasts into functional cardiomyocytes by defined
factors. Cell 142:375.
3. Qian L et al. (2012) In vivo reprogramming of murine cardiac
fibroblasts into induced cardiomyocytes. Nature 485:593.
RV
RV
LV
LV
Control
Gata4/Mef2c/Tbx5
4.Zhao Y et al. (2005) Serum
response factor regulates a musclespecific microRNA that targets
Hand2 during cardiogenesis. Nature
436:214.
5. Zhao Y et al. (2007) Dysregulation
of cardiogenesis, cardiac conduction, and cell cycle in mice lacking
miRNA-1-2. Cell 129:303.
Direct reprogramming of cardiac fibroblasts to iCM-like cells can be accomplished
by the expression of three developmental transcription factors — Gata4, Mef2c,
and Tbx5. In vitro, cells are only partially reprogrammed. However, in vivo delivery
of these three reprogramming factors into injured adult hearts resulted in more
fully reprogrammed iCMs and improved cardiac function. RV, right ventricle; LV,
left ventricle.
“We hope that
our investigations
shed light on how
extracellular signals
affect epigenetic
regulation of hiPS
cell differentiation.”
Kiichiro Tomoda, PhD
labs.gladstone.ucsf.edu/tomoda
• XaXa hiPS cell lines can be frequently generated using SNL
feeder cells
Our goal is to precisely control the X-inactivation
status in female hiPS cells as either XaXi or XaXa
by defined extracellular signals.
• LIF contributes to Xi reactivation
Promise and Challenges
• Culture conditions strongly affect
X-inactivation status in hiPS cells
Human induced pluripotent stem (hiPS)
cells hold great promise for disease modeling, toxicology, drug screening, and
future clinical applications. However,
hiPS cells exhibit variability in gene
expression, epigenetic status, and differentiation propensity, which may affect
applications of hiPS cells. Particularly,
female hiPS cell lines have substantial
variability in X-inactivation status. The
majority of hiPS cell lines maintain one
transcriptionally active X (Xa) and one
inactive X (Xi) chromosome from donor
somatic cells. Some reactivate the Xi
(XaXa) at a low frequency. Others lose
regulation of the Xi so that the X chromosome inactivation is eroded (XaXe).
Problematically, some XaXe hiPS cells
lose the ability to differentiate or have
detrimental effects on disease modeling.
Collectively, the X chromosome must be
kept in control to ensure high-quality
hiPS cells.
HIGHLIGHTS
LAB MEMBER
Mariselle Lancero
Our Studies
Several reports imply that culture conditions affect the X-inactivation status
in female human pluripotent stem
cells. Thus, we began to investigate the
X-inactivation status in female hiPS cell
lines derived by standard conditions
in the Yamanaka laboratory. By gene
expression profiling, we found that
X-linked genes are specifically expressed
at higher levels in our female hiPS cells
than in XaXi human embryonic stem
(hES) cells. Fluorescent in situ hybridization and single nucleotide polymorphism (SNP) sequencing revealed that
X-linked genes show biallelic expression
in our female hiPS cells but monoallelic
in XaXi hES cells, suggesting that most
of our female hiPS cell lines are XaXa.
Our conclusion that the majority of
female hiPS cell lines are XaXa (n=20/23
cell lines) is different from several reports
that most female hiPS cell lines are XaXi.
When considering reasons for this discrepancy, we noticed that our group uses
SNL feeder cells, immortalized mouse
fibroblast STO cells expressing neomycin-resistance and leukemia inhibitory
factor (LIF) genes, to derive hiPS cells,
while others mainly use non-SNL feeders,
such as mouse embryonic fibroblasts
(MEFs). To test whether feeders affect
Xi reactivation, we generated hiPS cell
lines on non-SNLs and found that in the
majority of lines on non-SNLs, X-linked
genes are monoallelically expressed at
levels similar to those in XaXi pluripotent stem cells. These data confirm that
most hiPS cell lines grown on non-SNLs
are XaXi.
Next we examined the effects of feeders
on Xi reactivation during culture. When
we cultured early passage hiPS cells on
SNLs, many X-linked genes were up-regulated, and X-linked genes became biallelic
even in hiPS cells originally reprogrammed
on non-SNLs. In contrast, when we cultured early passage hiPS cells on MEFs,
X-linked genes were not substantially
upregulated even in hiPS cells originally
reprogrammed on SNLs. These data indicate that the Xi reactivation occurs after
reprogrammed cells are initially picked
and cultured on SNLs.
Since SNLs produce LIF, we examined
the effects of LIF on Xi reactivation. When
we cultured early passage hiPS cells on
MEFs in medium containing recombinant
LIF, some X-linked genes were upregulated
and became biallelic. Therefore, LIF helps
to reactivate some X-linked genes on the
Xi, but SNLs provide LIF and additional
factors that may synergistically function in
ChrX/Autosomes
Ratio (Log2)
A
0.6
Xi reactivation. Collectively, we conclude
that culture conditions strongly affect the
X-inactivation status in female hiPS cells.
Publications
Future Directions
1. Tomoda K et al. (2012) Derivation
conditions impact X-inactivation
status in female human induced
pluripotent stem cells. Cell Stem
Cell 11:91.
Our goal is to precisely control the
X-inactivation status as either XaXi or XaXa
by defined extracellular signals. To achieve
our goal, we narrowed down the extracellular signals that affect the X-inactivation
status in mouse pluripotent stem cells and
are testing whether these signals affect the
X-inactivation status in female hiPS cells.
In addition, we hypothesize that the signals
that reactivate the Xi also reduce facultative heterochromatin regions on autosomes
and enhance differentiation potential of
hiPS cells. We are testing these hypotheses
in hiPS cell lines cultured with or without
the signals. We hope that our investigations
will shed light on how extracellular signals
shape the differentiation potential of hiPS
cells through epigenetic regulation.
Generated on MEFs
Feeders
SNLs
MEFs
+ rLIF
MEFs
0.4
0.2
0
Before
B
After
923M2
PGK1/XIST/HOECHST
Before
After
923M3
PGK1/XIST/HOECHST
SELECTED RECENT
TOP FIVE OVERALL
1. Takahashi K et al. (2007) Induction
of pluripotent stem cells from adult
human fibroblasts by defined factors. Cell 131:861.
2. Tomoda K et al. (1999) Degradation of the cyclin-dependent-kinase
inhibitor p27Kip1 is instigated by
Jab1. Nature 398:160.
3. Tomoda K et al. (2002) The cytoplasmic shuttling and subsequent
degradation of p27Kip1 mediated
by Jab1/CSN5 and COP9 signalosome complex. J. Biol. Chem.
277:2302.
Female hiPSC on Non-SNLs
Female hiPSC on SNLs
A) Gene expression ratios of X-linked and autosomal genes (X/A ratios). We
generated two female hiPS cell lines (923M2 and 923M3) on MEFs. Then, we
separated the lines into three groups and cultured them on either MEFs (red), MEFs
in medium containing rLIF (green), or SNLs (purple). We collected RNA before and
after separation, analyzed global gene expression, and calculated the X/A ratios.
Points above the top line have a 0.95 probability of being XaXa, while those below
the bottom line have a 0.95 probability of being XaXi. B) RNA FISH for PGK1 (green)
and XIST (red) in hiPS cells grown on non-SNLs or SNLs. (r)LIF, recombinant LIF.
4.Tomoda K et al. (2004) Multiple
functions of Jab1 are required for
early embryonic development and
growth potential in mice. J. Biol.
Chem. 279:43013.
5. Tomoda K et al. (2005) The Jab1/
COP9 signalosome subcomplex is
a downstream mediator of Bcr-Abl
kinase activity and facilitates cell
cycle progression. Blood 105:775.
Shinya Yamanaka, MD, PhD
Senior Investigator
labs.gladstone.ucsf.edu/yamanaka
HIGHLIGHTS
• Differentiated cells can be more
fully reprogrammed to iPS cells by
Oct4, c-Myc, Sox2, and Klf4
• LIN-41, a target of let-7 miRNA,
potently enhances cellular
reprogramming
• iPS cell generation can be used
to select against large chromosomal aberrations such as ring
chromosomes
LAB MEMBERS
Yohei Hayashi
Cody Kime
Mariselle Lancero
Timothy Rand
Salma Sami
Hayami Sugiyama
Kiichiro Tomoda
Kathleen Worringer
“Understanding the
steps of iPS cell
generation may
help us discover
new methods
to reprogram
therapeutically
relevant cell lines.”
In 2006, our laboratory used a defined set of transcription factors to reprogram fully differentiated adult cells
and return them to the embryonic stem cell state.
Because these reprogrammed cells,
called induced pluripotent stem (iPS)
cells, can be differentiated into any cell
type, we hope this technique will help
generate tissues and organs for treating
aging or damaged tissues. In the original protocol, we used retroviruses to
integrate four reprogramming factors
(Oct4, c-Myc, Sox2, and Klf4) into
the mouse genome. Since then, we have
improved the system by enhancing its
reprogramming efficiency (by depleting
p53 expression using short hairpin RNA
and improving its safety (by replacing
tumorigenic c-Myc with L-Myc). With
these improvements, we also created a
three-vector episomal reprogramming
system that is robust, easy to use, and
avoids the DNA integration pitfalls
caused by the original retroviral reprogramming system.
Understanding Reprogramming
Our recent projects focus on elucidating
the complex, month-long process of
cellular reprogramming. From the initial discovery of the iPS cell generation
protocol, we noted that only ~0.1% of
cells successfully complete the reprogramming process. If we could identify
the important steps in the process and
the functions of the molecules responsible for these steps, we could potentially
improve the frequency of complete conversion. In addition, this understanding
might shed light on other reprogramming methods — for example, direct
reprogramming of therapeutically useful
cell lines such as cardiac myocytes.
One interesting insight into the
mechanism of the reprogramming process has come from studies aimed at
understanding how let-7 microRNA
(miRNA) suppresses reprogramming.
These experiments focus on a particular
target of let-7 miRNA, the mRNA that
encodes the protein LIN-41. LIN-41 is
expressed by embryonic stem (ES) cells,
but it is quickly eliminated as the cells
differentiate from the pluripotent state.
When reprogramming fibroblasts, the
LIN-41 promoter is reactivated around
day 7 of reprogramming. LIN-41 performs better than c-Myc in reprogramming cocktails. But unlike other
reprogramming factors, LIN-41 is not a
nuclear factor. It resides in the cytoplasm
where it regulates RNA translation. We
are currently investigating how LIN-41
works, what transcripts it targets, and
why its expression so strongly affects cellular reprogramming.
Correcting Ring Chromosomes
We are also continuing to create novel
tools for regenerative medicine and cell
therapy-based treatments using cellular
reprogramming. One of our recent
contributions involves a new method
for correcting ring chromosomes.
Chromosomes are normally linear, like a
string. A ring chromosome occurs when
the ends of a chromosome fuse together,
often after large regions at one or both
ends have been lost. On karyotype analysis, the result is a ring-shaped chromosome. Ring chromosomes are associated
with growth retardation and mental
and physical disabilities. In our recent
work in collaboration with Dr. Anthony
Wynshaw-Boris’s laboratory at the
University of California, San Francisco,
we demonstrated that iPS cell generation
can be used to select against ring chromosomes in cells obtained from patients that
have such chromosomes, such as patients
with Miller-Dieker syndrome. This type
of selection often results in duplication of
the wild-type chromosome via a compensatory uniparental disomy mechanism.
Our study demonstrates the first viable
strategy for correcting large chromosomal aberrations and may inform the
development of novel treatments for disorders caused by ring chromosomes.
X Chromosome Reactivation
We are also working on another interesting process that intersects with cellular reprogramming — X chromosome
reactivation. In the body, this process
only happens when germ lines form,
making it exceedingly difficult to study.
We recently demonstrated that signals
originating from the feeder cell layer
influence the success or failure of X chromosome reactivation. Because the reactivation of the silenced X chromosome
may have consequences for the medical
applications of the resulting cell lines,
we are interested in further dissecting
the signals that influence the status of
X chromosomal activation and inactivation in human ES and iPS cells.
let-7 microRNAs
let-7
Inhibitor
OCT4
KLF4
SOX2
c-MYC
Differentiation
LIN-41
KLF4
SOX2
1. Bershteyn M et al. Cell-autonomous
correction of ring chromosomes in
human induced pluripotent stem
cells. Nature (in press).
2. Koyanagi-Aoi M et al. (2013) Differentiation-defective phenotypes
revealed by large-scale analyses of
human pluripotent stem cells. Proc.
3. Tanabe K et al. (2103). Maturation, not initiation, is the major
roadblock during reprogramming
toward pluripotency from human
fibroblasts. Proc. Natl. Acad. Sci.
USA 110:12172.
4.Tomoda K et al. (2012) Derivation
Cell 11:91.
5. Worringer K et al. (2014) The let-7/
LIN-41 pathway regulates reprogramming to human induced pluripotent stem cells by controlling
expression of prodifferentiation
genes. Cell Stem Cell 14:40.
TOP FIVE OVERALL
2. Miura K et al. (2009) Variation in
the safety of induced pluripotent
stem cell lines. Nat. Biotechnol.
27:743.
EGR1
OCT4
SELECTED RECENT
1. Hong H et al. (2009) Suppression
of induced pluripotent stem cell
generation by the p53-p21 pathway.
Nature 460:1132.
LIN-41
Pluripotency
Publications
let-7
EGR1
c-MYC
Four transcription factors (OCT4, KLF4, SOX2, and c-MYC) reprogram adult cells into
stem cells. Through our studies, we have learned that the microRNA let-7 is a barrier to
reprogramming that inhibits LIN-41, a strong reprogramming factor that is expressed in
pluripotent embryonic stem cells. We have found that LIN-41 inhibits the transcription
factor EGR1, which promotes cell differentiation and is another barrier to reprogramming. OCT4, octamer-binding transcription factor 4; KLF4, Kruppel-like factor 4; SOX2,
SRY-related HMG-box; LIN-41, lineage-41; EGR1, early growth response protein 1.
3. Okita K et al. (2011) A more
efficient method to generate integration-free human iPS cells. Nat.
Methods 8:409.
4.Takahashi K et al. (2006) Induction
of pluripotent stem cells from
mouse embryonic and adult fibroblast cultures by defined factors.
Cell 126:663.
5. Takahashi K et al. (2007) Induction
of pluripotent stem cells from adult
human fibroblasts by defined factors. Cell 131:861.
Director’s Report
“The efforts to stop HIV/AIDS
have moved very far, very fast, but
our work is far from done.”
Virology and Immunology Research
Although we do not yet have a cure for the 35.3 million people
infected with human immunodeficiency virus (HIV) nor a vaccine to prevent the uninfected, great progress has been made
in the fight against HIV and acquired immunodeficiency syndrome (AIDS). The creation of more than 30 different antiretroviral drugs will become a seminal accomplishment in the
history of modern medicine. These drugs are also reaching more
people in need-nearly 11 million people are now on therapy
who had no options before. Here at Gladstone, we remain fully
engaged in our battle against this defining infectious disease of
the last 100 years. Below, I will share a personal reflection about
this virus and the global epidemic it has spawned.
A New Disease
In mid-July 1981, I was completing my fellowship at the
National Institutes of Health (NIH) and was one of six fellows
who rotated to admit patients from all over the world and study
their immune system diseases. Because I was “second up” in
the rotation, I was not assigned responsibility for a young man
from New York City. But because of the mystery he posed, I
carefully followed his clinical course. His immune system literally disintegrated in front of our eyes — cause unknown. He
fell prey to one opportunistic infection after another, including
a parasitic infection in his brain and an aggressive cancer of
the skin (Kaposi’s sarcoma) that rapidly spread to his internal
organs. We had never seen anything like this full-f ledged
assault on the immune system. Of course, this young man had
AIDS, the first case seen at the NIH. We had no idea what was
driving his immunodeficiency. However, we did determine that
he had almost completely lost his helper CD4 T cells — this
key lymphocyte subset serves as the cellular “conductor” of the
immunological orchestra.
By the summer of 1991, the AIDS epidemic was in full
fury. HIV had been identified as the cause, but there was still
no effective treatment. Ryan White was banned from school
because he was infected with HIV. Rock Hudson had died of
AIDS after seeking treatment in France. I vividly recall my first
month on the ward at San Francisco General Hospital — ground
zero for the AIDS epidemic in San Francisco — leading my
medical team of two students, two interns, and a resident.
Every single one of our patients admitted for the entire month
was infected with HIV and dying from AIDS, and there was
nothing we could do.
Turning the Corner
Today, the landscape has radically changed. Full-blown AIDS
is rarely seen. HIV infection is no longer a death sentence due
to the advent of combination antiretroviral therapy (ART). It
is efficiently managed as a chronic disease in outpatient clinics.
Driven by basic and translational studies of HIV, weak points
in the HIV lifecycle involving viral enzymes have been successfully targeted with small-molecule inhibitors. These inhibitors
moved from clinical trials to full FDA approval in record time.
When used in combination, these drugs can suppress the virus
to undetectable levels and prevent the emergence of resistant
viruses. Their creation is a landmark advancement in modern
medicine. Thus far, ART has saved 6.6 million lives, and these
numbers will likely increase 5–10-fold. What a gratifyingly
different picture compared to Bethesda in 1981 or the wards of
San Francisco General Hospital in 1991.
The story is not yet complete. Magical as they are, these
drugs only beat the virus down. They don’t eliminate it. Once
infected, always infected, and the drugs must be taken for life.
Outlook for the Future
Great progress was made in 2013. While an effective HIV vaccine remains beyond our grasp, key insights into the conformation of the Env trimer have been gained, and four classes of
broadly neutralizing antibodies have been defined. Scientists
are attempting to build on what appeared to be an early protective response that waned over time in the RV144 vaccine
trial. Male circumcision provides 73% protection from the
acquisition of HIV from an infected woman in a real world
setting. Circumcision clinics are now scaling up, but only
3.2 million circumcisions expect to be performed by 2015,
well short of the desired goal of 20 million. Pre-exposure prophylaxis, select microbicides, and antiviral drugs have shown
promise for prevention. Trials are under way to determine if
combinations of these preventive interventions, such as combinations of antiviral drugs, will more capably stop the spread
of HIV. Advances in prevention have prompted discussion
of the concept of an “AIDS-free generation,” although most
regard this as an aspirational rather than a realistic goal over
the next 5–10 years.
Progress against HIV/AIDS is being made around the world.
As noted, 35.3 million people are living with the virus — an
increase of about 1 million since 2011. However, this increase
is driven mainly by the longer survival with ART. There were
2.3 million new infections in 2012, down 33% from 3.4 million annual infections in 2001. AIDS deaths also declined to
1.6 million in 2012, down from 2.3 million in 2005. Prevention
efforts are paying off. HIV infection rates, mainly driven by
heterosexual transmission, have decreased by more than 50% in
26 countries around the world, including 13 countries in subSaharan Africa. Africa is home to 70% of all new HIV infections, but even there, infection rates have fallen by 42% in the
most at-risk population, 15–24 year olds.
The situation is also rapidly improving for children. ART
coverage is now reaching 62% of pregnant HIV-infected
mothers, and HIV infection rates among children have fallen
by 35% since 2009. Botswana, Namibia, Ghana, and Zambia
have already met the Millennium Development Goal of providing antiretroviral medications to more than 90% of pregnant HIV-infected mothers. The President’s Emergency Plan
for AIDS Relief (PEPFAR), which will celebrate its 10th anniversary this year, has prevented over 1,000,000 new HIV
infections in children. However, much more needs to be done:
260,000 children were newly infected in 2012.
Hope for a Cure
A cure may be the only solution for the increasingly daunting
economic challenge posed by lifelong treatment of tens of
millions of HIV-infected individuals in the developing
world. Previously, no one dared use the “C” word; however,
recent progress has changed the landscape. For example, Mr.
Timothy Brown, “the Berlin patient,” has been completely
cured of his HIV infection after two stem cell transplants for
an underlying leukemia. The Visconti cohort of HIV-infected
individuals treated early in their disease no longer require
antiretroviral therapy and are thus “functionally cured” even
though the virus persists at low levels. During this past year,
the World Health Organization changed its guidelines for
treating HIV infections, raising the CD4 T-cell threshold
for starting therapy to 500 cells/mm 3. Under this guideline,
26 million people of the 35.3 million infected should start
ART. Treatment is reaching approximately 10 million people.
However, there is neither a plan nor the funding to expand
this effort. Additionally, for every 10 individuals put on ART,
16 individuals become newly infected. Clearly, we need a
better approach to preventing new infections and a cure for
those already infected. Such a cure would obviate the need for
lifelong ART — an economic game-changer. Three national
collaboratories have been funded by the National Institute
of Allergy and Infectious Disease (NIAID) to focus on an
HIV cure. Several Gladstone investigators are involved in the
CARE collaboratory, which comprises 20 leading scientists
from 12 different institutions, including Merck, a major pharmaceutical company.
The HIV/AIDS landscape has surely changed for the
better. However, our work is far from done. There is still no
workable vaccine, and a scalable HIV cure remains a distant
dream. Nevertheless, basic and translational science has convincingly moved the needle and will help save tens of millions
of lives around the world. We should be proud of what has
been accomplished but at the same time remain focused on
developing and delivering the tools that can truly transform
the vision of an “AIDS-free generation” into a reality.
Warner Greene,
Nevan Krogan, Robert Grant,
Phillip Berman,
Leor Weinberger; front row:
Eric Verdin, Marielle Cavrois,
JJ Miranda, Melanie Ott,
Shomyseh Sanjabi,
Gilad Doitsh.
Phillip W. Berman, PhD
“Therapeutics
have reduced the
morbidity of HIV-1
infection, but only an
effective vaccine will
eradicate AIDS .”
Visiting Investigator
labs.gladstone.ucsf.edu/berman
HIGHLIGHTS
• Determined that previous gp120
vaccines could not bind broadly
neutralizing antibodies
• Protective antibodies overlap a V1/
V2 epitope recognized by broadly
neutralizing antibodies
• New gp120s and V1/V2 scaffolds
are being designed to elicit protective antibodies
LAB MEMBERS
David Alexander
Gabriel Byrne
Rachel Doran
Ann Durbin
Kathryn Mesa
Javier Morales
Gwen Tatsuno
Richard Theolis, Jr.
Bin Yu
The goal of our research is to improve the protective
efficacy of vaccines based on the HIV-1 envelope
protein gp120.
Our lab began this work in 1985 and
championed this approach through multiple clinical trials. The RV144 clinical
trial, completed in 2009, showed for the
first time that immunization could prevent human immunodeficiency virus
(HIV)-1 infection in humans. This
trial involved a prime/boost immunization regimen with our bivalent gp120
subunit vaccine (AIDSVAX B/E) and
an engineered poxvirus-based vaccine
(vCP1521). However, the level of protection achieved (31.2%) was too low for
regulatory approval. Therefore, we redoubled our research efforts to improve the
gp120 vaccine concept, with the goal of
achieving the 60% or greater level of protection thought to be required for product
registration and clinical deployment.
