Michael, Marcia, and Christa Parseghian Scientific Conference for

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Michael, Marcia, and Christa Parseghian Scientific Conference for
Michael, Marcia, and Christa Parseghian Scientific Conference
for Niemann- Pick Type C Research
June 12-14, 2014
niemannpick.nd.edu
Table of Contents
Recently Funded APMRF Grants
1
Scientific Advisory Board Members
2
Program
3
Abstracts
12
Attendees
43
Community Resources
45
Save the Date
46
Ara Parseghian Medical Research Foundation
Scientific Advisory Board
6/2014
William Balch, Ph.D.
Professor
The Scripps Research Institute
Department of Cell & Molecular
La Jolla, CA
William Pavan, Ph.D.
Public Health Service
National Institutes of Health
Bethesda, MD
Michael S. Parmacek, M.D.
Herbert C. Rorer Professor of Medical Sciences
Chief, Division of Cardiovascular Medicine
The University of Pennsylvania
Philadelphia, PA 19104
Matthew Scott, Ph.D.
Stanford University School of Medicine
Stanford, CA
Marc C. Patterson, M.D.,
Mayo Clinic
Rochester, MN
2
2014 Michael, Marcia, and Christa Parseghian
Scientific Conference for
Niemann-Pick Type C Research
June 12-14th, 2014
THURSDAY, JUNE 12
11:00 a.m. – Noon
Registration
Jordan Hall Galleria
Noon – 1:15 p.m.
LUNCH
Jordan Hall Galleria
Jordan Hall of Science, Room 105
1:15 – 1:30 p.m.
Opening Remarks
Cindy Parseghian
Ara Parseghian Medical Research Foundation
SESSION I: (CLOSED; RESEARCHERS ONLY)
Jordan Hall of Science, Room 105
1:30 p.m.
DVT Presentation for Family Members in Jordan Hall of Science, Room 100
1:30 – 2:00 p.m.
Identification of Cerebrospinal Fluid Protein Biomarkers in
Niemann-Pick Disease, type C1
Stephanie M. Cologna1, Christopher A. Wassif1, Nicole M. Yanjanin1,
Peter S. Backlund2, Brian C. Searle3, Alfred L. Yergey22, and Forbes D.
Porter1
1Program in Developmental Endocrinology and Genetics, Eunice Kennedy
Shriver National Institute of Child Health and Human Development, National
Institutes of Health, 2Biomedical Mass Spectrometry Facility, Eunice Kennedy
Shriver National Institute of Child Health and Human Development, National
Institutes of Health, 3Proteome Software, Inc.
[Abstract #1, p. 12]
2:00 – 2:30 p.m.
A Drosophila Screen for Genes that Interact with Npc1
Luis A. Milla, Shivani Baisiwala, Karthik Ramasubramanian, Jian Cao,
and Matthew P. Scott
Departments of Developmental Biology, Genetics, and Bioengineering, Stanford
University
[Abstract #2, p. 13]
3 2:30 – 3:00 p.m.
A new formulation to treat neurological and systemic disease in Niemann
Pick Type C
Md. Suhail Alam1, 2, Michelle Getz1, 2 and Kasturi Haldar1, 2
1Center for Rare and Neglected Diseases, 2Department of Biological Sciences
University of Notre Dame, IN 46556, USA
[Abstract #3, p. 14]
3:00 – 3:15 p.m.
Discussion
3:15 – 3:30 p.m.
COFFEE BREAK
Jordan Hall Galleria
SESSION II: (CLOSED; RESEARCHERS ONLY)
Jordan Hall of Science, Room 105
3:30 – 4:00 p.m.
Progress Toward the Development of Water-°©‐soluble and Long
Circulating HP-°©‐β-°©‐CD Polyrotaxanes as Potential NPC Therapeutics
Chris J. Collins, Yawo A. Mondijou, and David H. Thompson
Department of Chemistry Purdue University West Lafayette, Indiana 47907
[Abstract #4, p. 15]
4:00 - 4:30 p.m.
Intracisternal cyclodextrin ameliorates neurological dysfunction,
increases survival time, and stops Purkinje cell death in feline NiemannPick type C1 disease.
C. H. Vite1, J.H. Bagel1, G. P. Swain1, M. Prociuk1, T. U. Sikora2, V. M.
Stein1, P. O’Donnell2, T. Ruane2, S. Ward1, A. Crooks1, S. Li1, E.
Mauldin2, S. Mellon3, D. S. Ory4, M. L. Kao5, M. De Meulder6, C.