Importance of the V1/V2 Domain
of Gp120
Two independent lines of investigation
suggested that the antibody response to
the V1/V2 domain of gp120 is of particular importance in developing a vaccine to prevent HIV-1 infection. First,
the correlates of protection study from
the RV144 trial showed that protection
correlated with an antibody response
to positions 167–171 in the V2 domain
of gp120. Second, studies of antibodies
recovered from rare individuals (termed
“elite neutralizers”) showed that the
binding of several potent, broadly neutralizing antibodies also depended on
amino acids 167–171. However, these
antibodies were unusual because their
binding also depended on contacts with
specific glycans (mannose-5) at positions
156 and 160. When we analyzed the vaccine immunogens within the AIDSVAX
B/E vaccine, we discovered that they
lacked the carbohydrate essential for
binding to these broadly neutralizing
antibodies. Therefore, we reasoned that
we might be able to improve the gp120
immunogens used in the RV144 trial
by producing them under conditions
that favor the incorporation of mannose-5 glycans. Over the past year, we
developed new gp120 immunogens that
incorporate the proper glycans and can
be produced in cell lines suitable for
human pharmaceuticals.
Engineering Glycopeptide Scaffolds
to Increase the Magnitude of the
Antibody Response to the
V1/V2 Domain
The HIV-1 envelope protein gp120 has a
complex structure, typically consisting of
~510 amino acids distributed among five
conserved (C) and five variable (V) regions;
it includes nine disulfide bridges and
26 N-linked glycosylation sites. Because
immunization with these scaffolds greatly
improves the antibody responses associated with protection when compared to
immunization with gp120 alone. Over the
next few years, we plan to carry out the
developmental work required to advance
these molecules into human clinical trials.
This work entails screening scaffolds prepared from worldwide isolates of HIV-1
as well as conducting formulation studies
with different adjuvants, stability studies,
and prime/boost immunization studies to
discover the most effective way to achieve
broadly protective antibody responses to
the V1/V2 domain.
of this complex structure, immunization
with gp120 elicits antibodies to dozens of
epitopes, of which only a small fraction
are associated with protective immunity. To maximize the antibody response
to regions that mediate protection and
to minimize the immune response to
regions unrelated to protection, we used
genetic engineering to create small glycopeptide scaffolds. These scaffolds possess
the structural features required to bind
to broadly neutralizing antibodies and to
antibodies that are similar to those that
correlated with protection in the RV144
trial. Preliminary results suggest that
P183
α4β7
Start of V1
K178
L176
End of V2
D
A
B
N156
N160
C
C
D
1. Haynes BF et al. (2012)
Immune-correlates analysis of an
HIV-1 vaccine efficacy trial. N. Engl.
J. Med. 366:1275.
2. Nakamura GR et al. (2012) Monoclonal antibodies to the V2 domain
of MN-rgp120: fine mapping of
epitopes and inhibition of α4β7
binding. PLoS One 7:e39045.
3. O’Rourke S et al. (2012) Sequences
in gp41, the CD4 binding site, and
the V2 domain regulate sensitivity
and resistance to broadly neutralizing antibodies. J. Virol. 86:12105.
4.Yu B et al. (2012) Glycoform and
net charge heterogeneity in gp120
immunogens used in HIV vaccine
trials. PLoS One 7:e43903.
5. Zolla-Pazner S et al. (2013) Analysis of V2 antibody responses
induced in vaccines in the ALVAC/
AIDSVAX HIV-1 vaccine efficacy
trial. PLoS One 8:e53629.
D167
K168
B
SELECTED RECENT
G166
E172
A
Publications
CVTLNCTDLRNTTNTTNSTDNNNSKSEGTIKGG
EMKNCSFNITTSI
GDKMQKEYALLYKLDIEPIDNDSTSY
RLISCN
Diagram of the four-stranded V1/V2 domain β-sheet structure. Locations of the
N-linked glycosylation sites with mannose-5 glycans, essential for binding with
the broadly neutralizing PG9 monoclonal antibody, are shown in red. Shaded ovals
indicate approximate locations of the two immunodominant epitopes. Amino acids
167–171 (recognized by antibodies that correlated with protection in the RV144 trial,
as well as by PG9) occur at the junction of the B and C strands.
TOP FIVE OVERALL
1. Berman PW et al. (1983) Detection
of antibodies to herpes simplex
virus with a continuous cell line
expressing cloned glycoprotein D.
Science 222:524.
2. Berman PW et al. (1985) Protection
from genital herpes simplex virus
type 2 infection by vaccination
with cloned type 1 glycoprotein D.
Science 227:1490.
3. Berman PW et al. (1990) Protection
of chimpanzees from infection by
HIV-1 after vaccination with recombinant glycoprotein gp120 but not
gp160. Nature 345:622.
4.Lasky LA et al. (1986) Neutralization of the AIDS retrovirus by
antibodies to a recombinant
envelope glycoprotein. Science
233:209.
5. Weiss RA et al. (1986) Variable and
conserved neutralization antigens
of human immunodeficiency virus.
Nature 324:572.
Marielle Cavrois, PhD
“HIV envelope is one
of the fastest evolving
proteins, but we do
not know how this
impacts its function.”
Staff Research Scientist
labs.gladstone.ucsf.edu/cavrois
HIGHLIGHTS
• HIV-1 species encoding Envs with
compact V1/V2 domains mediate
fusion efficiently
• Shortening V1/V2 domains
increases Env incorporation into
virions and fusion
• The number of glycosylation sites on
V1/V2 inversely correlates with Env
incorporation and fusion
HIV-1 adapts when pressured by the host’s immune
response, but adaptation occurs at a fitness cost, especially for Env, which mediates viral entry into cells.
Human immunodeficiency virus (HIV)-1
envelope glycoprotein (Env) constantly
evolves to escape the immune response,
but it retains the elements needed to
mediate viral and cellular membrane
fusion. The genetic diversity of HIV-1 is
particularly striking in Env hypervariable
domains, which shield Env functional
domains. HIV-1 virions that encode Env
with long hypervariable domains and
additional potential glycosylation sites are
less sensitive to antibody neutralization.
Acquiring resistance to antibodies seems
to develop at the expense of transmissibility. HIV-1 species with long hypervariable domains appear less fit to establish
HIV infection in a new host, where the
founder virus selectively encodes compact
Env with fewer glycosylation sites. Taking
advantage of the opposing selective pressures exerted by the immune response and
transmission bottleneck is a promising
avenue for developing a safe and effective
prophylactic vaccine.
My research focuses on investigating
the viral determinants of HIV-1 transmission. We adapted our fluorescenceactivated cell sorting (FACS)-based fusion
assay to measure the fusion of 37 subtype
C Envs from the Lusaka cohort of newly
infected hosts and their transmitting partners. We found that natural variations in
the length of the V1/V2 hypervariable
domains exerted a profound effect on
HIV-1 entry into resting or activated
CD4 T cells and dendritic cells. HIV-1
species that encoded compact V1/V2
domains mediated fusion with higher efficiencies than related Envs that encoded
longer V1/V2 domains (see figure). By
exchanging the V1/V2 domains between
Envs of the same infected person or
between two persons linked by a transmission event, we further demonstrated
that V1/V2 domains critically influence
both Env incorporation into viral particles and fusion. Shortening the V1/
V2 domains consistently increased Env
incorporation and fusion, whereas lengthening the V1/V2 domains decreased Env
incorporation and fusion.
Relevance to HIV-1 Transmission
and Vaccine Development
Our study points to fusion and the
incorporation of Env into virions as limiting steps in the transmission of HIV-1
to a new host, and it suggests that V1/
V2 hypervariable domains inf luence
these steps. These V1/V2 domains are
frequently targeted by both natural
and vaccine-elicited antibodies. In the
RV144 Thai vaccine trial — the first
HIV-1 vaccine trial to show some protection with vaccines — the presence of V1/
V2-reactive antibodies correlated with
protection from infection. These V1/
V2 antibodies may have preferentially
restricted HIV-1 species that had a V1/
V2 conducive to high-level Env incorporation and high-level fusion (i.e., the
compact V1/V2 Env).
The Next Step Forward
The expression of trimeric Env spikes —
the functional form of Env — a ppears
to be a delicate step in the HIV-1 lifecycle. Compared with other glycoproteins, Env folding is very slow, and
cleavage of the signal peptide occurs
only when the protein is fully synthesized. N-glycosylation plays a key role in
protein folding and trafficking. In our
study, the number of potential glycosylation sites on V1/V2 inversely correlated
with Env incorporation into viral particles, suggesting that there is a threshold
of glycans that HIV Env can tolerate to
ensure proper folding and assembling of
the trimeric Env spike. Defining glycan
signatures necessary for proper folding of
Env will help us design better immunogens to elicit antibodies targeting these
highly fusiogenic Env species.
Publications
SELECTED RECENT
1. Cavrois M et al. Enhanced fusion
and virion incorporation for HIV-1
subtype C envelope glycoproteins
with compact V1/V2 domains.
J. Virol. (in press).
Fusion (Relative to 81A Control)
102
101
100
TOP FIVE OVERALL
10–1
10–2
1. Cavrois M et al. (1996) Adult
T-cell leukemia/lymphoma on a
background of clonally expanding
HTLV-1 positive cells. Blood
88:4646.
0
60
70
80
90
100
V1/V2 Domains Length (aa)
Natural variations in the amino acid length (aa)of V1/V2 domains significantly
affect HIV-1 fusion. A total of 37 full-length Envs were subcloned in an NL4-3-based
provirus to produce virions containing BlaM-Vpr, the marker of fusion. These virions
were then used to infect CD4 T cells of healthy donors. Laboratory-adapted 81A
virions were used as internal controls, allowing normalization across multiple viral
preparations and blood donors. Note the inverse correlation between the length of
V1/V2 hypervariable domains and fusion.
2. Cavrois M et al. (2000) Common
human T cell leukemia virus type 1
(HTLV-1) integration sites in
cerebrospinal fluid and blood
lymphocytes of patients with
HTLV-1-associated myelopathy/
tropical spastic paraparesis indicate
that HTLV-1 crosses the blood-brain
barrier via clonal HTLV-1-infected
cells. J. Infect. Dis. 182:1044.
3. Cavrois M et al. (2002) A sensitive
and specific enzyme-based assay
detecting HIV-1 virion fusion in
primary T lymphocytes. Nat. Biotechnol. 11:1151.
4.Cavrois M et al. (2006) HIV fusion
to dendritic cells declines as cells
mature. J. Virol. 80:1992.
5. Cavrois M et al. (2007) In vitro
derived dendritic cells trans-infect
CD4 T cells primarily with surface-bound HIV-1 virions. PLoS
Pathog. 3:e4.
“We’ve identified
potentially
therapeutic
compounds for AIDS
that target the host
instead of the virus.”
Gilad Doitsh, PhD
labs.gladstone.ucsf.edu/doitsh
HIGHLIGHTS
• Abortive HIV infection of CD4
T cells triggers pyroptosis, a highly
inflammatory form of programmed
cell death
• Pyroptosis in lymphoid tissues promotes chronic inflammation, driving
HIV pathogenesis
• Caspase-1 inhibitors may represent
a novel therapy that targets the
host, not the virus
LAB MEMBERS
Nicole Galloway
Xin Geng
Kathryn Monroe
Isa Muñoz Arias
Zhiyuan Yang
After more than 30 years of study, the mechanisms
behind how HIV actually kills immune cells remained
unknown until recently.
Helper CD4 T cells are critically
important for the body’s immune
response against foreign pathogens. As
dramatically demonstrated in patients
with acquired immunodeficiency syndrome (AIDS), a person lacking CD4
T cells cannot fend off many common
microbes that are normally harmless.
AIDS is caused by the human immunodeficiency virus (HIV), which infects
and kills CD4 T cells.
CD4 T-Cell Death in HIV
Despite vigorous research over three
decades, the precise mechanism underlying CD4 T-cell death in AIDS has
remained poorly understood. To investigate how CD4 T cells die during HIV
infection, we utilized a physiologically
relevant human lymphoid aggregate culture (HLAC) system formed with fresh
human tonsil or spleen tissue and analyzed
fresh lymph nodes from consenting HIVinfected subjects. Surprisingly, we found
that CD4 T cells are markedly depleted in
HLACs, even though only ~5% of the cells
become productively infected. Rather,
>95% of the dying cells are abortively
infected with HIV, and death is caused by
a cellular innate immune response directed
against the incomplete reverse DNA transcripts that accumulate in the cytoplasm
of these cells (see also Dr. Warner Greene’s
laboratory report on page 40). While this
cellular response is probably designed to
be protective, HIV subverts and amplifies
it so effectively that it becomes a central
driver of HIV pathogenesis.
CD4 T-Cell Death Induces
Pyroptosis
A second surprise was our discovery that
the mechanism of CD4 T-cell death
was not by “silent” caspase-3-mediated
apoptosis. Instead, the innate immune
response in these cells triggers the production of type I interferon and activates
caspase-1 in inflammasomes, which promote the release of proinf lammatory
interleukin (IL)-1β and induce pyroptosis. Pyroptosis is a highly inf lammatory form of programmed cell death
where the entire cytoplasmic content of
the dying cell is released into the extracellular space. Pyroptosis in HIV-infected
lymphoid tissues triggers a vicious pathogenic cycle, in which dying CD4 T cells
release inflammatory signals that attract
more cells into the infected lymphoid
tissue. These recruited cells ultimately die
and produce additional inflammation.
The release of intracellular 5ʹ-ATP by
pyroptotic CD4 T cells may induce activation of caspase-1 in nearby CD4 T cells,
resulting in an avalanche of new rounds
of pyroptosis in a virus-independent
such non-pathogenic infections, caspase-3
may signal for most of the cell death
instead of caspase-1, thus avoiding local
inflammation and preserving the integrity
of the gut epithelium.
The identification of pyroptosis provides novel targets, such as caspase-1, for
potential therapeutic intervention. CD4
T-cell depletion and inflammation are efficiently blocked by VX-765, a small-molecule inhibitor of caspase-1 that was safe
in human clinical trials. Thus, VX-765 or
related compounds may form a new, firstin-class “anti-AIDS” therapy that targets
the host instead of the virus in a way that
preserves CD4 T cells and reduces inflammation. Such therapeutics may be sufficient as a temporizing measure for infected
subjects who cannot access antiviral drugs.
They may also synergize with antivirals,
resulting in improved long-term clinical
outcomes including reduced early appearance of diseases associated with aging.
manner. This may be particularly relevant for patients on antiretrovirals where
dying cells might propagate new rounds
of pyroptosis independent of highlevel HIV production. Pyroptosis may
also promote increased production of
IL-7 and IL-15, leading to survival and
homeostatic proliferation of memory
CD4 T cells, which may contribute to
maintaining the latent HIV reservoir.
CD4 T-Cell Death Linked with
Chronic Inflammation
Depletion of CD4 T cells and development
of chronic inflammation are signature
processes in HIV pathogenesis. Our
findings suggest that these two diseasepromoting processes are interlinked.
Simian immunodeficiency virus (SIV)
infection of its natural non-human
primate hosts also causes massive depletion
of CD4 T cells and high viral loads but not
inflammation or immunodeficiency. In
E
GC
HIV-1
p24gag
PC
GC
PC
GC
GC
PC
PC
GC
GC
E
PC
E
E
Bioactive
IL-1β
MZ
GC
E
200 µm
PC
PC
1. Doitsh G et al. (2014) Cell death
by pyroptosis drives CD4 T-cell
depletion in HIV-1 infection. Nature
505:509.
2. Geng X et al. Efficient delivery
of lentiviral vectors into resting
human CD4 T cells. Gene Ther.
(in press).
3. Monroe KM et al. (2014) IFI16
DNA sensor is required for death
of lymphoid CD4 T cells abortively
infected with HIV. Science 343:428.
TOP FIVE OVERALL
1. Doitsh G et al. (2003) A long HBV
transcript encoding pX is inefficiently exported from the nucleus.
Virology 309:339.
2. Doitsh G et al. (2004) Enhancer I
predominance in hepatitis B virus
gene expression. Mol. Cell. Biol.
24:1799.
E
Active
Caspase-1
SELECTED RECENT
E
PC
Active
Caspase-3
Publications
300 µm
Distinct regions of caspase-1 and caspase-3 activity in the lymph node of a chronically infected, untreated HIV patient. Inguinal lymph nodes were collected from a
50-year-old immunosuppressed HIV-1-infected subject during the chronic phase of
disease. Patient was identified with HIV in 1985 and displayed a CD4 count of 156/
μl and a viral load of 85,756 copies/ml at the time of lymph node resection. Note
the positive staining for caspase-1 and IL-1β in the paracortical zone (PC), while
caspase 3 and HIV-1 p24 gag are present in the germinal center (GC). MZ, mantle
zone; E, epithelium.
3. Doitsh G et al. (2010) Abortive
HIV infection mediates CD4
T-cell depletion and inflammation
in human lymphoid tissue. Cell
143:789.
4.Doitsh G et al. (2014) Cell death
505:509.
Robert M. Grant, MD, MPH
“The iPrEx study
provided proof of
concept that HIV
transmission can be
safely prevented by
daily medication.”
Senior Investigator
labs.gladstone.ucsf.edu/grant
HIGHLIGHTS
• PrEP is safe and effective for HIV
prevention
• Drug concentrations in blood are
highly predictive of the clinical
benefit of PrEP
• Ineffective use of PrEP typically
does not select for drug-resistant
HIV-1 strains
LAB MEMBERS
Ariceli Alfaro
Lloyd Bentley
Patricia Defechereux
Deirdre Devine
Christopher Eden
Pedro Goicochea
Robert Hance
Kim Koester
Jeanny Lee
Teri Liegler
Julia Marcus
James McConnell
Vanessa McMahan
Megha Mehrotra
Mohamed Moshen
Marc Solomon
The AIDS pandemic continues, particularly in the
developing world. Without an effective vaccine, other
methods of stopping HIV transmission are critical.
The overall mission of my 30-year career
in research is to find ways to end the
plague of human immunodeficiency virus
(HIV) and acquired immunodeficiency
syndrome (AIDS). AIDS was discovered
just before I started graduate studies in
Berkeley. The fear, grief, and urgency to
find solutions motivated me to develop
the skills and knowledge of science to
contribute to the end of HIV/AIDS.
Stopping Infection After the Fact
My research over the past 10 years has
focused on finding and evaluating the
value of an HIV prevention pill, to be
taken by people potentially exposed to
HIV infection. We initially focused on
prevention pills that could be taken after
exposure, a concept called post-exposure
prophylaxis, or PEP. PEP is used in some
urban centers, although uptake is limited
by how quickly people must recognize they
have been exposed. People must take action
within hours in order to obtain a prescription, and they must start the medication
within 72 hours. Furthermore, the effectiveness of PEP was never rigorously evaluated, leaving some doubt about whether
the effort to use PEP is worthwhile.
Stopping Infection in Advance
The concept of pre-exposure prophylaxis,
or PrEP, addressed the limitations of PEP.
Our goal was to identify highly effective
strategies that would engage people on a
day-to-day basis in planning for better
sexual health; to help people create daily
habits of remembering HIV in calm
moments when plans can be made.
Our PrEP trials included studies in
Asia, Africa, South America, and the
United States. The largest and most pivotal trial is called Global iPrEx; 2499 gay
men and transgender women were
enrolled at 11 sites in six countries on four
continents. These brave and committed
people were offered all the prevention services we were able to provide, including
HIV testing, counseling, condoms, community-building programs, management
of sexually transmitted infections, and a
chance to take the prevention pill (a combination of two antiviral drugs called
emtricitabine and tenofovir disoproxil
fumarate, sold as Truvada™ in the United
States and available in generic forms
throughout Africa). Overall, HIV infection rates dropped by 42% among those
offered PrEP; this benefit was in addition
to the benefits of intensively offering all
other known prevention interventions.
Among those who took the pill daily,
as recommended, HIV infection rates
dropped by more than 99%. It worked!
Our PrEP trials also showed that the
medications were safer than we thought.
Previously used only for treating existing
HIV infections, which are associated
with a wide range of ailments, the medications were perceived as being linked
to numerous concerns. In our studies of
PrEP, the majority of our uninfected participants reported no adverse effects at all.
Fewer than 1 in 10 noted some nausea in
the first few weeks, which resolved after the
first month. Overall, the HIV prevention
pill was remarkably safe, comparable to
medicines routinely used by healthy people
to prevent pregnancy and heart attacks.
What’s next?
Based in part on our research, the
US Food and Drug Administration
approved the preventative use of this
medicine, and the Centers for Disease
Control have issued guida nce for
its use. Where PrEP is available, its
demand is growing. Our research focus
has now turned to key related questions. Can the pill be used before and
after exposure rather than daily? What
is the best way to inform people about
how to protect themselves from HIV
infection? What is the best way to integrate PrEP programs into research and
practices aimed at providing treatment,
or even a cure, for HIV? There is still
much to do.
Estimated Incidence by Drug Concentration
STRAND Doses*: 2/Week
4/Week 7/Week
6
HIV Incidence/100 P-Y (iPrEx)
5
Placebo
Publications
SELECTED RECENT
1. Anderson PL et al. (2012) Emtricitabine-tenofovir concentrations and
pre-exposure prophylaxis efficacy
in men who have sex with men. Sci.
Transl. Med. 4:151ra125.
2. Baeten JM et al. (2013) Use of
antiretrovirals for HIV prevention:
what do we know and what don’t
we know? Curr. HIV/AIDS Rep.
10:142.
3. Grant RM (2013) The new revolution: HIV prevention in a pill.
Positively Aware 25:2.
4.Marcus JL et al. (2013) No evidence
of sexual risk compensation in
the iPrEx trial of daily oral HIV
preexposure prophylaxis. PLoS One
8:e81997.
5. Tangmunkongvorakul A (2013)
Facilitators and barriers to medication adherence in an HIV prevention study among men who have
sex with men in the iPrEx study in
Chiang Mai, Thailand. AIDS Care
25:961.
4
TOP FIVE OVERALL
3
1. Blower SM et al. (2000) A tale of
two futures: HIV and antiretroviral
therapy in San Francisco. Science
287:650.
2
1
TFV-DP
0
BLQ
10
20
30
TFV-DP Level (fmol/M)
40
50
Daily use of PrEP is recommended, but actual use varies. In the iPrEx study, the
rate of HIV infection was reduced 99% among people whose blood tests showed
that PrEP was used daily, 96% if PrEP was used four times per week, and 76%
if PrEP was used two times per week. Drug resistance did not occur in persons
infected with HIV while trying to use PrEP, and sexual behavior tended to be safer.
*Concentration achieved after 6 weeks (interquantile range). BLQ, below limits
of quantification; P-Y, person-years; TFV-DP, intracellular tenofovir-diphosphate.
Anderson et al. (2012) Sci. Trans. Med. 4:151ra125.
2. Deeks SG et al. (2001) Virologic
and immunologic consequences of
discontinuing combination antiretroviral-drug therapy in HIV-infected patients with detectable
viremia. N. Engl. J. Med. 344:472.
3. Grant RM et al. (1987) The infectivity of the human immunodeficiency virus: estimates from a
cohort of homosexual men. J. Infect.
Dis. 156:189.