Davidson7, M. T. Vanier8, S. U. Walkley7
1Department of Clinical Studies, School of Veterinary Medicine, University of
Pennsylvania, Philadelphia, Pennsylvania, 2Department of Pathobiology, School
of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania,
3Department of Obstetrics, Gynecology, and Reproductive Sciences, University
California, San Francisco, California, 4Diabetic Cardiovascular Disease Center,
School of Medicine, Washington University School of Medicine, St Louis,
Missouri, 5Janssen Research and Development, Janssen Pharmaceutical
Companies of Johnson and Johnson, Titusville, New Jersey, 6Janssen Research
and Development, Janssen Pharmaceutical Companies of Johnson and Johnson,
Antwerp, Belgium, 7Dominick P Purpura Department of Neuroscience, Rose F
Kennedy Intellectual and Developmental Disabilities Research Center, Albert
Einstein College of Medicine, Bronx, New York, 8Institut National de laSante´ et
de la Recherche Me´dicale (INSERM), Lyon, France
[Abstract #5, p. 16]
4:30 – 5:00 p.m.
Intermediate Size Patient Population IND for 2-HydroxypropylCyclodextrin (HP-CD) Outpatient Treatment of Siblings with Niemann-
4 Pick Type C1 (NP-C1) and Disparate Rates of Progression: Exploration of
Outcome Measures and Biomarkers
Elizabeth Berry-Kravis1, 2, 3, Joanne O’Keefe2, 4, Tamar Pounardjian1,
Jamie Chin1, Forbes Denny Porter5, Daniel Ory6, Suhail Alam7, Kasturi
Haldar7
1Departments of Pediatrics, 2Neurological Sciences, 3Biochemistry, and
4Anatomy, Rush University Medical Center, Chicago, IL; 5National Institute of
Child Health and Human Development, National Institutes of Health;
6Departments of Medicine and Cell Biology and Physiology, Washington
University, St. Louis, MO; 7Center for Rare and Neglected Diseases and
Department of Biological Sciences, University of Notre Dame, Notre Dame, IN
[Abstract #6, p. 17]
5:00 – 5:30 p.m.
Therapeutic trials for Niemann-Pick Disease, Type C1:
2-Hydroxypropyl-β-Cyclodextrin
Forbes D. Porter and the TRND Team (Nicole Yanjanin1, Aiyi Liu
(DESPR)1, Roopa Shankar1, Daniel Ory2, Bill Pavan3, Charles Vite4,
Russell Lonser5, John Heiss5, Steven Walkley6, Chris Austin7, John
McKew7, Nuria Carrillo7, Liz Ottinger7, Juan Marugan7, Pramod Terse7,
Xin Xu7, Wei Zeng7, Steven Silber8, Mark Kao8, Marcus Brewster8, Jon
Stocker9, Charles Finn10, Frank Hurley10, Joy Vanderwal10, Patrick
Frenchick10, Sandra Morseth10, Kimberly Lilly10, Carmen Brewer11, Beth
Solomon11, Naomi O’Grady11, Kelly King11, Ariane Soldatos5, 12
1NICHD, 2Washington University School of Medicine, 3NHGRI, 4University of
Pennsylvania, 5NINDS, 6Albert Einstein Collage of Medicine, 7TRND/NCATS,
8Johnson and Johnson, 9SAIC, 10RRD International, 11Clinical Center, 12NIH
[Abstract #7, p. 18]
5:30 – 5:45 p.m.
Discussion
6:30 – 7:30 p.m.
RECEPTION
Morris Inn Ball Room
7:30 – 9:00 p.m.
DINNER
Cindy Parseghian and Nadine Hill Presentations
Morris Inn Ball Room
FRIDAY, JUNE 13
SESSION III: (CLOSED; RESEARCHERS ONLY)
Jordan Hall of Science, Room 105
8:30 – 10:15 a.m.
Family Member Discussion and Continental Breakfast in the Jordan Hall of
Science Reading Room
8:00 – 8:30 a.m.
Tracing the Development of Histone Deacetylase Inhibitors as Potential
Therapeutic Agents for Niemann-Pick Type C Disease
5 Olaf Wiest and Paul Helquist
University of Notre Dame
[Abstract #8, p. 19]
8:30 – 9:00 a.m.
Testing Histone Deacetylase Inhibitors as NPC Therapeutics
Frederick R. Maxfield, Deepti Gadi, Shu Mao, and Nina Pipalia
Weill Cornell Medical College, New York, NY
[Abstract #9, p. 20]
9:00 – 9:30 a.m.
Proteostasis Regulators Reprogram Variant NPC1 Folding Environments
to Mitigate Cholesterol Homeostasis in Disease
Kanagaraj Subramanian1, Jason Gestwicki2, Fred Maxfield3 and
William E. Balch1, 4
1The Scripps Research Institute (TSRI), 2Departments of Molecular and Cell
Biology and Chemical Physiology, 4The Skaggs Institute of Chemical Biology, La
Jolla, California 92037; University California San Francisco (UCSF) School of
Medicine, 2Institute for Neurodegenerative Disease, San Francisco, California
94158; 3Weill Cornell Medical College, Department of Biochemistry, Weill
Cornell Medical College, New York, NY 10065
[Abstract #10, p. 21]
9:30 – 10:00 a.m.
A murine Niemann-Pick C1 (NPC1) I1061T knockin model recapitulates
the pathological features of the most prevalent human disease allele
Maria Praggastis1, Brett Tortelli1, Jessie Zhang1, Hideji Fujiwara1,
Rohini Sidhu1, Anita Chacko1, Zhouji Chen1, Andrew P. Lieberman2,
Cristin Davidson3, Steven U. Walkley3, Nina H. Pipalia4, Frederick R.
Maxfield4, Jean E. Schaffer1, and Daniel S. Ory1
1Washington University School of Medicine, St. Louis, MO 63110; 2University
of Michigan, Ann Arbor, MI; 3Albert Einstein College of Medicine, NY; 4Weill
Cornell Medical College, New York, NY
[Abstract #11, p. 22]
10:00 – 10:15 a.m.
Discussion
10:15 – 10:30 a.m.
COFFEE BREAK
Jordan Hall Galleria
SESSION IV: (CLOSED; RESEARCHERS ONLY)
Jordan Hall of Science, Room 105
10:30 – 11:00 a.m.
Inhibition of histone deacetylation in a mouse model of Niemann Pick
Type C disease
Andrew B. Munkacsi1, Natalie Hammond1, and Stephen L. Sturley2
1School of Biological Sciences and Centre for Biodiscovery, Victoria University of
Wellington, Wellington, New Zealand 6012, 2Department of Pediatrics,
Columbia University Medical Center, New York, NY 10032
[Abstract #12, p. 23]
6 11:00 – 11:30 a.m.
The Active Phosphorylated Form of FTY720 Is a Histone Deacetylase
Inhibitor that Enhances NPC1 Expression
Sarah Spiegel
VCU School of Medicine, Department of Biochemistry and Molecular Biology,
Richmond, VA
[Abstract #13, p. 24]
11:30 – 12:00 p.m.
Therapeutic trials for Niemann-Pick Disease, Type C1: Proof of Concept
HDAC Inhibitor Trial
Fred Maxfield1, Daniel Ory2, Forbes Porter3, 4, Paul Helquist5, Olaf
Wiest5, Edward Holson6, Xu Xin4, 7, Marc Patterson8, Mathew Berg5,
Cindy Parseghian9, Stephen Gately10
1Weill Cornell Medical College, 2Washington University, 3NICHD, 4NIH,
5Notre Dame, 6Broad Institute, 7NCATS, 8Mayo Clinic, 9APMRF, 10TD2
[Abstract #14, p. 25]
12:00 – 12:15 p.m.
Discussion
12:15 – 1:30 p.m.
LUNCH
SESSION V:
Jordan Hall of Science, Room 105
1:30 – 2:00 p.m.
rHSP70: A novel therapeutic opportunity for lysosomal storage diseases
Thomas Kirkegaard Jensen1, 4, James Gray2, Ole Dines Olsen1, 4,
Svetlana Drndarski3, Linda Ingemann1, Signe Humle Jørgensen1, Ian
Williams2, Kerri-Lee Wallom2, David A Priestman2, David Begley3,
Marja Jäättelä4, Frances M. Platt2
1Orphazyme ApS, Copenhagen, Denmark, 2Department of Pharmacology,
Oxford University, Oxford, UK, 3Institute of Pharmaceutical Science, King’s
College London, London, UK, 4Department of Cell Death and Metabolism,
Danish Cancer Society Research Center, Copenhagen, DK
[Abstract #15, p. 26] 2:00 – 2:30 p.m.
Rational drug discovery using patient-derived human-induced
pluripotent stem cell models of Niemann-Pick type C1
Lawrence S.B. Goldstein, PhD and Paulina Ordonez-Naranjo, MD
University of California San Diego
[Abstract #16, p. 27]
2:30 – 3:00 p.m.
Reactions of a CNS neuron to impaired intracellular cholesterol transport
Valérie Demais1, Nicole Ungerer2, Martine Perraut2, Frank W. Pfrieger2
1Plateforme Imagerie in Vitro, CNRS UPS 3156, 67084 Strasbourg, France,
2Institute of Cellular and Integrative Neurosciences, CNRS UPR 3212,
University of Strasbourg, 67084 Strasbourg, France
7 [Abstract #17, p. 28]
3:00 – 3:15 p.m.
Discussion
3:15 – 4:30 p.m.
POSTER SESSION
Jordan Hall Galleria
Heat shock protein 27 protects vulnerable neurons in NPC1 deficiency
Chan Chung, Matthew J. Elrick, Brittany Dixon and Andrew P.