4.Grant RM et al. (2002) Time trends
in primary HIV-1 drug resistance
among persons recently infected
in San Francisco. J. Am. Med. Assoc.
288:181.
5. Grant RM et al. (2010) Preexposure
chemoprophylaxis for HIV prevention in men who have sex with men.
N. Engl. J. Med. 363:2587.
“AIDS is
characterized by CD4
T-cell depletion that
is driven by cellular
suicide rather than
viral murder.”
labs.gladstone.ucsf.edu/greene
HIGHLIGHTS
• CD4 T-cell death during abortive
HIV infection involves viral DNAactivated pyroptosis
• Blood-derived CD4 T cells do not
die by pyroptosis: they lack the IFI16
DNA sensor
• Pyroptosis requires cell-to-cell
transmission of the virus; cell-free
virions do not induce this key pathogenic response
LAB MEMBERS
Joseph Callahan
Jonathan Chan
Holli Deval
Gilad Doitsh
Nicole Galloway
Xin Geng
Nargis Kohgadai
Jeremy Martin
Kathyrn Monroe
Mauricio Montano
Isa Muñoz Arias
Jason Neidleman
Nadia Roan
Debbie Ruelas
Stefanie Sowinski
Katsunari Tateiwa
Brian Webster
Zhiyuan Yang
Orlando Zepeda
Although HIV has been studied for nearly 30 years,
how it actually promotes CD4 T-cell loss remains
poorly understood.
Acquired immunodeficiency syndrome
(AIDS) is primarily caused by the progressive depletion of the numerous quiescent
“bystander” CD4 T cells that populate
lymphoid organs. These resting cells are
not used for viral replication. Instead, they
become abortively infected, resulting in
the accumulation of incomplete viral DNA
transcripts in the cytosol. These viral DNAs
are detected by a previously unidentified
DNA sensor triggering an innate immune
response that culminates in caspase-1-dependent pyroptosis, a highly inflammatory form of programmed cell death (see
the laboratory report by Dr. Gilad Doitsh
on page 36 for further details).
Identification of a Long Elusive
Single-Strand DNA Sensor
In collaboration with Dr. Nevan Krogan
and colleagues at the University of
California, San Francisco (UCSF), Drs.
Kathryn Monroe and Zhiyuan Yang in
my laboratory identified interferon-inducible protein 16 (IFI16) as the DNA
sensor that triggers the pyroptotic death
response to human immunodeficiency
virus (HIV). IFI16 recognizes both singleand double-stranded DNA and forms its
own inflammasomes. The IFI16 inflammasome recruits and activates caspase-1,
leading to the processing and release of
the proinflammatory cytokine interleukin
(IL)-1β and to the induction of pyroptosis.
These events provide an unexpected link
between two signature pathogenic processes in HIV infection — CD4 T-cell
depletion and chronic inflammation.
Blood and Tissue Have Striking
Biological Differences
Unexpectedly, resting blood CD4
T cells appear to be highly resistant to
pyroptosis. However, these cells are routinely used in >99% of in vitro HIV
experiments requiring primary cells.
Our findings highlight a fundamental
difference between blood and tissue
CD4 T cells that has important pathogenic implications. Isa Muñoz Arias, a
UC Berkeley graduate student in my laboratory, showed that blood CD4 T cells
resist pyroptosis, at least in part because
they lack expression of the IFI16 DNA
sensor. Even though abortive infection
occurs, the cytosolic viral DNA is never
sensed, and thus, the death response is
short-circuited. However, blood cells
can be rendered sensitive to pyroptosis
by co-culture with lymphoid tissue cells,
leading to a low-level activation sufficient
to induce IFI16 and possibly other key
factors. These studies emphasize how
AIDS is a disease of lymphoid tissues
and not blood. They also highlight a
“Goldilocks effect” where just the right
state of cellular activation is required for
CD4 T-cell pyroptosis — too much activation and the cell becomes productively
infected with virus, and with too little
activation the cells are entirely death
resistant. Optimized conditions for abortive infection and pyroptosis are present
in many lymphoid tissues, such as lymph
nodes, spleen, and tonsils, while gut-associated lymphoid tissue may be more
activated and support higher levels of
productive HIV infection. These productively infected cells ultimately die of
caspase-3-dependent apoptosis, a silent
and non-inf lammatory form of programmed cell death.
Importance of Cell-to-Cell
Transmission of HIV
Studies by Nicole Byers Galloway, a
UCSF Biomedical Sciences (BMS) graduate student working with Dr. Doitsh and
me, highlighted how pyroptosis requires
cell-to-cell transmission of the virus. Cellfree virions can establish productive viral
infections in activated CD4 T cells and
induce apoptosis, but these free viruses
cannot induce pyroptosis. Ms. Galloway
found that the virological synapse formed
by interactions of LFA1 (lymphocyte
function-associated antigen 1) and
ICAM1 (intercellular adhesion molecule
1) is important for inducing pyroptosis.
Disruption of this synapse with blocking
antibodies shuts down the pyroptotic
death response. Cell-to-cell transmission
of HIV is 10–100-fold more efficient than
transmission by cell-free virions. Highly
efficient transfer of virions may overwhelm
intrinsic defenses within the resting CD4
T cell and lead to the accumulation of sufficient viral DNA so that sensing occurs
and pyroptosis is activated. These findings
emphasize that virus-infected cells rather
than cell-free virions are the principal
“killing units” in HIV infection.
Publications
SELECTED RECENT
1. Doitsh G et al. (2014) Cell death
505:509.
2. Greene WC (2013) Solutions to
global health challenges: no more
Band-Aids. J. Clin. Invest. 123:4967.
4.Ruelas DS et al. (2013) An integrated overview of HIV-1 latency.
Cell 155:519.
5. Webster B et al. (2013) Evasion
of superinfection exclusion and
elimination of primary viral RNA by
an adapted strain of HCV. J. Virol.
87:13354.
HIV
TOP FIVE OVERALL
Resting CD4 T Cell
1. Chen LF et al. (2001) Duration of
nuclear NF-κB action regulated
by reversible acetylation. Science
293:1653.
Abortive
HIV Infection
IFI16
Caspase-1
Pyroptosis
IFNβ
Abortive HIV infection in resting lymphoid CD4 T cells leads to accumulation of
cytosolic DNA that is detected by the host IFI16 DNA sensor. IFI16 induces two
innate signaling pathways, one leading to production of interferon (IFN)-β and the
other to inflammasome formation, caspase-1 activation, and pyroptosis, a highly
inflammatory form of programmed cell death.
2. Doitsh G et al. (2010) Abortive
HIV infection mediates CD4
T cell depletion and inflammation
in human lymphoid tissue. Cell
143:789.
3. Leonard WJ et al. (1982) A monoclonal antibody that appears to
recognize the receptor for human
T cell growth factor partial characterization of the receptor. Nature
300:267.
4.Leonard WJ et al. (1984) Molecular cloning and expression of
cDNAs for the human interleukin-2
receptor. Nature 311:626.
5. Sun SC et al. (1993) NF-κB controls
expression of inhibitor IκBα: evidence for an inducible autoregulatory pathway. Science 259:1912.
“My research bridges
the gap between
systems biology
and more detailed,
mechanistic studies.”
Senior Investigator
labs.gladstone.ucsf.edu/krogan-givi
HIGHLIGHTS
• Our systematic and unbiased
analyses identify host proteins that
physically interact with all HIV-1
proteins
• We identified the new Vif co-factor
CBFβ
• A Vif-dependent dual hijack mechanism affects both host ubiquitination and transcription regulation
LAB MEMBERS
Stefan Bohn
Hannes Braberg
Si-Han Chen
Manon Eckhardt
Kathy Franks-Skiba
David Gordon
Ruth Huettenhain
Jeffrey Johnson
Tasha Johnson
Joshua Kane
Billy Newton
Assen Roguev
Priya Shah
Erik Verschueren
Ariane Watson
Jason Wojcechowskyj
Jiewei Xu
Our research involves using large-scale quantitative
genetic maps that show physical interactions to comprehensively study biological processes.
While many groups generate largescale biological datasets, extracting
deep insights from these data remains a
major challenge. By focusing powerful
genetic and mass spectrometry methods
on comprehensively defining proteinprotein and genetic interaction networks, we have discovered a wealth of
new functions, complexes, and networks
in a variety of organisms.
Focus on HIV-Human Interactions
Since pathogens normally have a limited
protein-coding capacity, they rely heavily
on the host’s cellular machinery. As such,
they target key nodes in virtually every
biological process. Therefore, the use of
pathogenic organisms as systems tools
allows us to discover important aspects of
mammalian biology. To this end, we used
affinity tag-purification mass spectrometry
(AP-MS) to systematically characterize the
human proteins that are physically associated with human immunodeficiency
virus (HIV)-1 proteins. We found 497
high-confidence HIV-human interactions
involving 435 human proteins and 16 viral
proteins. Remarkably, only 19 of these
interactions had been reported previously.
This work has established a fresh perspective on the HIV-human interactome and
has opened many new and exciting avenues of research relating to HIV biology.
We have been focusing our efforts
on the HIV protein Vif, which counteracts the host’s antiviral defenses by
hijacking a CUL5-containing ubiquitin
ligase complex and targeting for degradation the deaminase APOBEC3G,
which otherwise blocks viral replication.
Even though Vif is well studied (>1000
papers published) and represents a valuable drug target, the exact composition of
this ligase remains unknown. No group
has yet been able to reconstitute the complex, despite efforts in many laboratories
over several years. Through our proteomic studies, we identified one protein,
the transcription factor CBFβ, as a novel
component that Vif recruits to the ubiquitin ligase complex. In collaboration
with Dr. John Gross at the University of
California, San Francisco (UCSF), we
showed that CBFβ enables the reconstitution of the active ubiquitin ligase
complex — which is now being structurally characterized — that can degrade
APOBEC3G and preserve HIV-1 infectivity. Furthermore, we have uncovered
a dual hijack mechanism in which Vif
also impinges on the transcriptional regulation of CBFβ and its host-interacting
partner RUNX1 in a fashion that ultimately benefits viral replication. We are
now carrying out an evolutionary analysis
to further elucidate the functions of Vif
understand functionally the interactions
uncovered by the proteomic approaches.
by targeting this factor in several other
lentiviruses, both primate and non-primate. Disrupting the CBFβ-Vif interaction might restrict HIV-1 and provide a
supplement to current antiviral therapies
that primarily target viral proteins.
Furthermore, in collaboration with
Drs. Eric Verdin and Warner Greene
(both at Gladstone), we are analyzing
global changes in the host’s post-translational modifications in an HIVdependent manner, focusing our attention
on phosphorylation and ubiquitination.
Integration of this information with the
protein-protein interaction data permits a
deeper understanding of the pathways that
are perturbed in infected host cells. Finally,
we recently developed a genetic interaction mapping platform for mammalian
cells so that we can create an HIV-host
genetic interaction map, which will help us
Cross-Pathogen Analysis
Systems
We are also using these genetic and proteomic platforms to characterize several
other pathogenic organisms and determine how they infect their hosts. We are
focusing on hepatitis C in collaboration
with Dr. Melanie Ott (Gladstone), as
well as herpes, the flu, and Mycobacterium
tuberculosis in collaboration with Dr. Jeff
Cox (UCSF). Interestingly, we are finding
common nodes targeted by multiple
pathogens, and we are now focusing our
mechanistic studies on these complexes.
Ultimately, this cross-pathogen approach
will provide even greater insight into the
host pathways that are being routinely
hijacked and rewired, providing a clearer
vision for future therapeutic interventions.
VIF
Virology
CUL5
CUL5/RBX2/
Vif/CBFß/
28–
ELOBC
3
Mechanism
40
50
60
70
Vif
CBFß
ELOB
RBX2
ELOC
10–
1
80 90ml
2
3
Percent Infectivity
kDa
CUL5/RBX2 95–
12
25
ELOB
ELOC
20
15
10
CBFß
RUNX
Gene Expression
Vif
CBFβ
55 Å
0
3
5
7 10 12 14
Days
150 Å
CBFß
Vif
Vif
5
0
CBFβ
ELOC
ELOB
Vif
A3G
CUL5
Ub
Ub
Ub
E2
RBX2
1. Fraser J et al. (2013) From systems
to structure: bridging networks and
mechanism. Mol. Cell 49:222.
2. Jager S et al. (2012) Global landscape of HIV-human protein complexes. Nature 481:365.
3. Jager S et al. (2012) Vif hijacks
CBFβ to degrade APOBEC3G and
promote HIV-1 infection. Nature
481:371.
4.Kim DY et al. (2013) CBFβ stabilizes HIV Vif to counteract
APOBEC3 at the expense of RUNX1
target gene expression. Mol. Cell
49:632.
5. Roguev A et al. (2013) Quantitative genetic-interaction mapping
in mammalian cells. Nat. Meth.
10:432.
1. Beltrao P et al. (2010) Quantitative
genetic interactions reveal layers of
biological modularity. Cell 141:739.
Structure
Non-Silencing shRNA
CBFβ-specific shRNA
SELECTED RECENT
TOP FIVE OVERALL
CBFβ
Biochemistry
Publications
Ub
Ubiquitin Ligation
Systematic affinity purification-mass spectrometry (AP-MS) was carried out to identify
HIV-human protein-protein interactions (top). Using biochemical, genetic, and structural analyses, the interaction between Vif and CBFβ was characterized in more depth
(middle), leading to a dual hijack model where Vif impinges on host pathways involving
both ubiquitination and transcriptional regulation (bottom).
2. Collins SR et al. (2007) Functional
dissection of protein complexes
involved in yeast chromosome
biology using a genetic interaction
map. Nature 446:806.
3. Keogh MC et al. (2005) Cotranscriptional set2 methylation of
histone H3 lysine 36 recruits a
repressive Rpd3 complex. Cell
123:593.
4.Krogan NJ et al. (2006) Global
landscape of protein complexes in
the yeast Saccharomyces cerevisiae.
Nature 440:637.
5. Nagai S et al. (2008) Functional targeting of DNA damage
to a nuclear pore-associated
SUMO-dependent ubiquitin ligase.
Science 322:597.
“Novel mechanisms of
maintaining latency
identify virusspecific strategies
for reactivation.”
JJ Miranda, PhD
Assistant Investigator
labs.gladstone.ucsf.edu/miranda
HIGHLIGHTS
• Viral genomes associate with
human chromosomes in threedimensional space
• Unlike latent EBV genes, transcribed
lytic genes contain chromatin
without activated nucleosomes
• Small-molecule activation of key
signaling pathways selectively
reactivates EBV or HIV
LAB MEMBERS
Samantha Fernandez
Kristin Keck
Stephanie Liu
Stephanie Moquin
An Phan
Our team studies molecular determinants that control
the ease or difficulty with which a latent virus emerges
into active replication.
We have recently begun to compare the
molecular determinants of viral reactivation in the Epstein-Barr virus (EBV) and
human immunodeficiency virus (HIV).
EBV adopts a quiescent episomal state
during latency that involves transcription
of a few important genes. HIV integrates
into the human genome and suppresses
transcription in a manner that depends
on the chromosomal context. Latency
models of each virus have distinct technical and biological advantages and disadvantages. By leveraging the strengths
of one, we can design newly feasible and
novel experiments in the other.
Viral Genome Organization
The human genome is not a linear
sequence, but rather a contorted knot of
chromosomes in the three-dimensional
space of the nucleus. Chromosomes do
not work in isolation; long-range interactions are prevalent and interchromosomal
associations regulate gene expression. This
discovery has implications for viral infections. Pathogens have long been studied
for their ability to hijack host functions.
Viruses in particular have revealed much
about cell biology. But we still talk of
host and pathogen genomes as if they are
independent. If gene regulation occurs
between chromosomes, can it also occur
between genomes? I contend that host
and pathogen genomes are not separate,
but rather an intertwined entity we refer
to as a “hosthogen” genome.
The innovation of this idea relates to
distance. We understand how EBV and
HIV promoters regulate gene expression.
We also have good ideas about how the
chromatin structure of the viral genome
affects reactivation. But we have been
near-sighted in our focus on local regulatory elements. No one has yet asked,
“Does the virus hijack human genome
organization to regulate viral gene expression? Does distal DNA affect transcription? Are there regulatory elements on
other chromosomes?”
We used cutting-edge technology to
identify long-range interactions between
host and pathogen genomes in the
three-dimensional space of the nucleus.
Initial pilot experiments began with EBV
latency. We trapped chromatin, prepared
DNA for deep sequencing, and analyzed the data with bioinformatic tools
developed in-house. We learned that a
repressed gene of the EBV genome associates with repressed genes of the human
genome in three-dimensional space. The
spatial co-localization between host and
pathogen genes raises many fundamental
questions we hope to explore. Does colocalization specify co-regulation? Is this
organization disrupted upon reactivation?
Can perturbation of this organization
induce reactivation?
Our success with EBV-human inter
genome bridges encouraged us to study
HIV genome organization. Beginning
with latency, we will attempt to identify
long-range, possibly interchromosomal
interactions between HIV and human
genes. We will also examine local changes
in chromatin structure and quantitative
measurements of gene expression during
reactivation of both EBV and HIV.
Small-Molecule Reactivation
Many small-molecule drugs have been
used in laboratories and clinics to induce
reactivation of both EBV and HIV.
Histone deacetylase inhibitors, such as
SAHA and butyrate, are perhaps the best
known. Other classes of molecules also
disrupt the latency of either virus, but
3' LMP2A/B
EBER1 and
EBER2
EBNA Cp
their specificities have not been systemically studied. Specificity is of particular
concern for clinical use during coinfection. For example, lytic induction
therapy of EBV-driven cancer in AIDS
survivors must take care not to raise the
HIV viral load. Attempts to purge the
HIV reservoir must similarly avoid a
storm of reawakened herpesviruses.
By perturbing cell-culture models
of latency, we identified classes of compounds that specifically reactivate only
EBV or HIV. For example, Brd4 bromodomain inhibitors selectively induce HIV
replication. DNA-damaging agents and
glucocorticoid receptor agonists selectively induce EBV replication. We hope
to expand these studies to intelligently
inform translational experiments that
aim to improve the specificity of therapies that target latent viral reservoirs.
3' EBNA1/2/ BMRF1/2/
3A/3B/3C/LP BaRF1
BLLF1
BHLF1
BHRF1
Raji
Counts per
Million Reads
40
10
Counts per
Million Reads
Daudi
3. Martinez SR et al. (2010) CTCF
terminal segments are unstructured. Protein Sci. 19:1110.
TOP FIVE OVERALL
0
1. Miranda JJ (2000) Highly reactive cysteine residues in rodent
hemoglobins. Biochem. Biophys. Res.
Commun. 275:517.
30
20
10
Counts per
Million Reads
KemIII
2. Holdorf MM et al. (2010) Occupancy of chromatin organizers in
the Epstein-Barr virus genome.
Virology 415:1.
20
0
2. Miranda JJ (2003) Position-dependent interactions between cysteine
residues and the helix dipole. Protein Sci. 12:73.
30
20
10
0
Counts per
Million Reads
40
MutuI
1. Campbell AE et al. (2010)
Molecular architecture of CTCFL.
Biochem. Biophys. Res. Commun.
396:648.
30
40
3. Miranda JJ et al. (2005) Thermoglobin, oxygen-avid hemoglobin in
a bacterial hyperthermophile.
J. Biol. Chem. 280:36754.
30
20
10
0
4.Miranda JJ et al. (2005) The yeast
DASH complex forms closed rings
on microtubules. Nat. Struct. Mol.
Biol. 12:138.
Counts per
Million Reads
40
KemI
SELECTED RECENT
3' RPMS1/A73/
3' EBNA1
BARF0/5' BALF3
BKRF2/3/4
LMP1
BDLF1/2/3
5' RPMS1
LF3
40
30
20
10
0
5. Miranda JJ et al. (2007) Protein
arms in the kinetochore-microtubule interface of the yeast DASH
complex. Mol. Biol. Cell 18:2503.
Counts per
Million Reads
40
RaeI
Publications
30
20
10
0
0
30
60
90
Position (kbp)
120
150
180
RNA deep sequencing profiles of EBV transcriptomes expressed by different
latency programs. Each cell line provides subtly different gene regulatory programs
that may be directly compared with each other for changes in genome organization
and chromatin structure.
Melanie Ott, MD, PhD
Senior Investigator
labs.gladstone.ucsf.edu/ott
HIGHLIGHTS
• Paused RNA polymerase II is highly
acetylated within its CTD
• Lysines in the CTD cannot be
acetylated if neighboring serines are
phosphorylated and vice versa
• Transcription is regulated differently in simple and more complex
organisms
LAB MEMBERS
Dorothee Alatorre
Ibraheem Ali
Nina Baur
Daniela Boehm
Gregory Camus
Ryan Conrad
Mingjian Fei
Abigail Hintermeister
Mark Jeng
Andrew Kondratowicz
Gagandeep Kumar
Pao-Chen Li
Juan Martinez
Ankit Modi
Holly Ramage
Sebastian SchrÖder
William Tatlonghari
Chia-Lin Tsou
“We identified a new
signal that enables
the successful
transition from
transcription
initiation to
elongation.”
RNA polymerase II is the central enzyme that transcribes genetic information from DNA into RNA, a
key step in deciphering the genetic code.
Transcription initiates when the RNA
polymerase II complex assembles at the
transcription start site and the first few
base pairs of a gene are transcribed. In
complex organisms such as humans and
mice, the initiated polymerase complex
often pauses immediately downstream
of the transcription start site, awaiting
further signals to properly enter into the
elongation phase in which the bulk of
the genetic information is transcribed.
Polymerase pausing was initially described
for heat shock genes in fruit flies and for
the human immunodeficiency virus
(HIV) provirus, but has now emerged
as a central regulatory step in the coordinated expression of many mammalian
genes (i.e., during activation of immune
cells or differentiation of stem cells).
New Signal for Polymerase Pausing
We identified a new signal that is critical
for polymerase pausing and the successful
transitioning from transcription initiation to elongation. This signal comes in
the form of a new modification that is
added to the C-terminal domain (CTD)
of the largest polymerase subunit immediately after transcription is initiated. The
CTD is a highly repetitive stretch of seven
amino-acid repeats (heptad repeats) that
undergo a coordinated series of phosphorylation events during transcription.
We found eight heptad repeats in the
mammalian CTD that contain lysines
and are acetylated. Using biochemical
acetylation assays and RNA interference
(RNAi)-based knockdown approaches,
we demonstrated that the acetyltransferase
p300/KAT3B is a critical CTD acetyltransferase. Using mass spectrometry, we
further showed that p300/KAT3B cannot
acetylate heptad repeats that are phosphorylated and vice versa, demonstrating that
phosphorylation and acetylation events
within the CTD are spatially separated.