Lieberman
From the Department of Pathology, University of Michigan Medical School,
Ann Arbor, Michigan 48109, USA
[Abstract #18, p. 29]
Regulation of Cholesterol Homeostasis with GEX1A: A Potential Lead for
Niemann-Pick Type C Disease
Jarred R.E. Pickering, Eve A. Granatosky, Michael J. Ahlers, D. Cole
Stevens, and Richard E. Taylor
Department of Chemistry and Biochemistry, University of Notre Dame, Notre
Dame, IN, 46556, USA
[Abstract #19, p. 30]
The Active Phosphorylated Form of FTY720 Is a Histone Deacetylase
Inhibitor that Enhances NPC1 Expression
Sarah Spiegel
VCU School of Medicine, Department of Biochemistry and Molecular Biology,
Richmond, VA
[Abstract #13, p. 24]
Proteastatic rescue of a high fraction of human NPC1 mutations by
HDAC inhibitors
Shu Mao, Nina H. Pipalia, and Frederick R. Maxfield
Department of Biochemistry, Weill Cornell Medical College, Cornell University
[Abstract #20, p. 31]
Cholesterol lowering effect of c-Abl inhibitors, a new therapeutic tool for
Niemann-Pick C disease
Contreras P.1,2,3, González-Zuñiga M.1,2, Dulcey A4., Marugan J4.,
Alvarez A.R.1,2, Zanlungo S.3
1Cell Signaling Laboratory. Biological Sciences Faculty, Pontificia Universidad
Católica de Chile, Santiago, Chile, 2CARE-Chile-UC, 3School of Medicine,
Pontificia Universidad Católica de Chile, Santiago, Chile, 4 NCGC, National
Institutes of Health, Rockville, USA.
[Abstract #21, p. 32]
Hydrophobic amine inhibition of cholesterol trafficking can be reversed
by cathepsin B inhibitors
Joslyn Mills, Jerry Faust, and Laura Liscum
8 Program in Cellular and Molecular Physiology, Sackler School of Graduate
Biomedical Sciences, Tufts University School of Medicine, Boston, MA
[Abstract #22, p. 33]
Cerebellar neurobiology of Niemann-Pick type C1 disease
Ian M. Williams, Celine Cluzeau, Christopher A. Wassif and Forbes D.
Porter
[Abstract #23, p. 34]
Assessing Cholesterol Metabolism, Storage and Transport in Live Cells
and C. elegans by SRS Imaging of Phenyl-Diyne Cholesterol
Hyeon Jeong Lee1, 2, Wandi Zhang3, Yang Yang3, Delong Zhang3, Bin
Liu4, Eric L. Barker5, Kimberly K. Buhman6, Lyudmila V. Slipchenko3,
Mingji Dai3, Ji-Xin Cheng1, 3, 7
1Interdisciplinary Life Science Program, Purdue University, West Lafayette,
Indiana, USA, 2Department of Comparative Pathobiology, Purdue University,
West Lafayette, IN 47907, 3Department of Chemistry, Purdue University, West
Lafayette, Indiana, USA, 4National Key Laboratory of Science and Technology
on Tunable Laser, Harbin Institute of Technology, Harbin 150080, China,
5Medicinal Chemistry and Molecular Pharmacology, Purdue University, West
Lafayette, IN 47907, 6Department of Nutrition Science, Purdue University,
West Lafayette, IN 47906, 7Weldon School of Biomedical Engineering, Purdue
University, West Lafayette, IN 47907
[Abstract #24, p. 35]
6:00 – 7:00 p.m.
RECEPTION
The Mark
7:00 – 9:00 p.m.
DINNER
The Mark
SATURDAY, JUNE 14
SESSION VI:
Jordan Hall of Science, Room 105
8:00 – 8:30 a.m.
Site directed mutagenesis of NPC2 reveals sterol transfer and LBPAinteraction domains
Leslie McCauliff and Judith Storch
Department of Nutritional Sciences and Rutgers Center for Lipid
Research, Rutgers University, NJ, USA
[Abstract #25, p. 36]
8:30 – 9:00 a.m.
Phosphatidic acid and ubiquitin catabolism modify intracellular lipid
accumulation and neurodegeneration in Niemann Pick type C disease.
9 Andrew B. Munkacsi1, Lauren Csaki2, Katsumi Higaki4, Giselle
Domínguez-Gutiérrez3, Robin B. Chan5, Bowen Zhou5, Manjari
Vasanthan1, Noor Alsarrage1, Namal Coorey1, David N. Brindley6,
Daniel Finley7, Aaron D. Gitler8, Gilbert Di Paolo5, Karen Reue2, and
Stephen L. Sturley5, 9
1School of Biological Sciences and Centre for Biodiscovery, Victoria University of
Wellington, Wellington, New Zealand 6012, 2Department of Human Genetics,
University of California at Los Angeles, Los Angeles, CA 90095, 3Institute of
Human Nutrition, Columbia University Medical Center, New York, NY 10032,
4Division of Functional Genomics, Research Center for Bioscience and
Technology, Tottori University, Japan, 5Department of Pathology and Cell
Biology, Columbia University Medical Center, New York, NY 10032,
6Department of Biochemistry, University of Alberta Edmonton, Alberta T6G
2H7 Canada, 7Department of Cell Biology, Harvard Medical School, Boston, MA
02115, 8Department of Genetics, Stanford University School of Medicine,
Stanford, CA 94305, 9Department of Pediatrics, Columbia University Medical
Center, New York, NY 10032
[Abstract #26, p. 37]
9:00 – 9:30 a.m.
Determination of the Allelic Frequency in Niemann-Pick Type C by
Analysis of Massively Parallel Sequencing Data Sets
Christopher A. Wassif, Joanna L. Cross, James Iben, Forbes D. Porter
Program in Developmental Endocrinology and Genetics, Eunice Kennedy
Shriver National Institute of Child Health and Human Development, National
Institutes of Health, Department of Health and Human Services
[Abstract #27, p. 38]
9:30 – 9:45 a.m.