CTD Acetylation of Polymerase
Affects Pausing
A fter the successful generation of
acetylation-specific CTD antibodies,
we determined the genome-wide occupancy of acetylated polymerases in
mouse embryonic stem cells (in collaboration with the Bruneau laboratory at
Gladstone). These studies showed that
acetylation is a frequent polymerase
modification. It peaks at many genes
immediately downstream of the transcription start site where polymerases
pause or enter early elongation. To
study the functional relevance of this
observation, we generated mouse fibroblast cell lines stably expressing wildtype or mutated RNA polymerase II
where all lysines in the CTD were
replaced by arginines (8KR). Wild-type
and mutated constructs also carried
a single point mutation that rendered
the enzymes resistant to the cell toxin
α-amanitin, thus allowing the overexpression of the proteins in cells treated
with α-amanitin when endogenous
RNA polymerase II is degraded.
We tested the transcription of immediate-early genes regulated by polymerase
pausing in these cell lines and found that
the induction of paused gene expression
in response to epidermal growth factor
(EGF) treatment was completely lost in
8KR cells. No such changes were observed
at non-paused housekeeping genes. In
chromatin immunoprecipitation experiments, mutated polymerases after EGF
stimulation did not elongate properly at
paused genes. In addition, we observed a
decrease in polymerase signal at the pause
site itself. These results support the model
that CTD acetylation has a critical regulatory role during the release and possibly
also during the establishment of polymerase pausing, underscoring that both
processes are functionally linked.
Evolution of CTD Acetylation
An interesting aspect of CTD acetylation is that lysine residues in the CTD
are not present in yeast and are unique
to higher eukaryotes. In collaboration
with the Pollard laboratory at Gladstone,
we examined the evolution of lysinecontaining repeats across eukaryotes with
sequenced CTDs. Collectively, our studies
point to unique differences in transcription
regulation between simple and more complex organisms and illustrate how multicellular organisms, such as humans, adapted
to paracrine signaling to allow rapid and
coordinated gene expression during development and cell differentiation.
RPB1 CTD
Publications
SELECTED RECENT
1. Boehm D et al. (2013) BET bromodomain-targeting compounds
reactivate HIV from latency via a
Tat-independent mechanism. Cell
Cycle 12:452.
2. Camus G et al. (2013) Diacylglycerol acyltransferase-1 localizes hepatitis C virus NS5A protein to lipid
droplets and enhances interaction
of NS5A with the viral capsid core.
J. Biol. Chem. 288:9915.
3. Kwon HS et al. (2012) Three novel
acetylation sites in the Foxp3
transcription factor regulate the
suppressive activity of regulatory
T cells. J. Immunol. 188:2712.
4.Schröder S et al. (2013) Acetylation of
RNA polymerase II regulates growth
factor–induced gene transcription in
mammalian cells. Mol. Cell 52:314.
5. Vogt D et al. (2013) Lipid droplet-binding protein TIP47 regulates
hepatitis C Virus RNA replication through interaction with the
viral NS5A protein. PLoS Pathog.
9:e100332.
TOP FIVE OVERALL
P
P
Ac
Ac
YSPTSPS YSPTSPS YSPTSPS YSPTSPK YSPTSPK
1. Herker E et al. (2010) Efficient
hepatitis C virus particle formation
requires diacylglycerol acyltransferase-1. Nat. Med. 16:1295.
2. Kaehlcke K et al. (2003)
Acetylation of Tat defines a
cyclinT1-independent step in HIV
transactivation. Mol. Cell 12:167.
HDACs
p300
YSPTSPS YSPTSPS YSPTSPS YSPTSPK YSPTSPK
P
P
P
P
P
P
P
P
The mammalian CTD for RNA polymerase I (RPBI) contains 52 heptad repeats,
including eight (in red) in the distal CTD that contain a lysine at position 7 (K7).
These lysines are acetylated by p300, which prevents phosphorylation of neighboring serine residues and vice versa, and establishes a balance of phosphorylation
and acetylation within the mammalian CTD. HDAC, histone deacetylase.
3. Kwon HS et al. (2008) Human
immunodeficiency virus type 1 Tat
protein inhibits the SIRT1 deacetylase and induces T-cell hyperactivation. Cell Host Microb. 3:158.
4.Ott M et al. (1997) Immune hyperactivation of HIV-1-infected T cells
mediated by Tat and the CD28
pathway. Science 275:1481.
5. Schröder S et al. (2013) Acetylation
of RNA polymerase II regulates
growth factor–induced gene transcription in mammalian cells. Mol.
Cell 52:314.
“Understanding how
CD8 T-cell priming
occurs in mucosal
tissues holds the
key to developing
an effective HIV
vaccine.”
Shomyseh Sanjabi, PhD
labs.gladstone.ucsf.edu/sanjabi
HIGHLIGHTS
• Mucosal LCMV infection in mice
demonstrates a delayed and dampened CTL response
• Suboptimal innate immune activation may be responsible for the
delayed mucosal CTL response
• Low inflammation during mucosal
CD8 T-cell priming dampens the
CTL response
LAB MEMBERS
Timothy Borbet
Benjamin Cohn
Emily Deal
Ashley Hughey
Shahzada Khan
Julie Luong
Joey Pham
For an HIV-free world, we must embrace the challenge
of functionally curing infected individuals and generating an effective preventative HIV vaccine.
To achieve these goals, we must better
define the mechanisms and factors that
govern the innate and adaptive immune
responses in the female reproductive
tract and the rectum, the portals of viral
entry during sexual viral transmission.
Protective immunity requires pathogen-specific neutralizing antibodies and
resident memory CD8+ cytotoxic T lymphocytes (CTLs) in mucosal tissues to
immediately clear infected cells before the
virus systemically disseminates.
In non-human primates, the natural CTL response is “too little and too
late” to prevent systemic infection by
simian immunodeficiency virus (SIV).
Although the exact mechanism of this
delayed and dampened antiviral immune
response is unknown, vaccines that
induce mucosal responses — in particular,
strong CTL responses at the portal of viral
entry — control viral replication and limit
systemic dissemination. Thus, the goal
of a protective vaccine in humans is to
induce an immune response stronger than
that elicited against natural infections.
Elite Controllers
A small number of human immunodeficiency virus (HIV)-infected individuals
called “elite controllers” maintain low
levels of HIV RNA and high CD4+ T-cell
counts without antiretroviral therapy.
Genome-wide association studies have
correlated their immune response with
certain human leukocyte antigen (HLA)
class I alleles. As HLA class I molecules
activate CD8 T cells by presenting viral
peptides, the mechanisms governing elite
HIV control are likely mediated at the level
of antigen presentation and recognition
by the immune system. In support of this,
elite controllers have a more polyfunctional
repertoire of CTLs specific for early viral
antigens than other infected individuals.
Understanding how virus-specific CD8
T cells are primed and activated in the
mucosal tissues of these individuals will
inform the knowledge needed to develop
a vaccine that can elicit a similar immune
response in individuals without the protective genetic background. To this end, we
are generating small-animal models that
recapitulate the immune systems of the
elite controllers and non-controllers. We
hope to achieve this by generating human
induced pluripotent stem (hiPS) cells from
well-characterized HIV elite controllers
and non-controllers and differentiating the
hiPS cells into CD34+ hematopoietic precursor cells in vitro. The resulting cells will
be engrafted into a new humanized mouse
model, in which several mouse genes have
been replaced with their human counterparts to promote human hematopoietic
stem cell survival and differentiation. We
Eliciting protective immunity in mucosal
tissues is challenging, as these environments are inherently tolerogenic. As a first
step toward a better understanding of how
tolerance and immunity are induced at the
portals of entry for sexually transmitted
pathogens, we established vaginal and
rectal models of lymphocytic choriomeningitis virus (LCMV) infection in conventional mice. Using our mucosal LCMV
models, we demonstrated that, similar to
SIV infection in non-human primates,
mucosal LCMV infection generates a
delayed and dampened CTL response
when compared to a systemic infection.
We further showed that the delay in the
CTL response is caused by delayed and
suboptimal innate immune activation.
Based on these findings, we hypothesize that the tolerogenic cytokine environment in the mucosal tissues serves as an
immunological barrier for proper immune
activation against pathogens, and transforming growth factor β (TGFβ), an
abundant and immunosuppressive cytokine in mucosal tissues, serves as one of
these barriers. Understanding how antiviral immunity surpasses the inherent
mucosal tolerance and how CD8 T cells
are activated in this environment will
pave the way for enhancing mucosal
immune responses during vaccination
and for developing novel therapeutics
against sexually transmitted pathogens.
A
B
will dissect the early innate and adaptive immune responses that are elicited
after mucosal HIV transmission and
then determine how the latent reservoir
is differentially formed in these smallanimal models.
Is the tolerogenic mucosal environment a barrier to an effective
immune response?
8
6
4
2
0
3
5
7
9
11 13 15
Days Post Infection
450
400
350
300
250
200
150
100
50
0
8
1. Flavell RA et al. (2010) The polarization of immune cells in the
tumour environment by TGFβ.
Nat. Rev. Immunol. 10:554.
2
0
3
5
7
9
11
13
D
20
10
9
11
13
Days Post Infection
0
2. Li MO et al. (2006) Transforming
growth factor-β controls development, homeostasis, and tolerance
of T cells by regulatory T cell-dependent and -independent mechanisms. Immunity 25:455.
3. Sanjabi S et al. (2000) Selective
requirement for c-Rel during IL-12
P40 gene induction in macrophages. Proc. Natl. Acad. Sci. USA
97:12705.
35
7
2. Ishigame H et al. (2013) Truncated form of TGFβRII, but not its
absence, induces memory CD8+
T cell expansion and lymphoproliferative disorder in mice. J. Immunol.
190:6340.
TOP FIVE OVERALL
4
40
5
1. Ishigame H et al. (2013) Excessive
Th1 responses due to the absence
of TGFβ signaling cause autoimmune diabetes and dysregulated
Treg cell homeostasis. Proc. Natl.
Acad. Sci. USA 110:6961.
6
50
3
SELECTED RECENT
Uninfected
Intravaginal
Intraperitoneal
Days Post Infection
CD69 MFI on CD11bhiCD11c+
C
CD86 MFI on CD11bhiCD11c+
% CD11bhiCD11c+ in PBMC
% of P14s in PBMC
10
Publications
3
5
7
9
11
13
Days Post Infection
Using our new mucosal LCMV infection models in mice, we have shown that A) a
delayed and dampened virus-specific CD8 + T-cell (P14) response in vaginal LCMV
infections compared to systemic intraperitoneal infection, B) delayed and dampened expansion of inflammatory dendritic cells (CD11b hi CD11c+), C) absence of
co-stimulatory molecule CD86, and D) improper activation of these innate cells
are most likely responsible for the delay in CD8 T-cell response. PBMC, peripheral
blood mononuclear cell; MFI, mean fluorescence intensity.
4.Sanjabi S et al. (2005) A c-Rel subdomain responsible for enhanced
DNA-binding affinity and selective
gene activation. Genes Dev. 19:2138.
5. Sanjabi S et al. (2009) Opposing
effects of TGF-β and IL-15 cytokines
control the number of short-lived
effector CD8+ T cells. Immunity
31:131.
Eric Verdin, MD
labs.gladstone.ucsf.edu/verdin
HIGHLIGHTS
• Epigenetic regulators utilize cofactors/substrates that are intermediary metabolites
•β-Hydroxybutyrate is an endogenous inhibitor of HDACs and protects against oxidative stress
• Diets promoting ketogenesis may
combat aging and oxidative stress–
related diseases
LAB MEMBERS
Emilie Battivelli
Emilie Besnard
Vincenzo
Calvanese
Christopher
Carrico
Leonard Chavez
Matthew
Dahabieh
Philipp Gut
Shweta Hakre
Wenjuan He
Linh Ho
Jing-Yi Huang
Hyunsun Jo
Herbert Kasler
Intelly Lee
Hyungwook Lim
Philip
Merksamer
Kyung Jin Min
John Newman
Che Ping Ng
Yuya Nishida
Yong Pan
Kotaro
Shirakawa
Margaret Wang
“We recently identified
a novel example of
close integration
between intermediary
metabolism and
transcriptional
regulation via
epigenetic modifiers.”
New research shows an emerging connection between
epigenetic regulation of transcription and intermediary metabolism.
Living organisms continuously adapt
to changes in their environment. With
regard to metabolism, the cellular transcriptional machinery and its chromatin-associated proteins (the epigenetic
regulators) integrate changes in the type
or abundance of energy substrates and
mediate adaptive homeostatic responses.
Numerous connections between the
products of intermediary metabolism
and epigenetic regulation have recently
been identified.
The Role of Epigenetic Regulators
Epigenetic (meaning “above the gene”)
mechanisms determine, in part, which
genes are activated by transcription factors at a given time. The smallest building
block of chromatin is the nucleosome,
which consists of 147 base pairs of DNA
wrapped around a histone core. Epigenetic
modifications occur as a result of DNA
methylation and post-translational modification of histones, such as acetylation
and methylation. These epigenetic modifications change the structure of chromatin and recruit transcription factors
and other non-histone proteins to the
DNA. Importantly, epigenetic modifications persist as “transcriptional memories” through mitosis or meiosis and can
be transmitted to subsequent generations
(transgenerational epigenetics).
Intermediary Metabolites Influence
Epigenetic Regulation
Remarkably, almost all chromatinmodifying enzymes utilize cofactors
or substrates that are crucial metabolites in the core pathways of intermediary metabolism. These metabolites
include acetyl-CoA for acetyltransferases,
α-ketoglutarate for lysine demethylases,
and nicotinamide adenine dinucleotide
(NAD) for either lysine deacetylases or
S-adenosylmethionine, which is the universal methyl donor for lysine and DNA
methyltransferases. Since the cellular
concentrations of these metabolites fluctuate with the metabolic status of the cell,
the activity of the epigenetic regulators is
likely to change as a function of metabolic
status and thereby transduce a homeostatic transcriptional response.
The Metabolic Intermediate βOHB
Inhibits Histone Deacetylases
We recently identified a novel example of
close integration between intermediary
metabolism and transcriptional regulation via epigenetic modifiers. We found
that the ketone body β-hydroxybutyrate
(βOHB) is a novel, endogenous, and specific inhibitor of class I histone deacetylases
(HDACs). Remarkably, administration of
exogenous βOHB — or fasting and calorie
restriction, two conditions associated with
increased βOHB levels — induces global
histone hyperacetylation in mouse tissues.
HDAC inhibition by βOHB is also associated with global changes in the transcription of specific genes that are normally
repressed by class I HDACs. Interestingly,
these genes include the FOXO3A and
MT2 genes, which are related to aging
and to the longer lifespans associated
with calorie restriction. βOHB induces
histone hyperacetylation at the FOXO3A
and MT2 promoters, and treating mice
with βOHB confers significant protection
against exogenous oxidative stress.
Ketone bodies, which were once
referred to as the “ugly ducklings” of
metabolism, may in fact be crucial regulators of metabolic health and longevity via
their ability to mediate HDAC activity
and thereby epigenetic gene regulation.
Fasting Calorie
Restriction Exercise
Ketogenic diets provide a partial phenocopy of calorie restriction through their
effects on insulin, insulin-like growth
factor (IGF), forkhead box 3a (Foxo3a),
fatty acid metabolism, AMP-dependent
kinase (AMPK), and mammalian target
of rapamycin (mTOR). The finding that
βOHB inhibits histone deacetylases,
together with the coincidence of biological effects of ketone bodies, suggests the
fascinating possibility that βOHB is an
endogenous avenue for extending lifespans, as is seen with HDAC inhibition in
model organisms. Therefore, our research
continues to define the molecular targets
of HDAC inhibition by βOHB in specific
tissues and metabolic states and investigate whether βOHB regulates HDACtargeted pathways like autophagy in
order to determine if these effects culminate in enhanced longevity in mammals.
Publications
SELECTED RECENT
1. Gut P et al. (2013) The nexus of
chromatin regulation and intermediary metabolism. Nature 502:489.
2. Newman JC et al. (2013) Ketone
bodies as signaling metabolites.
Trends Endocrinol. Metab. 25:42.
3. Rardin MJ et al. (2013) Label-free
quantitative proteomics of the
lysine acetylome in mitochondria
identifies substrates of SIRT3 in
metabolic pathways. Proc. Natl.
4.Rardin MJ et al. (2013) SIRT5 regulates the mitochondrial lysine succinylome and metabolic networks.
Cell. Metab. 18:920.
5. Shimazu T et al. (2013) Suppression of oxidative stress by
β-hydroxybutyrate, an endogenous
histone deacetylase inhibitor. Science 339:211.
Hepatocyte
Acyl-CoA
β-Oxidation
TOP FIVE OVERALL
Acetyl-CoA
Acetoacetate
Ketogenesis
BDH
1. He W et al. (2012) Mitochondrial
sirtuins: regulators of protein
acylation and metabolism. Trends
Endocrinol. Metab. 23:467.
βOHB
βOHB
Mitochondrion
2. Hirschey MD et al. (2010) SIRT3
regulates mitochondrial fatty-acid
oxidation by reversible enzyme
deacetylation. Nature 464:121.
BDH
3. Hirschey MD et al. (2011) SIRT3
deficiency and mitochondrial protein hyperacetylation accelerate
the development of the metabolic
syndrome. Mol. Cell 44:177.
βOHB
NAD+
NADH
Acetoacetate
Acetyl-CoA
Peripheral Cell
Class I
HDACs
KATs
Nucleus
Ac
Ac
Ac
Foxo3a
Mt2
Histone Hyperacetylation
Resistance to
Oxidative Stress
4.Schwer B et al. (2006) Reversible lysine acetylation controls
the activity of the mitochondrial
enzyme acetyl-CoA synthetase 2.
5. Shimazu T et al. (2010) SIRT3
deacetylates mitochondrial
3-hydroxy-3-methylglutaryl CoA
synthase 2 and regulates ketone
body production. Cell Metab.
12:654.
Actions of the ketone body βOHB. Formed in the liver during fasting, βOHB functions both as a form of glucose-sparing energy during fasting and also as a signaling
molecule by inhibiting endogenous HDACs. BDH, butanol dehydrogenase; KAT, lysine
acetyltransferase; Mt2, melatonin membrane receptor 2.
Leor S. Weinberger, PhD
“Our goal is to exploit
HIV’s master circuits
to engineer the
next generation of
antiviral therapies.”
labs.gladstone.ucsf.edu/weinberger
HIGHLIGHTS
• Discovered a transcriptional accelerator circuit in CMV and the source
of noise in the HIV-1 promoter
• Predicted that interfering particles
can be engineered into resistance-proof HIV therapies
• Received the 2013 NIH Director’s
Pioneer Award
LAB MEMBERS
Katherine Aull
Lisa Bishop
Cynthia
Bolovan-Fritts
Roy Dar
Joseph
Gershony
Elena Ingerman
Seung-Yong Jung
Grayson Kochi
Brian Linhares
Jac Michael Luna
Kieran Mace
Timothy Notton
Anand Pai
Renee Ram
Luke Rast
Brandon Razooky
Igor Rouzine
Jose Sardanes
Cayuela
Jeffrey Sasaki
Colleen
Scheitrum
Jaime Tawney
Melissa Teng
Micha Titus
Yi Wen
Moses Xie
We couple a computational experimental approach
with fluorescence microscopy and mathematical
modeling to attack HIV.
Our lab aims to define the transcriptional “master circuits” that viruses use
to regulate the developmental bifurcation between viral latency and active
viral replication. We have long been
interested in human immunodeficiency
virus (HIV) transcriptional regulation,
and were the first to discover that HIV
latency is regulated by stochastic gene
expression, which was recently verified in
patient cells. In our latest work, we have
expanded our studies of master circuits
in human herpesviruses, specifically
cytomegalovirus (CMV) and employed
quantitative single-cell imaging and
computational approaches to probe the
kinetics of these gene regulatory circuits
in live cells. We are now actively translating our basic science discoveries into
novel classes of antiviral therapies.
Probing Transcriptional Regulation
in CMV
To probe CMV transcriptional regulation, we capitalized on the single-cell
imaging and computational approaches
we developed for HIV circuitry. With
single-cell analysis, we identified novel
transcriptional circuitry that accelerates
viral gene expression without amplifying
steady-state expression levels. The “accelerator circuit” maps to a highly selfcooperative transcriptional feedback
circuit generated by homomultimerization of the virus’s essential transactivator
protein IE2 at nuclear promyelocytic
leukemia (PML) bodies. The accelerator circuit confers a significant fitness
advantage for the virus. Accelerators may
provide a mechanism for signal-transduction circuits to respond quickly to
external signals without increasing the
steady-state levels of potentially cytotoxic molecules.
Transcriptional Noise
We also continued to study how transcriptional fluctuations (“noise”) regulate
viral fate decisions. Noise is ubiquitous
at the single-cell level and results from
random molecular collisions and reactions within the cell. In earlier studies,
we demonstrated that noise in gene
expression can act as a genetic “switch”
in mammalian systems. Noise-driven
switches participate in developmental
fate decisions that guide sporulation
and competence in bacteria as well as
induced pluripotency in stem cells.
Stochastic noise may be responsible for
latent reactivation of HIV.
Ou r pre v iou s work ident i f ied
the molecula r source of noise in
HIV-1 — transcriptional “bursting” from
the HIV-1 promoter — and determined
that the HIV-1 promoter is the noisiest
promoter yet characterized, being many
times noisier than yeast and other human
promoters. To extend our noise-imaging
work into clinically relevant primary cells,
we recently developed first-of-their-kind
microwell “finger” devices to study noise
and HIV latency in primary CD4+ T cells
and novel noise-analysis techniques that
can pinpoint noise sources in many different systems. The microfluidic fingers
restrict cell migration to a nearly onedimensional region, which allows us to
image and track HIV-infected primary
T cells for multiple days with no mechanical manipulation, thereby maintaining
cell viability. These devices overcome
the major obstacles (i.e., difficulties in
immobilization and tracking) that have
previously prevented time-lapse imaging
of primary CD4+ T cells. A number of
other laboratories at Gladstone are now
using these devices to study HIV-1 at
the single-cell level. We recently capitalized on HIV’s noise to develop a
novel genome-wide single-cell imaging
approach to probe sources of noise
across the human genome. This study
quantified transcriptional noise and
dynamics at more than 8000 different
chromosomal loci across the human
genome and provides an unprecedented
data set that can be used to further
explore gene regulation.
Therapeutic Interfering Particles
Finally, we are pioneering a new class
of anti-HIV molecular parasites called
therapeutic interfering particles (TIPs).
TIPs replicate only in HIV-infected
cells and parasitize HIV molecular
componentry to reduce viral load and
infectivity. As a result of this piggybacking, TIPs may transmit along
the same routes as HIV and have the
potential to be “resistance-proof ” single-administration therapies that can
automatically target patients who have
the highest HIV burdens, especially in
resource-limited settings such as subSaharan A frica. This research was
rec ent ly recogni z ed by t he N IH
Director’s Pioneer Award.
Viral Accelerator Circuit
MIEP
1. Dar RD et al. (2012) Transcriptional
burst frequency and burst size
are equally modulated across the
human genome. Proc. Natl. Acad.
Sci. USA 109:17454.
2. Rouzine IM et al. (2013) Design
requirements for interfering particles to maintain coadaptive stability with HIV-1. J. Virol. 87:2081.
3. Singh A et al. (2012) Dynamics
of protein noise can distinguish
between alternate sources of
gene-expression variability. Mol.