Discussion
9:45 – 10:30 a.m.
BREAK
10:30 – 11:00 a.m.
Regulation of NPC1-mediated Cholesterol export by the lysosome
glycocalyx
Maika Deffieu, Jian Li and Suzanne R. Pfeffer
Department of Biochemistry, Stanford University School of Medicine
Stanford, CA 94305
[Abstract #28, p. 39]
11:00 – 11:30 a.m.
Niemann-Pick C Type 1 With Severe Pulmonary Manifestations
Orna Staretz-Chacham1, Micha Aviram2, Eliyahu Hershkovitz3
1Soroka University Medical Center, Ben-Gurion University of the Negev, BeerSheva, Israel, Israel, 2Soroka University Medical Center, Ben-Gurion University
of the Negev, Beer-Sheva, Israel, Israel, 3Soroka University Medical Center, BenGurion University of the Negev, Beer-Sheva, Israel, Israel
[Abstract #29, p. 40]
11:30 – 12:00 p.m.
Effect of TFEB activation on NPC phenotype
Hiroko Nagase, Joslyn Mills and Laura Liscum
10 Program in Cellular and Molecular Physiology, Sackler School of Graduate
Biomedical Sciences, Tufts University, 136 Harrison Avenue, Boston, MA 02111
[Abstract #30, p.41]
12:00 – 12:30 p.m.
Plasma signature of neurological disease in the monogenetic disorder Niemann
Pick Type C
Md. Suhail Alam1, 2, Michelle Getz1, 2, Sue Yi1, 2, Forbes Dennis Porter3,
Nicole Farhat3, Tamar Pounardjian4, Elizabeth M Berry-Kravis4, Jeffrey
Kurkewich1, 2, Innocent Safeukui1, 2 and Kasturi Haldar1, 2
1
Center for Rare and Neglected Diseases, 2Department of Biological Sciences
University of Notre Dame, IN 46556, USA; 3National Institute of Child Health
and Human Development, National Institutes of Health; 4Rush University
Medical Center, 1725 West Harrison Street, Chicago, IL 60612.
[Abstract #31, p. 42]
12:30 – 12:45 p.m.
Closing comments
Gregory P. Crawford
Dean, College of Science
12:45 p.m.
LUNCH
Jordan Hall Galleria
11 Abstracts
1. Identification of Cerebrospinal Fluid Protein Biomarkers in Niemann-Pick Disease, type C1
Stephanie M. Cologna1, Christopher A. Wassif1, Nicole M. Yanjanin1, Peter S. Backlund2, Brian C. Searle3,
Alfred L. Yergey22, and Forbes D. Porter1
1Program
in Developmental Endocrinology and Genetics, Eunice Kennedy Shriver National Institute of Child
Health and Human Development, National Institutes of Health, 2Biomedical Mass Spectrometry Facility,
Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of
Health, 3Proteome Software, Inc.
Niemann-Pick Disease, type C1 (NPC1) is an autosomal recessive, lysosomal storage disorder. The most
prominent cellular phenotype of NPC1 is accumulation of unesterified cholesterol and glycosphingolipids in
the endosomal/lysosomal system. Patients with NPC1 display a range of clinical features including
progressive neurodegeneration. To address the neurological aspect of this disorder, we have sought to define
a panel of proteins that are altered in cerebrospinal fluid from NPC1 patients compared to controls. Mass
spectrometry-based proteomics was utilized for protein identification and quantification which included the
use of multiplex stable isotope labeling (iTRAQ® technology) for quantification. Initial experiments were
focused on generating an experimental platform for protein identification. We then focused on defining the
behavior of the iTRAQ chemistry to make robust quantitative and biological conclusions. Using a cell lysate
as the model complex mixture, we characterized the proteome including the peak areas of the iTRAQ labels
used for quantification. Evaluation of these data resulted in normally distributed peak area values which are
important for downstream statistical evaluations. This dataset was also used to establish criteria for peptide
and protein identification and quantification requirements in clinical samples. Following our analytical
evaluation, the cerebrospinal fluid proteome characterization and quantification of NPC1 patients and controls
has been carried out. To date we have analyzed 8 NPC1 patient samples and 6 control samples. Proteins that
are altered in the NPC1 cohort include those consistent with previously published findings as well as novel
proteins. Together, these data can be used to evaluate the potential efficacy of drugs during therapeutic trials.
Results from both studies will be presented.
12 2. A Drosophila Screen for Genes that Interact with Npc1
Luis A. Milla, Shivani Baisiwala, Karthik Ramasubramanian, Jian Cao, and Matthew P. Scott
Departments of Developmental Biology, Genetics, and Bioengineering, Stanford University
Niemann-Pick type C disease is caused by mutations in either the NPC1 or NPC2 gene, leading to sterol
accumulation in late endosomes and lysosomes. These problems cause progressive neurodegeneration
and liver disease. One approach to identifying therapeutic targets to treat NPC disease is to screen for
genes or proteins that either modulate NPC1 levels or change cholesterol trafficking and metabolism.