Syst. Biol. 8:607.
4.Teng MW et al. (2012) An endogenous accelerator for viral gene
expression provides a fitness
advantage. Cell 151:1569.
5. Weinberger AD et al. (2013)
Stochastic fate selection in HIVinfected patients. Cell 155:497.
1. Metzger V et al. (2011) Autonomous targeting of infectious
superspreaders using engineered
transmissible therapies. PLoS
Comput. Biol. 7:e1002015.
IE2
Virus Output
Input 1
Output
SELECTED RECENT
TOP FIVE OVERALL
Input 1 > Input 2
Acceleration
without Amplificaton
Publications
Input 2
Time
Transcriptional accelerator circuit. This mechanism allows signal-transduction
circuits to respond quickly to external signals without increasing steady-state levels
of potentially cytotoxic molecules. For CMV, transcriptional activators accelerate
viral gene expression in single cells without increasing expression. This generates
a significant replication advantage. The accelerator maps to a highly self-cooperative transcriptional feedback circuit generated by homomultimerization of the
virus’s essential transactivator protein IE2 at nuclear PML bodies. Eliminating this
feedback carries a heavy fitness cost. MIEP, major immediate early promoter.
2. Razooky B et al. (2012) Microwell
devices with finger-like channels for
long-term imaging of HIV-1 expression kinetics in primary human
lymphocytes. Lab Chip 12:4305.
3. Weinberger LS et al. (2005) Stochastic gene expression in a lentiviral positive-feedback loop: HIV-1
Tat fluctuations drive phenotypic
diversity. Cell 122:169.
4.Weinberger LS et al. (2007) An
HIV feedback resistor: auto-regulatory circuit deactivator and noise
buffer. PLoS Biol. 5:e9.
5. Weinberger LS et al. (2008) Transient-mediated fate determination
in a transcriptional circuit of HIV.
Nat. Genet. 40:466.
Director’s Report
Lennart Mucke, MD
“In these last 2 years, we
have made significant
advances toward our mission
of better understanding,
preventing, treating,
and — ultimately — curing
major neurological diseases.”
Neurological Disease Research
This report summarizes 2 years of advances made by the
Gladstone Institute of Neurological Disease (GIND). We made
major progress in fundamental discoveries and their translation into promising therapeutic strategies. These represent
unique accomplishments of individual investigators and collaborative team approaches. GIND investigators published over
57 papers in high-quality journals, and these have been cited
over 600 times, highlighting their influence on the field. Here I
describe only a selection of discoveries that may lead to paradigm
shifts and have important therapeutic implications.
Research Advances
Alzheimer’s disease. The proteins tau, apolipoprotein (apo) E4,
and amyloid-β (Aβ) peptides causally contribute to Alzheimer’s
disease (AD). Elucidating their actions and developing therapeutic strategies to block them are key objectives. The Gan
laboratory discovered that acetylation determines tau turnover and mediates cognitive deficits in experimental tauopathy
models. Compounds that inhibited acetylation protected animal
models against memory deficits and hippocampal volume loss.
The Mucke laboratory showed that physiologic brain activity
causes neuronal double-strand DNA breaks, pathologically
elevated Aβ levels increase the DNA damage and delay repair,
and reducing tau prevents these effects. They demonstrated ageappropriate cognitive functions in aged tau-deficient mice,
supporting the safety of tau-lowering therapeutic strategies. The Huang and Mahley laboratories showed that smallmolecule “structure correctors” reduce apo E4’s neurotoxic
effects in cultured neurons and brain levels of neurotoxic apoE4
fragments in mouse models. They found that these drugs might
ameliorate apoE4-dependent cognitive impairments. The Huang
laboratory demonstrated that apoE4 affects dendritic arborization and spine development and causes age- and sex-dependent
impairments of inhibitory interneurons in the hilus of the dentate gyrus (DG). The Mucke and Palop laboratories showed
that depletion of sodium channels in cortical interneurons
contributes to Aβ-induced abnormalities in cognitive function
and neural network activity and that an anti-epileptic drug
(levetiracetam) ameliorates dysfunctions in AD mouse models.
A clinical Phase IIa trial will begin in 2014. With the University
of California, San Francisco (UCSF) Memory and Aging Center
and Epilepsy Center, the Mucke laboratory characterized the
nature of epileptiform activity in early AD and identified biomarkers that should facilitate the clinical trial.
Parkinson’s disease. Parkinson’s disease (PD) is characterized by
loss of dopaminergic innervation of the striatum, which impairs
motor programming and movement control. The Kreitzer laboratory discovered that regulation of G-protein signaling is required
for dopaminergic control of striatal function and susceptibility
to PD-related motor deficits. The Nakamura laboratory discovered that loss of the mitochondrial fission regulator dynamin-related protein 1 depletes axonal mitochondria and causes loss of
nigrostriatal dopamine neurons, providing insights into how
mitochondrial impairments contribute to PD. With the Shokat
laboratory (UCSF), the Nakamura laboratory characterized a
new therapeutic strategy targeting the PD protein PINK1. They
found that kinetin, a small molecule used in humans, increases
the activity of mutant PINK1 and restores its function in PD
cell-culture models, forming the basis for new studies.
Huntington’s disease. Huntington’s disease (HD), a severe inherited movement disorder, is caused by polyglutamine-encoding
mutations in huntingtin. A favorite hypothesis is that mutant huntingtin forms poisonous aggregates. The Finkbeiner laboratory
discovered that mutant huntingtin is pathogenic even as monomers and characterized the structures involved. They showed that
neurons vary in handling polyglutamine-containing proteins and
this predicts their susceptibility to neurodegeneration.
Frontotemporal dementia, amyotrophic lateral sclerosis, and
Alzheimer’s disease. Mutations in the RNA-binding protein
TDP-43 cause frontotemporal dementia (FTD), amyotrophic
lateral sclerosis (ALS), or both. The Finkbeiner laboratory
showed that modulating stress granules reduces TDP-43 toxicity
in ALS cell-culture models. Mutations in progranulin also cause
FTD. The Gan laboratory revealed that progranulin modulates
innate immunity and protects against Aβ. Their findings support
enhancing progranulin to treat FTD and AD.
Multiple sclerosis and chronic inflammation. The bloodbrain barrier is disrupted early in multiple sclerosis (MS), causing
leakage of blood proteins (e.g., fibrinogen) into the brain or spinal
cord. The Akassoglou laboratory discovered how fibrinogen initiates axonal damage and developed a probe to detect MS lesions
early. They are also developing therapies to block disease-causing
molecular cascades triggered by fibrinogen or its components.
Chronic inflammation may be important in diseases of aging.
Microglia are innate immune cells of the brain and spinal cord
that mediate inflammation. The Gan laboratory discovered that
the cellular senescence of microglia is accelerated by SIRT1 deficiency and that microglia lacking SIRT1 contribute to cognitive
decline and neurodegeneration in mouse models, most likely by
upregulating IL-1β. Targeting aging microglia may prevent cognitive decline in neurodegenerative diseases.
Critical functions of the brain and other organs. The Akassoglou
laboratory discovered that the p75 neurotrophin receptor regulates
circadian and metabolic networks and controls insulin resistance.
The Finkbeiner laboratory showed that Arc regulates transcription
of glutamate receptors and homeostatic synaptic plasticity, which
are critical for learning, memory, and adaptive brain functions.
The Huang and Kreitzer laboratories demonstrated that interneurons in the hilus of the DG (modulated by optogenetics) control
learning and memory retrieval. The Huang laboratory found that
ADP-ribosylation factor 4 is involved in DG-mediated pattern
separation and regulates dendritic spine development. The Kreitzer
laboratory discovered that striatal cholinergic interneurons drive
GABA release from dopamine terminals in the striatum, which
may be important for diseases of disinhibition, such as Tourette’s
and dystonia. They also mapped the connections of the basal ganglia, providing a starting point for understanding how inputs are
rewired in diseases affecting this brain region.
Stem cells and cell therapy. Stem cell–related technologies have
exciting potential for improved disease models and innovative
therapies. The Huang laboratory directly reprogrammed fibroblasts into multipotent neural stem cells with a single factor. The
Huang and Finkbeiner laboratories used patient-derived induced
pluripotent stem cells to study novel human cell-culture models
of AD, PD, FTD, ALS, and HD. The Huang and Palop laboratories showed that interneuron precursors transplanted into
brains of AD mouse models develop into functional interneurons, integrate into microcircuits, improve brain rhythms, and
reduce cognitive dysfunction.
Translational Centers and Industrial Collaborations
Several Gladstone centers were critical to our translational efforts:
Center for Comprehensive Alzheimer’s Disease Research, which
is supported by a lead gift from the S.D. Bechtel, Jr. Foundation,
Center for In Vivo Imaging Research, Center for Translational
Research, Koret/Taube Center for Neurodegenerative Disease
Research, and Hellman Family Foundation Alzheimer’s Disease
Research Program. GIND investigators are engaged in collaborations with 15 pharmaceutical and biotechnology companies.
Education, International Exchange, Awards and Honors
GIND investigators trained 48–53 postdoctoral fellows and
11–20 graduate students each year. Fourteen students received
PhDs as a result of thesis work at the GIND. GIND investigators
organized well-received symposia and workshops that promoted
the international exchange of ideas in diverse disciplines of neuroscience and biomedicine. GIND investigators received 19 new
National Institutes of Health (NIH) awards, including a U01
and six R01 grants. Other honors include the MetLife Award for
Medical Research, the J. Elliott Royer Award for Excellence in
Academic Neurology, and the American Pacesetter Award, as well
as appointments to the Executive Committee of the American
Society for Pharmacology and Experimental Therapeutics, NIH
study sections, and the NIH Board of Scientific Counselors.
Conclusions
I could not be happier about our progress. I thank our investigators, trainees, research staff, administrators, and the supporters
of our mission for making these advances possible. Inspired by
this progress, we will continue to advance our understanding of
the nervous system and contribute ever more actively to developing better treatments for major neurological diseases.
Jorge Palop, Yadong Huang,
Steve Finkbeiner, Robert
Mahley; front row: Li Gan,
Ken Nakamura, Anatol
Kreitzer, Katerina Akassoglou,
Lennart Mucke.
Katerina Akassoglou, PhD
“Neurovascular
interactions
determine CNS
pathogenesis and can
be used to develop
novel treatments.”
Senior Investigator
labs.gladstone.ucsf.edu/akassoglou
HIGHLIGHTS
• Neurovascular interactions are critical determinants for immune and
degenerative processes in the CNS
• Upon BBB disruption, fibrinogen
induces microglial activation and
axonal damage
• Fibrinogen must bind to the CD11b/
CD18 integrin receptor to damage
CNS functions
LAB MEMBERS
Bernat Baeza-Raja
Sophia Bardehle
Catherine Bedard
Belinda Cabriga
Nicholas Castello
Dimitrios Davalos
Michael Machado
Mario Merlini
Mark Petersen
Victoria Rafalski
Jae Kyu Ryu
Catriona Syme
Eirini Vagena
We aim to understand the mechanisms dictated by BBB
disruption that regulate inflammatory, neurodegenerative, and cognitive processes after CNS injury or disease.
Rupture of the vasculature allows blood
proteins to enter the brain in various disorders, including multiple sclerosis (MS),
stroke, brain injury, HIV encephalitis,
Alzheimer’s disease, bacterial meningitis,
and glioblastomas. However, how those
blood proteins contribute to neuroinflammation and neuronal damage remains
largely unknown. We integrate animal
modeling, in vivo two-photon microscopy, tissue culture, and biochemical
techniques as a multifaceted approach to
address the biological complexity of disease and repair mechanisms in the central
nervous system (CNS).
The Importance of Fibrinogen
Fibrinogen, a critical component of
blood coagulation and inf lammation,
crosses through the damaged bloodbrain barrier (BBB) and accumulates
as fibrin deposits at specific injury sites.
We have shown that fibrinogen is more
than a marker of BBB disruption; the
protein also induces inflammation and
mediates neurodegenerative processes
in the nervous system. We found that
persistent deposition of fibrinogen in
the CNS creates a deleterious environment that exacerbates disease processes
and inhibits natural regenerative mechanisms. We also showed that fibrinogen is
a potent inducer of microglial activation,
which is required for the development of
axonal damage in the CNS, a major contributor to disability in MS. Moreover,
we showed that fibrinogen must bind to
the CD11b/CD18 integrin receptor to
damage the CNS.
The Next Step Forward
Our view of fibrinogen is evolving from
a mere component of blood clots and a
marker of vascular rupture to a multifaceted signaling molecule that is involved
in hemostasis and thrombosis, coagulation and fibrosis, and protection from
infection and extensive inflammation.
Importantly, the wide spectrum of known
molecular targets and cellular partners
for fibrinogen appears to require distinct
non-overlapping epitopes on fibrin(ogen).
Taking advantage of this unique aspect of
the structural/functional segregation of
fibrinogen has already proven to inhibit
a specific interaction of fibrinogen with
a given receptor, without affecting the
ability of fibrinogen to bind other receptors, or the vital function of fibrinogen
in the coagulation cascade. In an effort
to discover new treatments for neurologic
diseases, we are developing novel strategies to pharmacologically target the deleterious actions of fibrin in the nervous
system without increasing the risk of
bleeding or thrombotic events.
p75 Neurotrophin Receptor
In our efforts to understand the complexities of neurological disease, we
have found that bidirectional cross-talk
between the brain and the periphery contributes to disease pathogenesis. We discovered an unexpected function for the
p75 neurotrophin receptor in regulating
circadian rhythms and insulin homeostasis. We expect that understanding
how nervous system receptors affect
metabolic functions will reveal novel
mechanisms of communication between
the brain and the periphery and identify
new targets for therapeutic intervention
in neurological and metabolic diseases.
Center for in Vivo Imaging Research
The neurovascular interface is a dynamic
structure that determines communications between the brain and the peripheral immune signals that affect brain
functions. We use in vivo two-photon
microscopy to study immune processes in
the CNS of living animals. We developed
novel methods to image the neurovascular
interface and, in particular, microglia,
the innate immune cells of the CNS. The
Gladstone Center for In Vivo Imaging
Research develops technologies to investigate the interactions between the brain
and the immune and vascular systems to
better understand the sequence of events
and causative relationships that lead to
neurologic disease. In vivo two-photon
microscopy allows us to answer fundamental questions of disease etiology and
pathogenesis and to identify interactions
between the brain and the periphery.
Publications
SELECTED RECENT
1. Baeza-Raja et al. (2012) p75 neurotrophin receptor regulates glucose
homeostasis and insulin sensitivity.
2. Baeza-Raja B et al. (2013) p75 neurotrophin receptor is a clock gene
that regulates oscillatory components of circadian and metabolic
networks. J. Neurosci. 33:10221.
3. Davalos et al. Early detection of
thrombin activity in neuroinflammatory disease. Ann. Neurol. (in press).
4.Davalos D et al. (2012) Fibrinogeninduced perivascular microglial
clustering is required for the
development of axonal damage in
neuroinflammation. Nat. Commun.
3:1227.
5. Merlini M et al. (2012) In vivo
imaging of the neurovascular interface. Intravital. 1:87.
Vasculature
BBB Disruption
Glia, Neurons
Br
Immune Cells
Innate Immunity
Autoimmunity
Microglial Activation
Neurite Outgrowth
Oxidative Stress
Axonal Damage
Scar Formation
Remyelination
TOP FIVE OVERALL
S cle rosis , S t ro
ltipl e
M u r aum a , N e u r o d e g e n eke ,
T
rat
io
a in
n
Fibrinogen
Imaging
In Vivo Imaging
Molecular Probe
Development
Therapeutics
CNS Targeting
New Animal Model
Biomarkers
Fibrinogen, a critical clotting factor, can penetrate a damaged BBB, which occurs
in several neurologic diseases and after trauma. But fibrinogen does not only mark
BBB disruption. It also affects the CNS by inducing inflammation and mediating
neurodegenerative processes. In my lab, we use imaging techniques and animal
models to better understand fibrinogen’s role in these systems, and we develop
novel fibrinogen therapeutics that target its damaging functions in the brain
without affecting its beneficial effects in blood clotting.
1. Adams RA et al. (2007) The
fibrin-derived γ377–395 peptide
inhibits microglia activation and
reverses relapsing paralysis in central nervous system autoimmune
disease. J. Exp. Med. 204:571.
2. Akassoglou K et al. (1998) Oligodendrocyte apoptosis and primary
demyelination induced by local
TNF/p55TNF receptor signaling in
the CNS of transgenic mice: models
for multiple sclerosis with primary
oligodendrogliopathy. Am. J. Pathol.
153:801.
3. Akassoglou K et al. (2002) Fibrin
inhibits peripheral nerve regeneration by arresting Schwann cell
differentiation. Neuron 33:861.
4.Davalos D et al. (2012) Fibrinogen-induced perivascular microglial
clustering is required for the
development of axonal damage in
neuroinflammation. Nat. Commun.
3:1227.
5. Passino MA et al. (2007) Regulation of hepatic stellate cell differentiation by the neurotrophin receptor
p75NTR. Science 315:1853.
“We study Arc
because it teaches us
how memory works
and because it is
elegant, intricate, and
truly fascinating.”
Steve Finkbeiner, MD, PhD
labs.gladstone.ucsf.edu/finkbeiner
HIGHLIGHTS
• Without Arc, mice can learn and
form new memories, but they
cannot retain them
• Arc is involved in scaling neuronal
responses to dampen the system
“noise” while maintaining the original memory
• Without Arc, this elegant system
cannot consolidate memories of
new experience
LAB MEMBERS
Dale Ando
Sue-Ann Mok
Arpana Arjun
Kien-Thiet
Nguyen
Rebecca Aron
Sami Barmada
Matthew
Campioni
Ana Cristina
Osorio Marinho
Oliveira
Aaron Daub
Jeannette
Osterloh
Dylan Edmunds
Maya Overland
Lisa Elia
Sarah Robinson
Samantha
Esselmann
Nicole Ruiz
Kimia Etemadi
Krystal Lauren
Smith
Kelly Haston
Gaia Skibinski
Ashkan
Javaherian
Tina Tran
Julia Kaye
Marc Vimolratana
Erica Korb
Hui Wang
Ian Kratter
Kurt Weiberth
Eva LaDow
Hengameh Zahed
Yuan-Hung Lin
Andre Zandona
Juila Margulis
Adam Ziemann
Amanda Mason
Mario Zubia
Andrey Tsvetkov
The principal goal of our laboratory is to understand
how the nervous system works and how neurodegenerative diseases lead to neuronal dysfunction and death.
We are most interested in how the nervous system forms and stores stable memories. To gain a better understanding of
how memories are created and retained,
we have been studying Arc, an activity-regulated, cytoskeletal-associated
protein. Arc is important for memory
retention. Mice that do not express the
Arc protein can learn and form new
memories, but they cannot retain them
for longer than a day or two. We believe
that studying how this protein works in
mouse memory consolidation will help us
to better understand the memory process.
Forming Memories
The current paradigm is that memories
are principally represented in the brain
through changes in the number and
function of synapses, the physical contacts between two nerve cells that allow
neurons to communicate messages to
one another. When a new memory is
formed, the strength of the synapse is
changed. If this excitation process is left
unregulated, a vicious cycle may lead to
the neuron becoming highly excitable,
so that all of the synapses are activated
by even a small stimulation. To prevent
this cycle, neurons trigger a scaling process that lowers the overall excitation of
the cell but preserves the relative differences in synaptic strength that were
introduced during memory formation.
Thus, scaling contributes to the consolidation of the memory while readying the
neuron to store new memories. Without
scaling, the high levels of excitability
would create noise that would prevent us
from distinguishing the strength of the
synapses that were part of the memory
from those that were not.
The Role of Arc
Scientists have been focused on understanding whether Arc has a direct action
at synapses. Recently, we discovered a
completely new and unexpected insight
into the role of Arc in the regulation
of memory formation. We learned that
after Arc travels out to synapses, it travels
back into the cell body and translocates
to the nucleus. In fact, we found that
about 30 minutes after a new memory is
formed, when the critical consolidation
events are occurring, more Arc protein
is in the nucleus than at the synapse.
Within the nucleus, Arc performs an
unexpected function: Arc regulates critical genes that are important for mediating the overall excitability of neurons.
Specifically, Arc participates in the
consolidation of new memories by regulating key genes important for the critical scaling process rather than through
actions at the synapses.
Interestingly, several studies have
suggested that scaling may be disrupted
in a variety of human conditions,
including autism, schizophrenia, and
Alzheimer’s disease. As the Arc protein
is now known to mediate the scaling
process, further investigations into the
functions of Arc may lead to the development of novel therapies for these
neurological diseases.
Ongoing projects in my laboratory
also involve a high-throughput screening
platform that we continue to refine. That
platform is based on robotic microscopy (R M), an automated live-cell
imaging technology that monitors functions in individual cells. Longitudinal
single-cell data are analyzed statistically as in clinical trials, and RM is
100 –1000-fold more sensitive than
any commercial system. We have combined RM and human induced pluripotent stem cell-derived neurons to study
human genes that affect neuronal function and survival as a means to identify
mechanisms involved in neurodegeneration. However, the same approach can
measure a large array of different cellular
functions simultaneously in single cells.
Using fluorescent biosensors, we previously assessed hundreds of functions
and pathways in neurons, including
electrical activity and Ca2+ mobilization,
gene expression, flux through biological
pathways (e.g., autophagy and proteasome), organelle morphology, microtubule-based transport, and receptor
trafficking, just to name a few.
Arc / Hoechst
0h
0.5 h
Publications
SELECTED RECENT
1. HD iPS Consortium (2012) Induced
pluripotent stem cells from patients
with Huntington’s disease show
CAG-repeat-expansion-associated
phenotypes. Cell Stem Cell 11:264.
2. Korb E et al. (2013) Arc in the nucleus
regulates synaptic PML-dependent
GluA1 transcription and homeostatic
plasticity. Nat. Neurosci. 16:874.
3. Peters-Libeu C et al. (2012) Diseaseassociated polyglutamine stretches
in monomeric huntingtin adopt
a compact structure. J. Mol. Biol.
421:587.
4.Serio A et al. (2013) Astrocyte
pathology and the absence of noncell autonomy in an induced pluri
potent stem cell model of TDP-43
proteinopathy. Proc. Natl. Acad. Sci.
USA 19:4697.
5. Tsvetkov AS et al. (2013) Proteostasis of polyglutamine varies among
neurons and predicts neurodegeneration. Nat. Chem. Biol. 9:586.
TOP FIVE OVERALL
1. Arrasate M et al. (2004) Inclusion
body formation reduces levels of
mutant huntingtin and the risk of
neuronal death. Nature 431:805.
4h
8h
2. Arrasate M et al. (2005) Automated microscope system for
determining factors that predict
neuronal fate. Proc. Natl. Acad. Sci.
USA 102:3840.
3. Miller J et al. (2011) Identifying
polyglutamine protein species in
situ that best predict neurodegeneration. Nat. Chem. Biol. 7:925.
4.Rao VR et al. (2006) AMPA receptors regulate transcription of the
plasticity-related immediate early
gene Arc. Nat. Neurosci. 9:887.