We are taking advantage of the fruit fly Drosophila to serve as a platform to identify suppressing
modifiers of the NPC1 phenotype.
Flies with loss of function of npc1a, a homolog of NPC1, exhibit aberrant cholesterol accumulation
similar to NPC-diseased mammalian tissue. The npc1a mutants have impaired synthesis of the steroid
hormone ecdysone, which is required for molting during transitions between the larval stages of fly
development. We have used a sensitized RNAi screen in which npc1a is inhibited in the Drosophila
ring gland, the neuroendocrine gland that produces ecdysone. This tissue-specific inhibition avoids
general damage to the health of the animal, but severely reduces successful molting. Providing sterols,
especially cholesterol precursors or oxysterols, to the fly larvae in their food rescues the larvae to
adulthood. We titrate the amount of sterol and choose thresholds useful for for enhancer or suppressor
screens. We find that enhancers (making damaged cells worse) tend to be non-specific, while
informative suppressors can be found. Suppressors constitute candidates for targets of future
therapies, since drugs could be used to inhibit suppressing loci instead of the RNAi used in the screens.
We began by screening a set of candidate genes whose protein products are involved in trafficking,
sterol metabolism, and cell death functions.
Genes that reverse the NPC phenotype in flies and human cells are involved with two cellular proceses:
early autophagy and endoplasmic reticulum-Golgi communication. Using the information from this
initial screening, we are testing other proteins involved in those processes to delineate the membrane
trafficking events and mechanisms that underlie the NPC phenotype. Inhibition of mammalian
homologs of several of the genes that suppress in Drosophila suppress sterol accumulation in mutant
npc1/npc1 mammalian cultured cells. The information from the screen and further analyses may be
useful for understanding NPC disease and to design new strategies for treatment.
13 3. A new formulation to treat neurological and systemic disease in Niemann Pick Type C
Md. Suhail Alam1, 2, Michelle Getz1, 2 and Kasturi Haldar1, 2
1Center
for Rare and Neglected Diseases, 2Department of Biological Sciences University of Notre Dame, IN
46556, USA
In a Balb/c model of Niemann Pick Type C, we have developed new formulation of an existing oral antiinflammatory drug plus an older formulation of a well-known compound. Both compounds have already been
given to target patients and were well tolerated.
A new dosing regimen, using both combined in treatments given once weekly i.p. at a specified time regimen
allowing a low dose of the existing oral anti-inflammatory drug, increased mouse life span by greater than
100% eliminating other effects of each drug given singly. The first signs of neurodegeneration were delayed
till adult hood and animals routinely became aged ( > 6 months). The presentation will describe the effects of
the formulation, routes of application and dosing that confer significant potential to treat neurodegeneration,
systemic disease and prolong life.
14 4. Progress Toward the Development of Water-°©-­‐‑soluble and Long Circulating HP-°©-­‐‑β-°©-­‐‑CD
Polyrotaxanes as Potential NPC Therapeutics
Chris J. Collins, Yawo A. Mondijou, and David H. Thompson
Department of Chemistry Purdue University West Lafayette, Indiana 47907
A family of multivalent magnetic resonance imaging agents, based on a 2---­‐‑hydroxypropyl---­‐‑β---­‐‑ cyclodextrin (HP--­‐‑â---­‐‑CD):Pluronic® polyrotaxanes [1,2], have been synthesized and its blood pool contrast properties
3+
characterized. These Gd :DOTA---­‐‑HPCD/Pluronic polyrotaxane constructs are shown to circulate for more
3+
than 30 min and provide substantial vascular enhancement relative to the monomeric Gd :DOTA---­‐‑HPCD
---­‐‑1 ---­‐‑1
control that is rapidly cleared via the kidney. The high r1 relaxivity at 37°C (23.83 mM s
at 1.5T),
extended blood circulation, and well---­‐‑known pharmacology of the polyrotaxane precursors make it a
highly attractive candidate for biodegradable blood pool contrast agents. ICP---­‐‑MS findings indicate that a
significant fraction of the materials remain in circulation 24 h after tail vein injection in normal Balb/c mice,
although there are differences in the observed circulation times as a function of polyrotaxane threading
extent and average molecular weight.
Parallel efforts have been undertaken toward improving the water---­‐‑solubility of the HP---­‐‑â---­‐‑CD---­‐‑ based polyrotaxane
TM
carriers by co---­‐‑including Captisol
(4---­‐‑sulfobutylether---­‐‑â---­‐‑cyclodextrin, SBE---­‐‑â---­‐‑ CD) during the rotaxanation
reaction. Our findings show that HP---­‐‑â---­‐‑CD:Pluronic® L81 polyrotaxanes display aqueous solubilities at
TM
concentrations greater than 50 mg/mL when blended with >10 wt% Captisol
in the feed mixture [3].