Arc becomes enriched in neuronal nuclei in mice after exposure to a novel environment. Immunohistochemical staining of Arc (yellow) and Hoechst nuclear staining
(blue) in coronal sections of mouse hippocampi after exposure to a novel environment for 0 – 8 h. At 0.5 h, most Arc is in the cytoplasm. By 4 h, a significant amount
has moved into the nucleus, and by 8 h, most is in the nucleus. Scale bar, 10 μm.
5. Tsvetkov AS et al. (2010) A
small-molecule scaffold induces
autophagy in primary neurons and
protects against toxicity in a Huntington disease model. Proc. Natl.
Li Gan, PhD
“As the population
ages, neurodegenerative diseases have
emerged as a major
health challenge
facing our society.”
labs.gladstone.ucsf.edu/gan
HIGHLIGHTS
• Microglia in aging mice exhibit
senescence that is accelerated by
SIRT1 deficiency
• Microglial SIRT1 deficiency selectively upregulates IL-1β, worsening
memory deficits
• Progranulin, a causal factor in FTD,
enhances microglial function and
protects against Aβ deposition
and toxicity
LAB MEMBERS
Meredith Chabrier
Robert Chen
Xu Chen
Seo Hyun Cho
Grietje Krabbe
David Le
Yaqiao Li
Sang-Won Min
Sakura S. Minami
Peter Sohn
Tara Tracy
Chao Wang
Michael Ward
Yungui Zhou
We study two interconnected mechanisms of neurodegenerative processes: the accumulation of protein aggregates and miscommunication between neurons and glia.
Aging is the largest risk factor for neurodegenerative diseases, including
Alzheimer’s disease (AD) and frontotemporal dementia (FTD). As the brain
ages, an aberrant innate immune response
causes chronic inflammation in the brain,
which is associated with neurodegenerative disease development. However, little
is known about how chronic inflammation occurs and whether it contributes to
cognitive decline. Our research focuses
on these two questions using multidisciplinary approaches that combine
transcriptome and pathway analyses,
cell-type-specific genetic modulation of
neurodegenerative mouse models, and
detailed mechanistic dissections, with
virus-mediated gene therapy.
Accelerating Microglial Aging and
Senescence Causes AD-Related
Memory Deficits
We are investigating whether dysfunction in aging microglia — the resident
immune cells in the brain — helps cause
neurodegeneration. Using unbiased
transcriptome analyses of microglia
isolated from aging brains, we showed
that these immune cells have highly
altered gene expression compared with
those from young brains. These changes
include increased levels of cell senescence
genes and reduced expression of SIRT1,
a gene implicated in anti-aging. To evaluate the link between SIRT1 expression and senescence, we induced SIRT1
deficiencies in three independent aging
and neurodegenerative mouse models,
which showed accelerated microglial
aging and exacerbated memory deficits.
These findings establish a role for SIRT1
in microg lial cell senescence associated
with aging. Our findings also implicate
senescent microglia as major contributors to cognitive deficits associated with
aging and neurodegeneration.
In our studies aimed at identifying
how SIRT1 affects cell senescence, we
found that SIRT1-deficient microglia
had selectively higher levels of interleukin-1β (IL-1β). The increased expression of IL-1β also positively correlated
with the extent of aging and AD-related
memory def icits. A lthough NF-κB
activation is known to be required for
IL-1β upregulation, NF-κB’s target
genes tumor necrosis factor-α and IL-6
were not enhanced in SIRT1-deficient
microglia both in vivo and in vitro.
Instead, we found that SIRT1 deficiencies mediate IL-1β regulation using an
epigenetic mechanism of CpGs methylation located in IL-1β’s proximal promoter. These initial results support a
novel epigenetic mechanism that contributes to cognitive deficits in aging
microglia a nd neurodegeneration,
which may offer new therapeutic avenues for neurodegenerative diseases.
Progranulin: A Protective Signal
in Microglia
A common cause of FTD, the second
most common cause of dementia, is
mutations on a gene (GRN) that encodes
progranulin. Mutations in this gene
reduce progranulin levels and exacerbate microglial activation, promoting
an aberrant innate immune response.
Interestingly, patients with loss-of-function GRN mutations exhibit AD-related
pathological and clinical phenotypes,
suggesting a potential protective role for
microglial progranulin in AD.
To investigate this protective role, we
used three independent AD mouse models
to show that microglial progranulin is one
of the most potent natural inhibitors of
amyloid-β (Aβ) plaque deposition, likely
through regulating phagocytosis. More
specifically, selectively ablating microglial
progranulin in A D mice impaired
phagocytosis and increased plaque load
three-fold. In contrast, progranulin overexpression significantly lowered plaque
load in AD mice with aggressive amyloid
plaque pathology. Moreover, the extent
of Aβ plaque load correlated negatively
with levels of hippocampal progranulin.
These findings reveal that progranulin
has a dose-dependent inhibitory effect on
plaque deposition and phagocytosis.
In addition, we discovered that
progranulin protects against Aβ toxicity. Reducing microglial progranulin
augmented cognitive deficits, whereas
progranulin overexpression in the hippocampus prevented spatial memory
deficits in AD mice. Remarkably, progranulin overexpression also prevented
hippocampal neuron loss in these mice.
These findings suggest that progranulin
regulates both Aβ deposition and toxicity, which may have important therapeutic implications for AD and other
neurodegenerative diseases.
Aberrant Protein
Modification and
Accumulation
↑Acetylation
↓Turn-over
Neurodegeneration
Chronic
Microglial
Activation
Aging
↑Senescence
Epigenetic Δ
Cognitive
Deficits
Diagram depicts common mechanisms in neurodegenerative diseases, which are
interconnected and strongly influenced by the aging process. Accumulated toxic
protein leads to chronic inflammation and microglial activation. Aberrant microglial activation impairs their ability to clear protein aggregates and contributes to
pathogenic accumulation.
Publications
SELECTED RECENT
1. Grinberg LT et al. (2013) Argyrophilic grain disease differs from
other tauopathies by lacking tau
acetylation. Acta Neuropathol.
125:581.
2. Kauppinen TM et al. (2013)
Poly(ADP-ribose) polymerase-1induced NAD+ depletion promotes nuclear factor-κB transcriptional activity by preventing p65
de-acetylation. Biochim. Biophys.
Acta 1833:1985.
3. Martens LH et al. (2012) Progranulin deficiency promotes neuro
inflammation and neuron loss in
toxin-induced CNS injury. J. Clin.
Invest. 122:3955.
4.Minami SS et al. (2012) Selective
targeting of microglia by quantum
dots. J. Neuroinflammation 9:22.
5. Wang C et al. (2012) Cathepsin B
degrades amyloid-b in mice
expressing wild-type human amyloid precursor protein. J. Biol. Chem.
287:39834.
TOP FIVE OVERALL
1. Chen J et al. (2005) SIRT1 protects
against microglia-dependent beta
amyloid toxicity through inhibiting NF-κB signaling. J. Biol. Chem.
280:40364.
2. Min SW et al. (2010) Acetylation
of tau inhibits its degradation and
contributes to tauopathy. Neuron
67:953.
3. Mueller-Steiner S et al. (2006)
Anti-amyloidogenic and neuroprotective functions of cathepsin
B: implications for Alzheimer’s
disease. Neuron 51:703.
4.Sun B et al. (2008) Cystatin
C-cathepsin B axis regulates
soluble amyloid beta and associated neuronal deficits in an animal
model of Alzheimer’s disease.
Neuron 60:247.
5. Sun B et al. (2009) Imbalance
between GABAergic and glutamatergic transmissions impairs adult
neurogenesis in an animal model of
Alzheimer’s disease. Cell Stem Cell
5:624.
Yadong Huang, MD, PhD
labs.gladstone.ucsf.edu/yadong
HIGHLIGHTS
• Imbalanced excitatory and inhibitory neuronal activity is related to
AD pathogenesis
• GABAergic interneurons are inhibitory neurons that help balance excitatory neuronal activity
• Inhibiting GABAergic interneuron
activity causes spatial learning and
memory deficits in mice
LAB MEMBERS
Maureen
Balestra
Jessie Carr
Andrew Chen
Jessica Dai
Helen Fong
Anna Gillespie
Natalie
Grant-Villegas
Dah-eun Jeong
Jerome Kahiapo
Johanna Knoferle
Mihir Kshirsagar
Laura Leung
Victoria Lin
Philip Nova
Karen Ring
Adrienne Stormo
Leslie Tong
Joe Udeochu
David Walker
ChengZhong
Wang
Max Wang
Qin Xu
Sachi Yim
Seo Yeon Yoon
“GABAergic
interneuron loss or
dysfunction may
contribute to learning
and memory deficits
in Alzheimer’s
disease.”
We have sought to explore how hilar GABAergic
interneurons function in learning and memory using a
variety of integrated approaches.
The hippocampus plays a key role in spatial learning and memory and is highly
susceptible to Alzheimer’s disease (AD)
pathology. The dentate gyrus is the
gateway to the hippocampus, which consists of >95% excitatory granule neurons
and <5% inhibitory GABAergic interneurons concentrated in the hilus. The
balance of excitatory and inhibitory neuronal activity is thought to be required
for normal learning and memory, while
imbalances have been implicated in the
pathogenesis of amnesia in AD. Although
extensive research has demonstrated the
importance of excitatory granule neurons
in learning and memory, the roles of hilar
GABAergic interneurons remain unclear.
Optogenetic Control of Hilar
GABAergic Interneuron Activity
To explore the roles of hilar GABAergic
interneurons in learning and memory, we
took an optogenetic approach to selectively control hilar GABAergic interneuron activity in mice. Specifically, we
expressed the enhanced Natronomonas
pharaonis halorhodopsin (eNpHR) protein in hilar GABAergic interneurons.
The eNpHR protein is a chloride pump
that can be activated by yellow light
(~590 nm), which then inhibits neuronal
activity. Image analysis of mouse brain sections revealed numerous eNpHR-positive
interneurons in the hilus but not in other
regions of the hippocampus. In active
mice, the firing in dentate granule neurons increased during laser illumination
because the hilar inhibitory interneuron
activity was suppressed. Importantly, the
elevated firing rate returned to baseline
within 1.5 seconds after stimulation, suggesting that optically inhibiting the hilar
interneurons resulted in transient overactivation of dentate granule neurons.
Inhibiting Hilar GABAergic
Interneuron Activity Impairs Spatial
Learning and Memory
We assessed the effect of inhibiting hilar
GABAergic interneuron activity on
learning and memory in behaving mice
using the Morris water maze test. Mice
expressing eNpHR in hilar interneurons
were divided into two groups: one in
which the hilus was illuminated with a
laser (laser on), and one where the hilus
was not illuminated (laser off ) during
each hidden-platform learning trial.
Illuminating the hilus, which inhibits
hilar GABAergic interneuron activity,
slowed the mice’s learning process, suggesting that inhibiting hilar GABAergic
interneuron activity impairs spatial
learning. Laser illumination during the
probe (memory) trials also impaired
memory at 24 hours, suggesting that
memory 24 hours later while under laser
illumination, and they showed memory
impairment. Interestingly, the same group
of mice had normal memory when tested
again without illumination at 72 hours,
suggesting normal memory retention.
Thus, inhibiting hilar GABAergic interneuron activity impaired spatial memory
retrieval but not memory retention.
Our study demonstrates precisely
that impairing hilar GABAergic interneuron function causes spatial learning
and memory deficits. Because impairments of hilar GABAergic interneurons
have been observed in AD, drugs that
enhance hilar GABAergic interneuron
function might be beneficial for treating
amnesia in AD.
Inhibiting Hilar GABAergic
Interneuron Activity Impairs Spatial
Memory Retrieval but Not Retention
To determine whether inhibiting hilar
interneuron activity impairs spatial
memory retention or retrieval, mice
expressing eNpHR in hilar GABAergic
interneurons were randomly divided into
two groups, both of which were trained
without illumination during the hidden-platform learning trials. The first
group was then tested 24 and 72 hours later
for spatial memory without laser illumination, and they showed normal memory.
The second group was tested for spatial
B
CA1
Laser
Recording
40
30
30
20
0
Probe (24h)
Target
Others
45
50
10
60
50uV
H0 H1 H2 H3 H4 H5
0
60
0
G
Probe
60
Target
Others
**
30
*
15
Laser Laser
Off
On
0
Probe
72h
Off
30
0
2. Bien-Ly N et al. (2012) Reducing
human apolipoprotein E levels
attenuates age-dependent Aβ
accumulation in mutant human
amyloid precursor protein transgenic mice. J. Neurosci. 32:4803.
3. Fong H et al. (2013) Genetic correction of tauopathy phenotypes
in neurons derived from human
induced pluripotent stem cells.
Stem Cell Reports 1:226.
4.Huang Y et al. (2012) Alzheimer
mechanisms and therapeutic strategies. Cell 148:1204.
1. Andrews-Zwilling Y et al. (2010)
Apolipoprotein E4 causes ageand tau-dependent impairment of
GABAergic interneurons, leading to
learning and memory deficits in
mice. J. Neurosci. 30:13707.
Target
Others
*
15
24h
Off
1. Andrews-Zwilling Y et al. (2012)
Hilar GABAergic interneuron
activity controls spatial learning
and memory retrieval. PLoS One
7:e40555.
TOP FIVE OVERALL
45
**
SELECTED RECENT
1
Time (sec)
45
15
P < 0.05
F
% Time Spent
60
E
Laser On
Laser Off
% Time Spent
Escape Latency (s)
70
Publications
5. Ring KL et al. (2012) Direct reprogramming of mouse and human
fibroblasts into multipotent neural
stem cells with a single factor. Cell
Stem Cell 11:100.
500µs
0
–1
eNpHR/NeuN
D
LASER
5
DG
Dentate Gyrus
C
Firing Rate
(Hz)
A
% Time Spent
inhibiting hilar GABAergic interneuron
activity also impairs spatial memory.
24h
On
72h
Off
Hilar GABAergic interneuron activity controls spatial learning and memory
retrieval. A) Expression of eNpHR in hilar GABAergic inhibitory interneurons
(green) in mouse hippocampus; neuronal nuclei are stained positive for NeuN
(red). B) Optical stimulation and recording in the mouse dentate gyrus (DG).
C) DG granule neurons increased their firing in response to yellow laser illumination
because hilar inhibitory interneuron activity was suppressed. D) Learning curves
(from Morris water maze tests) of mice expressing eNpHR in hilar interneurons
with or without laser illumination (H, hidden platform trial). E) Time spent in the
target quadrant in the memory trial. F, G) Time spent in the target quadrant in the
memory retention trial. *p < 0.05, **p < 0.01.
2. Brecht WJ et al. (2004) Neuronspecific apolipoprotein E4 proteolysis is associated with increased tau
phosphorylation in brains of transgenic mice. J. Neurosci. 24:2527.
3. Brodbeck J et al. (2011) Structuredependent impairment of intracellular apolipoprotein E4 trafficking
and its detrimental effects are rescued by small-molecule structure
correctors. J. Biol. Chem. 286:17217.
4.Harris FM et al. (2003) Carboxylterminal-truncated apolipoprotein
E4 causes Alzheimer’s disease-like
neurodegeneration and behavioral
deficits in transgenic mice. Proc.
5. Xu Q et al. (2006) Profile and
regulation of apolipoprotein (apo) E
expression in central nervous
system in mice with targeting of
green fluorescent protein to the
apoE locus. J. Neurosci. 26:4985.
“We aim to develop
new circuit-based
treatments to rewire
the brain and regain
functions lost to
disease.”
Anatol Kreitzer, PhD
labs.gladstone.ucsf.edu/kreitzer
HIGHLIGHTS
• Basal ganglia circuits regulate movement, but they go awry in Parkinson’s disease
• Different circuits within the basal
ganglia have unique forms of plasticity that mediate distinct functions
• Treatments targeting specific neural
circuits may help ameliorate disease
symptoms
LAB MEMBERS
Lisa Gunaydin
Mattias Karlsson
Arnaud Lalive D’Epinay
Andrew Lee
Diane Nathaniel
Alexandra Nelson
Scott Owen
Phillip Parker
Thomas Roseberry
Delanie Schulte
We focus on neural circuits and plasticity to determine
how disorders such as Parkinson’s disease impact largescale networks controlling movement.
The brain consists of billions of interconnected cells that form neural circuits.
Determining how the underlying circuit
architecture, the functions of different
circuits, and the mechanisms of neural
plasticity within these circuits mediate
learning and memory is vital to understanding how the brain functions both
normally and when degraded by disease.
Our research tackles each of these key
areas to understand how neurological diseases ultimately cause their characteristic
symptoms. Our goal is to develop new
circuit-based treatments to “rewire” the
brain and regain functions lost to disease.
Parkinson’s Disease and
Movement Disorders
The primary focus of our research is in
Parkinson’s disease (PD) — a neurodegenerative disease affecting millions of
people worldwide that is caused by a loss
of neurons that produce dopamine. PD
usually begins with a tremor and then
progresses to include slowed movements,
rigid muscles, and difficulty standing
upright and walking. Although drug
treatment regimens are initially effective, their use becomes limited over time
by side effects such as dyskinesia, which
results in uncontrolled movements that
are as disruptive as those that occur with
PD. We seek to understand and treat both
PD and dyskinesia using a variety of new
methods to selectively target neural circuits that control movement.
Motor Control Circuits
In PD and related movement disorders,
the disrupted motor control circuits are in
the basal ganglia, a set of interconnected
regions beneath the cortex. Within this
region, two important direct and indirect
pathway circuits have been described. Our
research focuses on these two circuits and
how their anatomical structure, inputs,
type of neuron plasticity, and function
and dysfunction affect health and disease.
Defining the structure of the direct
and indirect pathway circuits and their
inputs requires sophisticated brainmapping methods that were developed
only recently. As the direct and indirect
pathways are intermingled, selectively
targeting these cell types and labeling
their inputs requires a combination of
genetic, viral, and neuroanatomical
approaches. Using these new methods,
we discovered that the direct pathway
circuit receives preferential inputs from
sensory regions of the brain, whereas
the indirect pathway circuit receives
inputs from motor regions. The next
step will be to define how the inputs to
these circuits change in disease, which
will provide insight into how neuronal
dysfunction and degeneration lead to
circuit rewiring.
Circuit Plasticity
Another major focus of our lab is on the
function and plasticity of these circuits.
Plasticity is central to brain function and
allows us to adapt to our environment,
learn, form memories, and become
skilled at any number of tasks. In the
basal ganglia — the region of the brain
controlling motivation, action, and
movement — plasticity allows us to learn
how to coordinate precise movements or
actions to achieve successful outcomes,
such as hitting a golf ball straight or
picking up a cup of coffee. Diseases of
the basal ganglia disrupt our ability to
Wildtype
coordinate and perform movements and
actions, in part due to dysregulation of
plasticity in specific neural circuits. For
example, we recently discovered that
a protein called RGS4 (Regulator of
G-protein Signaling 4) disrupts normal
plasticity after dopamine loss, as occurs
in PD. Eliminating the function of
RGS4 allowed mice to better cope with
the effects of losing dopamine neurons (see figure). As we achieve a better
understanding of the neural circuits that
regulate movement — and the mechanisms of plasticity that govern their
rewiring — we will be able to discover a
host of new targets for treating different
aspects of PD and other neurodegenerative diseases.
RGS4–/–
Percentage of Time
70
**
40
Ambulation
**
3. Lerner TN et al. (2012) RGS4 is
required for dopaminergic control
of striatal LTD and susceptibility to
parkinsonian motor deficits. Neuron
73:347.
4.Nelson AB et al. Striatal cholinergic
interneurons drive GABA release
from dopamine terminals. Neuron
(in press).
20
Fine
Movement
10
Saline 6-OHDA
1. Gittis AH et al. (2011) Rapid
target-specific remodeling of fastspiking inhibitory circuits after loss
of dopamine. Neuron 71:858.
2. Kravitz AV et al. (2010) Regulation
of parkinsonian motor behaviours
by optogenetic control of basal
ganglia circuitry. Nature 466:622.
30
0
2. Kravitz AV et al. (2012) Distinct roles for direct and indirect
pathway striatal neurons in reinforcement. Nat. Neurosci. 15:816.
TOP FIVE OVERALL
60
50
1. Freeze BS et al. (2013) Control of
basal ganglia output by direct and
indirect pathway projection neurons. J. Neurosci. 33:18531.
Freezing
90
80
SELECTED RECENT
5. Wall NR et al. (2013) Differential
innervation of direct- and indirect-pathway striatal projection
neurons. Neuron 79:347.
110
100
Publications
Saline 6-OHDA
Eliminating RGS4 reduces motor deficits after dopamine loss in parkinsonian mice.
Wild-type (WT) or RGS4 knockout mice (RGS4–/–) were administered an inert saline
solution or the dopamine neurotoxin 6-OHDA, which caused the death of >90% of
dopamine neurons. Evaluation of the behavior of these mice revealed that RGS4–/– mice
treated with 6-OHDA spent less time immobile (“freezing”) and more time walking
around (“ambulation”) than WT mice, indicating that they were less susceptible to
developing motor impairments after dopamine loss. Asterisks indicate a significant
change in behavior (**p < 0.01).
3. Kravitz AV et al. (2012) Distinct roles for direct and indirect
pathway striatal neurons in reinforcement. Nat. Neurosci. 15:816.
4.Lerner TN et al. (2012) RGS4 is
required for dopaminergic control
of striatal LTD and susceptibility to
parkinsonian motor deficits. Neuron
73:347.
5. Wall NR et al. (2013) Differential
innervation of direct- and indirect-pathway striatal projection
neurons. Neuron 79:347.
President Emeritus and Senior Investigator
labs.gladstone.ucsf.edu/mahley-gind
HIGHLIGHTS
• Due to its abnormal structure,
apoE4 undergoes proteolysis in
neurons, generating neurotoxic
fragments
• ApoE4 structure correctors prevent
apoE4-related neurotoxicity in vitro
and in vivo
• ApoE4 structure correctors are
used as probes to elucidate apoE4
neuropathology
LAB MEMBERS
Yaisa Andrews-Zwilling
Walter Brecht
Dennis Miranda
Earl Rutenber
“Apolipoprotein E4 is
the greatest known
genetic risk factor
for developing
Alzheimer’s disease
and, thus, potentially
the key to its
treatment.”
A major goal in our laboratory is to reverse the detrimental effects of apoE4 by correcting its structure to
convert it into an apoE3-like molecule.
Several diseases, such as cystic fibrosis and
cancer, have been linked to proteinopathies associated with misfolded proteins
that can be caused by minor changes in
conformation that profoundly alter protein structure and function. Due to the
effects of proteinopathies, scientists are
developing small-molecule structure correctors capable of reversing the mishandling, sorting, and functional defects of
misfolded proteins. Of particular importance to our lab is the abnormal conformation of apolipoprotein (apo) E4 and
its subsequent pathological consequences
on neurodegenerative diseases, such as
Alzheimer’s disease, traumatic brain
injury, and multiple sclerosis.
ApoE Isoforms and Alzheimer’s
Disease
ApoE3, the most common isoform,
differs from apoE4 by a single amino
acid residue at position 112, which profoundly alters both the structure and
function of apoE4. That single substitution (cysteine to arginine) is essential for
converting apoE from a molecule that
supports neuronal maintenance, promotes neurite outgrowth and neuronal
repair, and protects neurons (apoE3) to
a molecule that becomes neuropathological (apoE4). Due to the substitution, the
structure of apoE4 is altered to a form
that is more susceptible to proteolytic
cleavage in neurons, resulting in the formation of highly toxic fragments. The
major neurotoxic fragment is apoE4
(1–272), which lacks the carboxylterminal 27 amino acids. This fragment,
and others subsequently generated,
escape the secretory pathway in neurons
and gain access to the cytosol, where they
can access intracellular organelles to produce toxicity. In our lab, we showed that
mitochondria are a major target of the
neurotoxicity associated with apoE4 and
apoE4(1–272) (see figure). Specifically,
apoE4 decreases levels of cytochrome c
oxidase subunit 1 (COX1) in neurons.
Small Molecules Control the Effects
of ApoE4
Two types of small molecules that protect neurons from apoE4-associated
neurotoxicity are (1) apoE4 structure
correctors (apoE4SCs) and (2) mitochondrial protectors. Previously, we
identified apoE4SCs that block the
apoE4 pathological conformation, converting it to an apoE3-like structure
and preventing the detrimental effects
of apoE4. In cultured neurons in vitro,
apoE4SCs restored neurite outgrowth,
rescued mitochondrial function and
normal cellular transport, and prevented
the generation of neurotoxic fragments.
Likewise, the prototypic apoE4SC,
PY-101, reduced neurotoxic fragment
generation and rescued COX1 levels in
the brains of apoE4 transgenic mice. In
addition, preliminary data establish that
PY-101 can reverse the impaired learning
and memory observed in apoE4 transgenic mice.
To identify small molecules that are
apoE4 mitochondrial protectors, we
screened 31,000 compounds by an automated, high-throughput system that
identifies molecules that prevent the
apoE4-induced reduction in COX1
levels in Neuro-2a cells. Approximately
116 potentially active compounds capable of enhancing COX1 levels were identified (0.37% hit rate). We have focused
on three initial classes of compounds
that display EC 50 of 400–1000 nM:
1) sulfonyl-imidazoles, 2) benzoxazoles,
and 3) isoxazoles. We are currently
determining whether these positive compounds act to rescue the apoE4 impaired COX1 levels by 1) serving as a
structure corrector to prevent fragment
formation, 2) blocking the interaction of
apoE4 fragments, 3) inducing COX1
transcription, or 4) increasing the
number of mitochondria.
Defining Mechanisms
All of these types of molecules are of
great interest for preventing or delaying
the detrimenta l ef fects of apoE4.
Elucidating the mechanism whereby
these compounds are active in enhancing
COX1 levels is under way. In addition,
determining the precise mechanism
whereby apoE4 causes mitochondrial
dysfunction is of critical importance to
our future endeavors.
“Mitochondrial
Protector”
ApoE
Fragment
Golgi
E3
E4
Protease
Mitochondrion
Energy
Disruption
ER
Stressor,
Injurious
Agents
ApoE
Synthesis
• Aging
• Oxidative Stress
• Trauma
• Aβ Deposition
SELECTED RECENT
1. Chen HK et al. (2012) Small molecule structure correctors abolish
detrimental effects of apolipoprotein E4 in cultured neurons. J. Biol.
Chem. 287:5253.
2. Hodoğlugil U et al. (2012) Turkish
population structure and genetic
ancestry reveal relatedness among
Eurasian populations. Ann. Hum.
Genet. 76:128.
3. Mahley RW et al. (2012) Apolipoprotein E sets the stage: response
to injury triggers neuropathology.
Neuron 76:871.
4.Mahley RW et al. (2012) Smallmolecule structure correctors
target abnormal protein structure
and function: the structure corrector rescue of apolipoprotein E–
associated neuropathology. J. Med.
Chem. 55:8997.
5. Naylor MD et al. (2012) Advancing
Alzheimer’s disease diagnosis,
treatment, and care: recommendations from the Ware Invitational
Summit. Alzheimers Dement. 8:445.
ApoE Secretion
ApoE4
Publications
Neurodegeneration
TOP FIVE OVERALL
1. Huang Y et al. (2001) Apolipoprotein E fragments present in
Alzheimer’s disease brains induce
neurofibrillary tangle-like intracellular inclusions in neurons. Proc.
2. Ignatius MJ et al. (1986) Expression
of apolipoprotein E during nerve
degeneration and regeneration.
3. Mahley RW (1988) Apolipoprotein
E: Cholesterol transport protein
with expanding role in cell biology.
Science 240:622.
Nucleus
Neuron
4.Nathan BP et al. (1994) Differential effects of apolipoproteins E3
and E4 on neuronal growth in vitro.
Science 264:850.
5. Rall SC Jr et al. (1982) Human
apolipoprotein E. The complete
amino acid sequence. J. Biol. Chem.
257:4171.
Pathological effects of apoE4 on mitochondria as a potential therapeutic target.
Neurons synthesize apoE in response to injury. ApoE4 undergoes proteolytic
cleavage, generating neurotoxic fragments that interact with mitochondria and
impair their function. ER, endoplasmic reticulum.
Lennart Mucke, MD
“This is a time of
unprecedented
challenges and
opportunities in
Alzheimer’s research.”
labs.gladstone.ucsf.edu/mucke
HIGHLIGHTS
• Non-convulsive epileptic activity
occurs in early AD; in AD models,
antiepileptic drugs lessen cognitive
deficits
• Blocking aberrant network activity
prevents and reverses neuronal
DNA damage
• Tau reduction is antiepileptogenic and prevents Aβ-induced
impairment
LAB MEMBERS
Biljana Djukic
Dena Dubal
Vira Fomenko
Makoto Furusawa
Ania Gheyara
Weikun Guo
Kaitlyn Ho
Erik Johnson
Jing Kang
Daniel Kim
Joseph Knox
Sumihiro Maeda
Takashi Miyamoto
Meaghan Morris
Anna Orr
Pascal Sanchez
Elsa Suberbielle
Praveen Taneja
Keith Vossel
Xin Wang
Gui-Qiu Yu
Lei Zhu
If we can prevent aberrant network activity in patients
with epilepsy and Alzheimer’s disease, we will be able
to kill two birds with one stone.
Alzheimer’s Disease and Epilepsy
Epileptic activit y a ssociated with
Alzheimer’s disease (AD) is harmful
for patients, can go unrecognized, and
may contribute to the illness. With the
Memory and Aging Center and the
Epilepsy Center at the University of
California, San Francisco, we investigated 54 patients with amnestic mild
cognitive impairment (aMCI) or early
AD who also had epilepsy or subclinical epileptiform activity. aMCI/AD
patients with epilepsy showed cognitive
decline roughly 5–7 years before those
without epilepsy. Patients with AD and
subclinical epileptiform activity also
experienced early cognitive decline. The
timing of seizure onset in patients with
aMCI and AD was non-uniform, clustering near the onset of cognitive decline.
Epilepsies were most often complex partial seizures, and, importantly, 55% were
non-convulsive. Serial or extended EEG
monitoring was more effective than routine electroencephalography (EEG) for
detecting interictal and subclinical epileptiform activity. Treatment outcomes
appeared to be better for lamotrigine and
levetiracetam (LEV) than for phenytoin.
Carefully identifying and treating epilepsy in patients with aMCI or AD may
improve their clinical course.
To explore this further, we examined
human amyloid precursor protein (hAPP)
transgenic mice, which simulate key
aspects of AD. We treated hAPP mice with
different antiepileptic drugs, but only LEV
reduced abnormal spike activity. Several
weeks of LEV treatment reversed hippocampal remodeling, behavioral abnormalities, synaptic dysfunction, and deficits in
learning and memory in hAPP mice (see
figure). Our findings suggest that aberrant network activity contributes causally
to synaptic and cognitive deficits in hAPP
mice. To evaluate LEV in people, we are
about to launch a clinical Phase IIa trial.
Neuronal DNA Integrity in Health
and Disease
Brains of AD patients show an abnormal
increase in DNA double-strand breaks
(DSBs) in neurons, but the underlying
mechanisms are unknown. We found
that hAPP mice also have increased neuronal DSBs in various brain regions, suggesting that Aβ accumulation is sufficient
to cause such DNA damage. Interventions
that suppress aberrant neuronal activity
and improve learning and memory in
hAPP mice, such as LEV treatment and
tau reduction, normalized DSBs. Blocking
extrasynaptic N-methyl-D-aspartate
(NMDA)-type glutamate receptors prevented amyloid beta (Aβ)-induced DSBs
We showed that tau ablation prevents
synaptic, network, and cognitive dysfunctions in hAPP mice and makes mice
more resistant to chemically induced
seizures. However, others reported that
aging Tau knockout mice develop a parkinsonian phenotype, raising concerns
about tau reduction as a therapeutic
approach. We assessed cognition and
motor functions in Tau+/+, Tau+/–, and
Tau–/– mice at 1 and 2 years of age. Tau
ablation did not impair cognition and
caused only minor motor deficits. Tau
ablation caused a mild increase in body
weight, which correlated with and might
have contributed to some of the motor
deficits. However, tau ablation did not
cause significant dopaminergic impairments, and dopamine treatment did not
improve the motor deficits, suggesting
that they do not reflect extrapyramidal
dysfunction. These findings encourage
us to pursue our quest to identif y
tau-lowering drugs with unabated vigor
and enthusiasm.
in neuronal cultures. Surprisingly, we
found that a natural behavior — exploring
a novel environment — caused DSBs in
neurons of young adult wild-type mice.
DSBs occurred in multiple brain regions,
were most abundant in the dentate gyrus
(involved in learning and memory), and
were repaired within 24 hours. Increasing
neuronal activity by sensory or optogenetic
stimulation increased neuronal DSBs in
relevant but not irrelevant networks. Thus,
transient increases in neuronal DSBs result
from physiological brain activity. Notably,
hAPP mice had more severe and prolonged
DSBs after exploration. We hypothesize
that Aβ peptides exacerbate neuronal DSBs
by eliciting synaptic dysfunction, activating endonucleases, and/or disrupting
DNA repair. Ongoing studies focus on
dissecting the underlying mechanisms and
evaluating the (patho)physiological roles of
neuronal DSBs in learning and memory.
Targeting Tau
Tau is expressed most highly in neurons.
Normalized fEPSP Slope (%)
A
NTG/Saline
NTG/LEV
hAPP/Saline
hAPP/LEV
NTG/Saline
200
hAPP/Saline
150
hAPP/LEV
100
0
1 mV
–20
0
20
C
Distance (cm)
1200
1000
800
600
0
40
NTG/Saline
NTG/LEV
hAPP/Saline
hAPP/LEV
1
2
3
Days
4
5
50
40
10 ms
60
Time (Minutes)
1400
400
SELECTED RECENT
1. Morris M et al. (2013) Age-appropriate cognition and subtle dopamine-independent motor deficits in
aged Tau knockout mice. Neurobiol.
Aging 34:1523.
2. Sanchez PE et al. (2012) Levetiracetam suppresses neuronal network
dysfunction and reverses synaptic
and cognitive deficits in an Alzheimer’s disease model. Proc. Natl.
Acad. Sci. USA 109:E2895.
3. Suberbielle E et al. (2013) Physiologic brain activity cause DNA
double-strand breaks in neurons,
with exacerbation by amyloid-β.
Nat. Neurosci. 16:616.
4.Verret L et al. (2012) Inhibitory
interneuron deficit links altered
network activity and cognitive
dysfunction in Alzheimer model.
Cell 149:708.
5. Vossel KA et al. (2013) Seizures
and epileptiform activity in the
early stages of Alzheimer’s disease.
JAMA Neurol. 70:1158.
TOP FIVE OVERALL
NTG/LEV
Time in Target Quadrant (%)
B
250
Publications
2. Hsia A et al. (1999) Plaque-independent disruption of neural circuits in
Alzheimer’s disease mouse models.
***
**
*
30
20
10
0
***
Saline LEV
NTG
1. Buttini M et al. (2002) Modulation
of Alzheimer-like synaptic and cholinergic deficits in transgenic mice
by human apolipoprotein E depends
on isoform, aging and overexpression of Aβ but not on plaque formation. J. Neurosci. 22:10539.
Saline LEV
hAPP
Antiepileptic drug treatment ameliorates functional deficits in an AD mouse model.
hAPP and non-transgenic (NTG) mice treated with LEV or saline for ~3 weeks
were assessed for synaptic and cognitive functions. A) Long-term potentiation, a
measure of synaptic plasticity, in the dentate gyrus of acute hippocampal slices
was impaired only in saline-treated hAPP mice. LEV, but not saline, treatment also
improved B) spatial learning and C) memory of hAPP mice in a water maze task.
*p < 0.05, **p < 0.005, ***p < 0.0005 vs 25%.
3. Palop JJ et al. (2007) Aberrant
excitatory neuronal activity and
compensatory remodeling of inhibitory hippocampal circuits in mouse
models of Alzheimer’s disease.
Neuron 55:697.
4.Roberson ED et al (2007) Reducing
endogenous tau ameliorates
amyloid β-induced deficits in an
Alzheimer’s disease mouse model.
Science 316:750.
5. Roberson ED et al. (2011) Amyloid-β/Fyn–induced synaptic, network, and cognitive impairments
depend on tau levels in multiple
mouse models of Alzheimer’s disease. J. Neurosci. 31:700
“Ultimately, we aim
to use our insights
into mitochondrial biology at the
synapse to therapeutically target
mitochondria.”
Ken Nakamura, MD, PhD
labs.gladstone.ucsf.edu/nakamura
HIGHLIGHTS
• We created new assays to measure
ATP requirements of the synaptic
vesicle cycle
• Vulnerable dopamine neurons
require mitochondrial fission to
transport mitochondria to the
nerve terminal
• Our new therapeutic approach
boosts the function of the PD
protein PINK1
LAB MEMBERS
Amandine Berthet
Dominik Haddad
Weiye Lin
Bryce Mendelsohn
Divya Pathak
Lauren Shields
Our research focuses on two related areas of mitochondrial biology at the synapse: bioenergetics and
mitochondrial dynamics.
The vast majority of mitochondria in
neurons are thought to be in axons,
where they play vital roles in supporting
synaptic function. Because synapse loss
occurs early in neurodegenerative diseases that involve mitochondria, such as
Parkinson’s disease (PD) and Alzheimer’s
disease (AD), changes in synaptic mitochondria may play a key role in the development of these diseases. Unfortunately,
the underlying mechanisms and nature
of the mitochondrial changes are poorly
characterized, especially at the synapse
and in vivo. To understand the relevance
of changes in axonal mitochondria to
disease progression, we must first understand the normal behavior and function
of mitochondria in this compartment.
Mitochondrial Bioenergetics
Although synaptic vesicle release may
be compromised early in degeneration,
we know very little about its energy
requirements because appropriate tools
and high-sensitivity assays have not been
available. To determine the dependence
of synaptic vesicle cycling on mitochondria-derived adenosine triphosphate
(ATP), we developed live-imaging assays
to measure ATP in individual synaptic
boutons. We found that basal ATP
levels and synaptic vesicle release can
be supported by aerobic or anaerobic
respiration, even in boutons lacking
mitochondria. This may explain why
mitochondria-based disease models often
fail to show the expected level of neuronal dysfunction and/or degeneration.
Interestingly, mitochondria are necessary
for maintaining energy requirements
when g lycolysis is compromised.
Under these conditions, pharmacologic
or genetic mitochondrial inhibition
decreases ATP below the threshold for
endocytosis. We are currently using the
aforementioned assays to determine if
PD and AD proteins decrease ATP and
whether this is sufficient to compromise
synaptic function and/or survival.
Mitochondrial Fission
Changes in mitochondrial dynamics —
the balance between mitochondrial fission and fusion — may have an important
role in neurodegenerative diseases. Most
of the central PD and AD proteins
affect this balance in cellular models,
but whether these changes contribute to
degeneration is unknown. To understand
this, we investigated the normal functions
of mitochondrial dynamics in susceptible
neuronal populations in vivo. We generated mouse models in which the key
mitochondrial fission protein Drp1 was
deleted in disease-relevant neuronal populations, thus circumventing the early
lethality that occurs when Drp1 is globally deleted from the brain. Drp1 loss
produced a robust and selective pattern
of degeneration of nigrostriatal dopamine neurons that began with the loss of
axonal mitochondria and early degeneration of synapses. We also defined a subset
of dopamine neurons with characteristic
electrophysiologic properties that survived in the adjacent ventral tegmental
area, despite losing their axonal mitochondria, suggesting that these neurons
need less energy. These findings suggest
that the depletion of axonal mitochondria
results from both an unexpected global
loss in mitochondrial mass and impaired
mitochondrial transport due to poorly
coordinated mitochondrial movements.
In hippocampal neurons, Drp1 loss produced markedly different results. CA1
hippocampal neurons survived without
Drp1. However, the hippocampal neurons did not fire normally and exhibited
changes in synaptic morphology, indicating mitochondrial dysfunction. These
model systems provide insight into the
distinct requirements for mitochondrial
fission in different neuronal types, and
they form the basis for ongoing studies
on how changes in the fission-fusion balance may influence the toxicity of key
PD and AD proteins in vivo.
Correcting Mitochondrial
Dysfunction
Ultimately, we aim to use our insights
into mitochondrial bioenergetics and
fission to therapeutically target mitochondria. We are starting to define new
molecular pathways for restoring and
boosting energy. In specific circumstances, we can directly target known
mitochondrial insults. For instance,
we made important contributions to
work in Dr. Kevan Shokat’s lab at the
(UCSF) characterizing a new therapeutic
strategy to boost the activity of the mitochondrial PD protein PTEN induced
putative kinase 1 (PINK1). These studies
are advancing into preclinical animal
models of sporadic PD, and possibly into
preliminary human studies of patients
with known PINK1 mutations.
Energy Failure in Individual Neurons
Does it occur in susceptible
neurons (cell body and/or processes)?
NO
YES
Does it occur sufficiently to
impair function and/or survival?
NO
YES
Continue to the following
areas of research.
Understanding
Disease Pathogenesis:
Does it have a primary or
secondary role in disease
pathogenesis?
Focus on other
mitochondrial
functions in neurons;
consider primary
bioenergetic changes
in glia.
Focus on other
mitochondrial
functions that may
have secondary
effects on energy levels.
Therapeutic Development:
Does restoring energy block
neuronal death?
Does the mechanism by which it
is reversed determine success?
Publications
SELECTED RECENT
1. Hertz NT et al. (2013) A neosubstrate that amplifies catalytic
activity of Parkinson’s disease
related kinase PINK1. Cell 154:737.
2. Manzanillo PS et al. (2013) The
ubiquitin ligase PARKIN is required
for autophagy and host resistance
to intracellular pathogens. Nature
501:512.
3. Nakamura K (2013) α-Synuclein
and mitochondria: partners in
crime? Neurotherapeutics 10:391.
4.Pathak D et al. (2013) Energy
failure — does it contribute to
neurodegeneration? Ann. Neurol.
74:506.
5. Skibinski G et al. (2014) Mutant
LRRK2 toxicity in neurons depends
on LRRK2 levels and synuclein but
not kinase activity or inclusion
bodies. J. Neurosci. 34:418.
TOP FIVE OVERALL
1. Hertz NT et al. (2013) A neosubstrate that amplifies catalytic
activity of Parkinson’s diseaserelated kinase PINK1. Cell 154:737.
2. Nakamura K et al. (2001) Tetrahydrobiopterin scavenges superoxide
in dopaminergic neurons. J. Biol.
Chem. 276:34402.
3. Nakamura K et al. (2008) Optical
reporters for the conformation of
α-synuclein reveal a specific interaction with mitochondria. J. Neurosci. 28:12305.
4.Nakamura K et al. (2011) Direct
membrane association drives mitochondrial fission by the Parkinson’s
disease-associated protein α-synuclein. J. Biol. Chem. 286:20710.
5. Pathak D et al. (2013) Energy
failure: does it contribute to neurodegeneration? Ann. Neurol. 74:506.
The schematic illustrates an algorithm of critical but challenging questions to determine if
energy failure occurs in individual neurons, including their processes, if and how it contributes
to degeneration, and how it might be targeted therapeutically. Figure reproduced from Pathak
et al. (2013) Energy failure — does it contribute to neurodegeneration? Ann. Neurol. 74:506.
Jorge J. Palop, PhD
“We are working to
restore brain rhythms
and cognition in
Alzheimer’s models
by enhancing
interneuron function.”
labs.gladstone.ucsf.edu/palop
• Inhibitory interneuron deficits
alter brain rhythms in Alzheimer’s
models
Impaired inhibitory interneurons and oscillatory
activity cause cognitive decline, but their manipulation
can improve function in Alzheimer’s disease models.
• Low levels of the Nav1.1 ion channel
underlie inhibitory interneuron
dysfunction
Brain Rhythms and Cognitive
Disorders Linked to Network
Instability
• Restoring inhibitory activity by
overexpressing Nav1.1 or transplanting interneurons improves
brain rhythms
Alzheimer’s disease (AD) results in the
deterioration of cognitive functions and
abnormal patterns of neuronal network
activity, but the underlying mechanisms
that drive AD pathogenesis are poorly
understood. Inhibitory neurons are critical in brain functioning because they
produce oscillatory rhythms that the brain
uses to organize information and precisely
time the neuronal firing required for cognitive processing. Because inhibitory
neurons play such a critical role in information processing, questions arise about
their role in AD. Does altered rhythmic
oscillatory activity contribute to memory
impairment? Can we prevent cognitive
decline by targeting inhibitory-dependent oscillatory network activity? Recent
findings from my laboratory indicate that
impaired inhibitory interneurons and
altered oscillatory activity contribute to
cognitive decline, and oscillatory network
activity can indeed be experimentally
manipulated to improve cognitive functions in a mouse model of AD (hAPPJ20
mice). Our findings were featured on the
cover of Cell, with the caption “Repairing
rhythms in Alzheimer’s models,” highlighting the mechanistic and therapeutic
HIGHLIGHTS
LAB MEMBERS
Abdullah Khan
Maria Martinez Losa
Laure Verret
value of our experimental approach.
Interestingly, alterations in inhibitory
neurons and oscillatory activity are also
associated with other neurological and
psychiatric disorders linked to network
instability, including cognitive aging,
epilepsy, schizophrenia, and autism.
Therefore, our research has implications
for multiple cognitive disorders.
Nav1.1-Dependent Inhibitory
Interneuron Dysfunction in
Alzheimer’s Disease
Inhibitory-excitatory loops generate specific oscillatory patterns of brain activity
(“brain rhythms”) that can be detected
by electroencephalography (EEG) in
behaving mice. In my laboratory, we
discovered that when parvalbuminexpressing cells (PV cells) — which are
inhibitory interneurons — are impaired,
they contribute to altered gamma oscillatory activity, network hyperexcitability,
and memory deficits in hAPPJ20 mice.