Collectively, these findings validate the strategy of using polyrotaxane scaffolds as long---­‐‑circulating, high
payload carriers of HP---­‐‑â---­‐‑ CD drug for potential use as an NPC therapeutic.
References
[1] Y. A. Mondjinou, L. McCauliff, A. Kulkarni, L. Paul, S.---­‐‑H. Hyun, Z. Zhang, Z. Wu, M. Wirth, J.
Storch, D. H. Thompson, “Synthesis of 2---­‐‑Hydroxypropyl---­‐‑â---­‐‑Cyclodextrin and Pluronic Based
Polyrotaxanes in Heterogeneous Reactions for Niemann---­‐‑Pick Type C Therapy”, Biomacromolecules 2013 14,
4189---­‐‑4197.
[2] C. J. Collins, L. McCauliff, S.---­‐‑H. Hyun, Z. Zhang, L. N. Paul, A. Kulkarni, K. Zick, M. Wirth, J.
Storch, D. H. Thompson, “Synthesis, Characterization, and Evaluation of Pluronic---­‐‑based â---­‐‑Cyclodextrin
Polyrotaxanes for Mobilization of Accumulated Cholesterol from Niemann---­‐‑Pick Type C Fibroblasts”,
Biochemistry 2013 52, 3242---­‐‑3253.
[3]
Y. A. Mondjinou, S.---­‐‑H. Hyun, M. Xiong, P. L. Thong, D. H. Thompson, “Impact of Mixed â---­‐‑
Cyclodextrin Feeds on Pluronic® Rotaxanation Efficiency and Product Solubility”, submitted for
publication.
‡
These authors contributed equally to this work.
15 5. Intracisternal cyclodextrin ameliorates neurological dysfunction, increases survival time, and stops
Purkinje cell death in feline Niemann Pick type C1 disease.
C. H. Vite1, J.H. Bagel1, G. P. Swain1, M. Prociuk1, T. U. Sikora2, V. M. Stein1, P. O’Donnell2, T. Ruane2, S. Ward1,
A. Crooks1, S. Li1, E. Mauldin2, S. Mellon3, D. S. Ory4, M. L. Kao5, M. De Meulder6, C. Davidson7, M. T. Vanier8,
S. U. Walkley7
1Department
of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, 2Department
of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, 3Department of Obstetrics,
Gynecology, and Reproductive Sciences, University California, San Francisco, California, 4Diabetic Cardiovascular Disease Center,
School of Medicine, Washington University School of Medicine, St Louis, Missouri, 5Janssen Research and Development, Janssen
Pharmaceutical Companies of Johnson and Johnson, Titusville, New Jersey, 6Janssen Research and Development, Janssen
Pharmaceutical Companies of Johnson and Johnson, Antwerp, Belgium, 7Dominick P Purpura Department of Neuroscience, Rose F
Kennedy Intellectual and Developmental Disabilities Research Center, Albert Einstein College of Medicine, Bronx, New York, 8Institut
National de laSante´ et de la Recherche Me´dicale (INSERM), Lyon, France
In 2005 we began study of a feline model of NPC disease in which we could repeatedly administer HPßCD either subcutaneously or
intrathecally and repeatedly sample cerebrospinal fluid (CSF) and blood to evaluate mechanistic, pharmacologic, and toxicity issues.
This model also allowed for validation of biochemical markers of disease severity and therapeutic effects that are specific to CNS
disease (Porter et al., 2010, Ward et al., 2010). Feline NPC disease results from a single missense mutation in the NPC1 gene (p.C955S)
that is evolutionarily conserved and in a cysteine-rich region commonly mutated in patients (Somers et al., 2003). Disease progression
in this model recapitulates both the neuropathological and biochemical abnormalities observed in human patients, with the closest
parallels to the juvenile form of NPC disease (Walkley and Suzuki, 2004, Vite et al., 2008, Stein et al., 2012). In the present study, we
showed that, notably, administration of HPßCD into the subarachnoid space of affected cats completely resolved the clinical
neurological signs of disease and Purkinje cell loss up to at least 24 wks of age (the median age when untreated cats die). Studies in the
feline animal model have provided critical data on efficacy and safety of drug administration directly into the CNS which were central
to advancing HPßCD into the current clinical trial for NPC patients (Ottinger et al., 2014). We will give an update of these studies that
were funded first, and continue to be funded in part, by the Ara Parseghian Medical Research Foundation.
NPC cats were treated with 120 mg IC HPßCD beginning at 3 wks of age and repeated every 14 days thereafter. Remarkably, these cats
were neurologically normal at 24 wks of age and showed only mild ataxia at 76 wks of age. However, no improvement in serum
albumin and only small significant decreases, but still abnormal concentrations, of ALT and cholesterol were found when compared to
untreated NPC cats. Liver histology and lipid biochemistry were similar to those found in untreated NPC cats. In the brain, these cats
showed a marked reduction in storage of filipin-stained unesterified cholesterol, of GM2 ganglioside immunostaining as well as no
evidence of Purkinje cell loss. Biochemical study of the ganglioside patterns demonstrated drastically reduced levels of gangliosides
GM2 and GM3, with unchanged concentrations of the major brain gangliosides.