We also identified a molecular mechanism that impairs inhibitory function:
decreased levels of the voltage-gated
sodium channel Nav1.1 subunit, which
is specific to interneurons and predominates in PV cells. Voltage-gated
sodium channels control neuronal and
network excitability by modulating
synaptic activity in specific neuronal
subtypes. Reduced Nav1.1 levels are
also found in AD patients. Importantly,
restoring Nav1.1 levels by overexpressing
Nav1.1BAC (bacterial artificial chromosome) increased PV cell–dependent
gamma oscillatory activity and cognitive performance in hAPPJ20 mice (see
figure), revealing key functional roles for
Nav1.1- and PV cell–dependent gamma
oscillatory activity in cognition. Overall,
our data support the hypothesis that
impaired neuronal inhibition critically
contributes to network alterations (e.g.,
in oscillatory brain rhythms and cortical
hyperexcitability) and cognitive deficits
in hAPPJ20 mice. Thus, we are testing
therapeutic approaches to improve
inhibitory functions in hAPPJ20 mice.
Cell-Based Therapy for
Alzheimer’s Disease
We are also pioneering cell-based therapies to treat AD. We are using embryonic
interneuron precursors from the medial
ganglionic eminence (MGE) as a source
of inhibitory interneurons for cell-based
Control
Mouse
AD
Mouse
therapy in mouse models of AD. During
brain development, interneuron precursors are generated in the MGE and
migrate into the cortex and hippocampus. Unlike most other embryonic
neurons, MGE-derived precursors can
migrate and integrate into neonatal and
adult host brains where they mature into
functional and synaptically active inhibitory interneurons. Thus, MGE-derived
precursors are excellent candidates for
cell-based therapies to treat neurological
disorders linked to impaired inhibitory
function, such as AD, epilepsy, schizophrenia, and autism. Our data indicate
that genetically modified transplants of
MGE-derived interneuron precursors can
functionally restore brain rhythms and
memory in mouse models of AD. Using
MGE-derived interneurons in models of
complex diseases and genetically modifying MGE precursors to enhance their
therapeutic value represent groundbreaking advancements in the development of cell-based therapies for AD and
other neurodegenerative diseases.
AD Mouse
+ Nav1.1BAC
AD Mouse
+ MGE-derived Nav1.1BAC
Transgenic Inhibitory Cells
Inhibitory
PV Cells
Nav1.1
Nav1.1BAC
SELECTED RECENT
1. Heng MY et al. (2013) Lamin
B1 mediates cell-autonomous
neuropathology in a leukodystrophy mouse model. J. Clin. Invest.
123:2719.
2. Sanchez PE et al. (2012) Levetiracetam suppresses neuronal network
dysfunction and reverses synaptic
and cognitive deficits in mouse
model of Alzheimer’s disease. Proc.
Natl. Acad. Sci. USA 109:E2895.
3. Verret L et al. (2012) Inhibitory
network activity and cognitive dysfunction in Alzheimer model. Cell
149:708.
TOP FIVE OVERALL
1. Palop JJ et al. (2003) Neuronal
depletion of calcium-dependent
proteins in the dentate gyrus
is tightly linked to Alzheimer’s
disease-related cognitive deficits.
2. Palop JJ et al. (2006) A network
dysfunction perspective on neurodegenerative diseases. Nature
443:768.
Gamma
Oscillatory
Activity
Network
Stability
Cognitive
Function
Publications
+++
+
++
++
Inhibitory dysfunction and cognitive impairment in AD models. Reduced Nav1.1
levels in inhibitory PV cells critically contribute to abnormalities in neuronal
network activity and cognitive decline in AD mice (red). Enhancing inhibitory cell
activity by overexpressing Nav1.1 BAC (yellow) or by implanting genetically modified
embryonic interneuron precursors (green) in mouse brains improves cognition
in AD mice. We propose that increasing PV cell function may be therapeutically
beneficial in AD. MGE, medical ganglionic eminence.
3. Palop JJ et al. (2007) Aberrant
excitatory neuronal activity and
compensatory remodeling of inhibitory hippocampal circuits in mouse
models of Alzheimer’s disease.
Neuron 55:697.
4.Palop JJ et al. (2010) Amyloid-βinduced neuronal dysfunction in
Alzheimer’s disease: from synapses
toward neural networks. Nat. Neurosci. 13:812.
5. Verret L et al. (2012) Inhibitory
network activity and cognitive
dysfunction in Alzheimer model.
Cell 149:708.
The Roddenberry Center for Stem Cell Biology
and Medicine at Gladstone
The Roddenberry Center for Stem Cell Biology and Medicine
at Gladstone was inaugurated in 2012 with a generous gift from
the Roddenberry Foundation, which was established to honor
the legacy of Eugene Roddenberry, creator of Star Trek. The
Center aims to harness the tremendous potential of stem cell
biology and regenerative medicine to address some of mankind’s most intractable diseases. We have interlaced the Stem
Cell Center with Gladstone’s deep knowledge of human diseases of the brain, heart, and immune system to make key
discoveries related to our areas of expertise. Over half of all
Gladstone investigators utilize stem cell biology and regenerative medicine approaches, with a major emphasis on cellular
reprogramming technology pioneered by Gladstone investigator Dr. Shinya Yamanaka. Drs. Deepak Srivastava and Sheng
Ding have developed “next-generation” cellular reprogramming approaches described in more detail below.
To celebrate the new Center and to highlight the particular
strengths of our program, we held an International Symposium
on Cellular Reprogramming in October 2012, which was
co-sponsored by the International Society for Stem Cell
Research. Over 450 attendees celebrated not only the Center,
but also the newly announced Nobel Laureates Dr. John
Gurdon and Dr. Yamanaka, who were together for the first
time since the Nobel Prize announcement a few weeks earlier.
Over the last 2 years, the Gladstone stem cell program has
made a number of important advances in three broad areas of
cellular reprogramming.
Reprogramming Cells to Pluripotency
At Gladstone, the laboratories of Drs. Yamanaka and Ding
have made new insights into how human fibroblasts are
reprogrammed into induced pluripotent stem (iPS) cells and
refined the production of such cells. Dr. Yamanaka described
barriers that can be overcome to increase the quality and efficiency of iPS cell reprogramming, while Dr. Ding identified
chemical cocktails that promote iPS cell reprogramming.
Several Gladstone investigators have improved the differentiation of human iPS cells into cardiomyocytes, endothelial cells,
and multiple specific neuronal cell types.
Direct Reprogramming of Cell Fate
Gladstone investigators have been at the forefront of the
next generation of cellular reprogramming, which involves
switching a cell’s fate without first passing through a pluri
potent state. Dr. Srivastava’s laboratory directly reprogrammed cardiac fibroblasts into cardiomyocyte-like cells
in vivo, which represents a new approach, to regenerate
damaged hearts. Drs. Ding and Yadong Huang developed
approaches to convert human fibroblasts directly into neurons. Dr. Ding has pioneered an approach, to partially reprogram fibroblasts toward pluripotency using factors identified
by Dr. Yamanaka, then redirect them toward specific cell
types using growth factors and chemicals. He used this
approach to directly reprogram cells into cardiomyocytes,
neurons, endothelial cells, liver cells, and insulin-producing
pancreatic cells.
Human Disease Modeling
Gladstone investigators have leveraged the power of human iPS
cells derived from patients to discover new mechanisms of disease, building on their deep knowledge of specific human disease processes and robust animal models. Dr. Steve Finkbeiner
has used unique robotic microscopy to reveal pathology in
iPS–derived neurons from patients with Huntington’s and
Parkinson’s disease, and amyotrophic lateral sclerosis (ALS),
which are crippling neurodegenerative diseases with no cures.
Dr. Huang used iPS cells to model Alzheimer’s disease and
deployed gene-editing technologies to correct mutations in
human cells. Drs. Srivastava, Bruce Conklin, and Benoit
Bruneau have generated iPS cells from many patients with
cardiovascular disorders, revealing new mechanisms underlying pathologic processes, and Dr. Conklin has generated new
methods to engineer the genome in human iPS cells. Gladstone
scientists are now pioneering the use of direct reprogramming
approaches to model human diseases.
The ability to reprogram human cells offers unprecedented
opportunity to identify new targets for treating human disease
and to regenerate damaged tissues that underlie currently
intractable diseases. The Center continues to drive this field
forward to achieve its full potential.
Director, The Roddenberry Center
for Stem Cell Biology and Medicine at Gladstone
SELECTED PUBLICATIONS
1. Bershteyn M et al. Cell-autonomous
correction of ring chromosomes in
human induced pluripotent stem cells.
Nature (in press).
6. Miyaoka Y et al. Isolation of single-base genome-edited human iPS
cells without antibiotic selection.
Nat. Methods (in press).
2. Fong H et al. (2013) Genetic correction of tauopathy phenotypes in
neurons derived from human induced
pluripotent stem cells. Stem Cell
Reports 1:226.
7. Qian L et al. (2012) In vivo reprogramming of murine cardiac fibroblasts
into induced cardiomyocytes. Nature
485:593.
3. Fu J et al. (2013) Direct reprogramming of human fibroblasts toward a
cardiomyocyte-like state. Stem Cell
Reports 1:235.
4. HD iPS Consortium (2012) Induced
pluripotent stem cells from patients
with Huntington’s disease show
CAG-repeat-expansion-associated
phenotypes. Cell Stem Cell 11:264.
5. Li K et al. Small molecules facilitate
the reprogramming of mouse fibroblasts into pancreatic lineages. Cell
Stem Cell (in press).
8. Ring KL et al. (2012) Direct reprogramming of mouse and human fibroblasts into multipotent neural stem
cells with a single factor. Cell Stem Cell
11:100.
9. Serio A et al. (2013) Astrocyte
pathology and the absence of non-cell
autonomy in an induced pluripotent
stem cell model of TDP-43 proteinopathy. Proc. Natl. Acad. Sci. USA
110:4697.
10.Tomoda K et al. (2012) Derivation
Cell 11:91.
and coordinated epigenetic regulation
of developmental transitions in the
cardiac lineage. Cell 151:206.
12. Wang H et al. Small molecules enable
cardiac reprogramming of mouse
fibroblasts with a single factor, Oct4.
Cell Reports (in press).
13. Worringer K et al. (2014) The let-7/
LIN-41 pathway regulates reprogramming to human induced pluripotent
stem cells by controlling expression
of prodifferentiation genes. Cell Stem
Cell 14:40.
14.Zhu S et al. Mouse liver repopulation
with hepatocytes generated from
human fibroblasts. Nature (in press).
Lennart Mucke, Kiichiro
Tomoda, Bruce Conklin,
Steve Finkbeiner; middle row:
Sheng Ding, Warner Greene,
Robert Mahley, Shinya
Yamanaka, Yadong Huang;
front row: Eric Verdin,
Benoit Bruneau, Kathryn Ivey,
Deepak Srivastava. Not
pictured: Shomyseh Sanjabi.
Gladstone Center for Translational Research
The goal of the Gladstone Center for Translational Research
(GCTR) is to facilitate and expedite the process that leads from
unravelling the biology of disease to improving the lives of
patients. The head of the GCTR is Dr. Stephen Freedman, who
has over 25 years of experience leading research and development
efforts within multinational pharmaceutical and entrepreneurial
biotechnology companies. He brings an industrial perspective
to Gladstone programs that has been an asset in facilitating the
formation of strategic alliances and partnerships focused on
the translation of Gladstone discoveries to the patient setting.
Dr. Freedman is supported by a cadre of external experts in all
aspects of drug discovery and development. Together, the GCTR
team remains actively involved in collaborations, utilizing their
knowledge and skills to promote success. The GCTR also creates tactical collaborations that bring together the unique disease
understanding of Gladstone scientists with specific foundations,
pharmaceutical and biotechnology companies, venture investors, and philanthropists, to create a more globally unified path
toward better patient care. Recognizing that the most successful
partnerships are where all parties benefit, the GCTR is adept at
identifying creative and flexible solutions to help bring scientific
discoveries to fruition.
As an integral part of the Gladstone Institutes, the GCTR
has made many successful contributions, as evidenced by the
number, breadth, and diversity of alliances developed. The following selection is illustrative of these collaborations, many of
which have been renewed or expanded.
Corporate Sponsored Research Agreements with
Pharmaceutical and Biotechnology Companies
A partnership between Bristol-Myers Squibb and Drs.
Lennart Mucke and Li Gan is identifying and validating targets related to a protein called tau, which has been implicated
in the pathogenesis of Alzheimer’s disease (AD).
Seminal work on the mechanisms of human immunodeficiency virus (HIV) latency by Dr. Warner Greene led to a
partnership with JT Pharma to determine methods of eradicating the latent virus.
A research collaboration with Takeda Pharmaceuticals led
by Dr. Mucke is focused on the evaluation of new drugs for
neurological diseases.
Coupling pioneering research on the role of blood proteins
in neurovascular disease with newly created imaging technologies, Dr. Katerina Akassoglou has forged an alliance with
H. Lundbeck A/S to promote translation of these discoveries
into new therapeutic agents benefiting patients with multiple
sclerosis and other neuroinflammatory diseases.
In addition to the above, the GCTR has supported the
establishment of a number of productive industrial collaborations over the last 18 months, which include Genentech,
Amgen, Acumen, Envoy/Takeda, and Ono.
Alliances with Foundations
Dr. Steve Finkbeiner is using induced pluripotent stem (iPS)
cell technology to create neurons derived from a specific set of
patients with amyotrophic lateral sclerosis (ALS). These patients
harbor a gene mutation that produces a particular protein implicated in the disease called TDP-43. This work is supported by
the ALS Therapy Development Institute and is searching for
drugs that suppress neurodegeneration.
Dr. Robert Mahley has worked with the Alzheimer’s Drug
Discovery Foundation to identify potential small-molecule
therapeutics to prevent the mitochondrial dysfunction that
underlies the neurotoxicity of apolipoprotein E4, the main
genetic risk factor for AD.
Gladstone scientists have established a number of productive relationships using California Institute for Regenerative
Medicine (CIRM) translational funding, including the laboratories of Drs. Deepak Srivastava and Finkbeiner. This mechanism remains an important contributor to the exploration of
our expertise in stem cell biology.
Strategic Partnerships among Multiple Entities
The National Multiple Sclerosis Society’s Fast Forward,
LLC and EMD Serono (an affiliate of Merck KGaA) recently
completed a collaboration with Dr. Akassoglou focused on
blocking the ability of fibrinogen to directly activate brain
immune cells called microglia, a strategy that could aid in the
treatment of multiple sclerosis.
Drs. Mahley and Yadong Huang are working with the
chemoinformatics company Numerate to identify smallmolecule therapeutics to prevent the neurotoxicity of apolipoprotein
E4 in patients with AD. This research is funded by the Wellcome
Trust under their Seeding Drug Discovery Award program.
The GCTR has also supported the establishment of strategic relationships with chemistry companies that complement
the biological expertise within the Gladstone Institutes. These
include Numerate, Nanosyn, and JS Innopharm, Inc. Funding
for these has come from a variety of mechanisms, including the
CIRM and the Small Business Innovation Research program.
Support of Transformative Translational Initiatives
The GCTR is fully integrated with and supports a number of
translational centers and programs that were initiated through
the vision of Gladstone investigators and the generosity of private donors.
The Taube-Koret Center for Neurodegenerative Disease
Research, led by Dr. Finkbeiner, was founded to develop
better treatments for Huntington’s disease. Its mission has
been expanded to identify common threads between this and
other crippling neurodegenerative diseases that might allow
for the identification of drugs with broad therapeutic potential. Closely related to this center is the Hellman Family
Foundation Alzheimer’s Research Program, also directed
by Dr. Finkbeiner, which helps leverage discoveries made in the
Finkbeiner laboratory or the Taube-Koret Center into better
treatments for AD.
The Roddenberry Center for Stem Cell Biology and
Medicine (see page 74), led by Dr. Srivastava, builds on the
pioneering work of Gladstone’s 2012 Nobel laureate, Dr.
Shinya Yamanaka. Its aim is to create more relevant and informative human disease models from patient-derived iPS cells.
This approach is expected to improve the likelihood of successfully translating research findings to patients.
The Center for Comprehensive Alzheimer’s Disease
Research, directed by Dr. Mucke, was initiated through a
lead gift from the S.D. Bechtel, Jr. Foundation. It currently
supports 11 projects focused on the development and assessment of better drug treatments for AD. Like the other centers
focused on neurodegenerative disorders, this center collaborates
closely with the Memory and Aging Center at the University
of California, San Francisco (UCSF) and aims to advance the
drug discovery process to a stage where industrial partners can
be attracted and engaged for further development.
Robotic microscopy in the laboratory of Dr. Steve Finkbeiner can follow the fate of
specific cells over time. In this image of artificially colored cells, the red cell eventually dies and breaks up, while the blue cell continues growing.
Facilitating Research: Gladstone’s Core Laboratories
LAB MEMBERS
Behavioral
Assessment Core
Pascal Sanchez (acting)
Ravi Ponnusamy (former)
Ryan Craft
Allyson Davis
Iris Lo
Daniel Zwilling
Bioinformatics Core
Alisha Holloway
Mariel Finucane
Kristina Hanspers
Tim Laurent
Samad Lotia
Justin Nand
Alex Pico
Anders Riutta
Sean Thomas
Alexander Williams
Electron Microscopy Core
Jinny Wong
Flow Cytometry Core
Marielle Cavrois
Marianne Gesner
Genomics Core
Robert Chadwick
Yanxia Hao
James McGuire
Linda Ta
Histology and Light
Microscopy Core
Caroline Miller
Kate Bummer
Ana Robles
Stem Cell Core
Kathryn Ivey
Joshua Arnold
Toni Chau
John Chunko
Uma Lakshmanan
Transgenic Gene-Targeting Core
Junli Zhang
Carlisa Benitez
Isidro Espineda
Richard MacDonald
John Taylor
The techniques and instrumentation
used in biomedical research have become
ever more sophisticated and expensive,
placing them out of reach for many
laboratories. Gladstone has solved this
problem by concentrating resources and
skilled personnel in core laboratories that
serve all Gladstone investigators.
The Behav iora l Core (Acting
Director: Pascal Sanchez, PhD) analyzes neurobehavioral functions such as
motor coordination, social interactions,
and learning and memory in mice.
Core staff phenotype transgenic mouse
models of human neurologic and psychiatric diseases and evaluate the effects
of environmental, pharmacological, and
genetic interventions.
The Bioinformatics Core (Director:
Alisha Holloway, PhD) provides expertise in experimental design, biostatistics,
high-throughput data analysis, data integration, and pathway and network analysis and visualization.
The Electron Microscopy Core
(Director: Jinny Wong) provides consultation and services for sample fixation,
embedding, ultramicrotomy, and transmission electron microscopy of tissues,
cells, organelles, and viruses. The Core
also offers negative staining of subcellular
and particulate samples, and assists with
the analysis and interpretation of images.
The Flow Cy tomet r y Core
(Director: Marielle Cavrois, PhD) provides flow cytometry and cell-sorting
services. The Core maintains several
instruments that give Gladstone investigators a range of experimental options.
The Core also offers assistance with data
analysis, advice on specific applications,
and training for users.
The Genomics Core (Director:
Robert B. Chadwick, PhD) specializes in characterizing large numbers of
genes simultaneously with microarrays
and library preparation for next-generation sequencing instruments. The
Core offers whole-transcript microarrays and microRNA arrays. The Core
offers Paired-end Genomic, RNA-Seq
libraries, and ChIP-Seq libraries for
next-generation sequencing. Sequencing
services are also offered for both the
Illumina MiSeq and Ion Torrent Proton
next-generation sequencing instruments.
The Histolog y a nd Light
Microscopy Core (Director: Caroline
Miller) provides investigators the expertise, instrumentation, and training to
generate and capture research data as
images. Services include paraffin and
cryo preparation of tissues, sectioning, a
wide selection of special stains, and
immunohistochemistry and histochemistry techniques. The Core offers brightf ield, phase, epif luorescence, and
confocal light microscopy equipment,
services, training, and support. Core
staff also assists with the quantitation,
analysis, interpretation, and presentation
of the images, providing researchers with
support and advice from tissue collection
to figure presentation.
The Stem Cell Core (Director:
Kathryn Ivey, PhD) is a shared research
facility open for use by scientists who
wish to employ mouse and human stem
cell research techniques. The Core offers
comprehensive training, all necessary
equipment and reagents, and technical
support for studying human and mouse
induced pluripotent stem (iPS) cells and
embryonic stem cell lines. They also provide human iPS cell derivation services
and teratoma formation to test cell lines
for pluripotency.
The Transgenic Gene-Targeting
Core (Director: Junli Zhang, MD)
has produced important mouse models
of human disease for investigators at
Gladstone. The service involves the injection of DNA fragments to produce transgenic mice or, alternatively, injection of
embryonic stem cells to create chimeric
or knockout mice.
Gladstone Institutes Publications List
CARDIOVASCULAR DISEASE PUBLICATIONS
Almeida S, Zhang Z, Coppola G, Mao W, Futai K, Karydas A,
Geschwind MD, Tartaglia MC…Farese RV Jr, Gao FB (2012)
Induced pluripotent stem cell models of progranulin-deficient frontotemporal dementia uncover specific reversible neuronal defects. Cell
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Ring KL, Tong LM, Balestra ME, Javier R, Andrews-Zwilling
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Direct reprogramming of mouse and human fibroblasts into
multipotent neural stem cells with a single factor. C ell Stem Cell
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Sanchez PE, Zhu L, Verret L, Vossel KA, Orr AG, Cirrito JR,
Devidze N, Ho K…Palop JJ, Mucke L (2012) Levetiracetam
suppresses neuronal network dysfunction and reverses synaptic and
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and the absence of non-cell autonomy in an induced pluripotent stem
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neurodegenerative disease. FEBS Lett.587:1139–1146.
Skibinski G, Nakamura K, Cookson M, Finkbeiner S (2014) Mutant
LRRK2 toxicity in neurons depends on LRRK2 levels and synuclein
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8:1931–1938.
THE GLADSTONE INDEX
Year established:
1979
Total investigators:
28
Total scientific staff:
323
Percentage of NIH grant applications
funded at Gladstone in 2012:
37.9%
Percentage of NIH grant applications
funded nationally in 2012:
20.0%
Total Gladstone publications, 2012–2013:
374
Number of years in The Scientist top-10
list of Best Places to Work in Academia:
8 of the last 8
Number of years in The Scientist top-10
list of Best Places to Work for Postdocs:
7 of the last 8
Average number of annual consulting
agreements with private industry:
20
Leverage factor of private philanthropy
to additional funding:
5:1
Election to societies and major prizes awarded
National Academy of Sciences,
Institute of Medicine, and American
Academy of Arts and Sciences:
7 are members of one or more
Association of American Physicians:
9 are members; 1 is president
Major Prizes:
Nobel, Lasker, Wolf, Shaw, Gairdner,
Potamkin, Passano, Millennium
Technology, Mead Johnson,
Scientific American 50, Time 100,
MetLife, Presidential Early Career,
Life Sciences Breakthrough, New
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Scientific Report 2014

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