Additionally, one cohort of NPC cats was treated with a combination of both 120 mg IC and 1000 mg/kg SC. These cats were
neurologically normal at 24 wks of age and either remained normal or showed only mild ataxia at 76 wks of age. However, they also
maintained serum ALT and albumin at levels found in normal cats, accompanied by significant decreases in serum cholesterol when
compared to untreated NPC cats. Biochemical analysis of liver showed a marked decrease of cholesterol, sphingomyelin, GM3
ganglioside, neutral glycolipids, and free sphingosine concentrations. In the brain, similar results as described for IC treatment alone
were observed, with strong decreases in filipin-stained cholesterol, GM2 and GM3 gangliosides (but not of the major gangliosides),
lactosylceramide, sphingosine, and no loss of Purkinje cells.
The above studies illustrate the ability of 120 mg IC HPßCD to ameliorate neurological disease, brain biochemical abnormalities, and
Purkinje cell loss when treatment is initiated prior to the onset of neurological deficits. To determine the effects of instituting therapy
when neurological dysfunction is already present, a cohort of NPC cats was administered 120 mg HPßCD IC every 14 days beginning
at 16 wks of age, an age at which moderate ataxia and tremor exist. Eight NPC cats treated in this manner showed either no
progression or slowed progression of clinical signs when evaluated at 24 wks of age. Histological and biochemical evaluation of these
cats showed an accumulation of cholesterol, gangliosides, lactosylceramide, and sphingosine which was greater than that seen in NPC
cats that began treatment at this dose at 3 wks of age, but less than that seen in untreated NPC cats at end-stage disease. The level of
GM2 ganglioside was clearly less than at the age when treatment began, and not much higher than in 4-week old untreated cats.
Subjective evaluation of Purkinje cell numbers also strongly suggested that loss of these cells was not a pronounced as that found in
untreated cats.
16 6. Intermediate Size Patient Population IND for 2-Hydroxypropyl-Cyclodextrin (HP-CD) Outpatient
Treatment of Siblings with Niemann-Pick Type C1 (NP-C1) and Disparate Rates of Progression:
Exploration of Outcome Measures and Biomarkers
Elizabeth Berry-Kravis1, 2, 3, Joanne O’Keefe2, 4, Tamar Pounardjian1, Jamie Chin1, Forbes Denny Porter5, Daniel
Ory6, Suhail Alam7, Kasturi Haldar7
1Departments
of Pediatrics, 2Neurological Sciences, 3Biochemistry, and 4Anatomy, Rush University Medical
Center, Chicago, IL; 5National Institute of Child Health and Human Development, National Institutes of
Health; 6Departments of Medicine and Cell Biology and Physiology, Washington University, St. Louis, MO;
7Center for Rare and Neglected Diseases and Department of Biological Sciences, University of Notre Dame,
Notre Dame, IN
An intermediate size patient population IND (21CFR 312.315) was obtained for intrathecal hydoxypropyl-ßcyclodextrin (HP-ß-CD) treatment of 14- and 15-year old siblings with NP-C1 who were unable to be enrolled
in the concurrent phase 1 clinical trial at NIH. The siblings were discordant for clinical severity: sibling 1 had
symptom onset at age 6 with substantial cognitive and motor skills impairment, poor balance, limited
language, choking with swallowing, vertical gaze palsy and intractable seizures, while sibling 2 had onset at
age 13 (after genetic diagnosis) with subtle deterioration in executive function and academic performance, and
a minimal vertical gaze abnormality. Diagnosis of NP-C was made by filipin staining of fibroblasts (sibling 1),
and molecular testing showing compound heterozygosity for mutations in NP-C1 (c410C>T and c2000C>T) in
both siblings. The HP-ß-CD treatment protocol was created as a modified version of the NIH phase 1 trial
protocol and those from several individual INDs. Modifications were directed at providing outpatient
treatment with adequate safety monitoring, allowing dose escalation based safety and response data, and
allowing reduction of some safety monitoring based on lack of safety concerns after multiple infusions. After
IRB approval and informed consent, treatment was initiated by lumbar puncture (LP) with intrathecal
infusion. An initial saline infusion tested for changes in biomarkers related to the LP and infusion process
itself, and was followed by infusions every two weeks of HP-ß-CD 200 mg for 6 infusions, followed by dose
escalation to 300 mg, then 400 mg. Safety and efficacy outcomes, including those in the NIH trial to the extent
possible, were assessed at baseline and monitored throughout the treatment protocol. These included ABRs,
swallow evaluations, language and cognitive testing, NP-C rating scale, neurological exam, parent-reported
PROMIS, pre-infusion audiology; pre- and post-infusion EKGs, blood chemistries, hematology, lipid panels,
urinalysis, and blood biomarkers including 24-hydroxycholesterol and lysozyme levels; pre-infusion CSF
glucose, protein, cell counts, immunoglobulins, cultures and biomarkers. Gait and balance performance has
been tracked with a Neurocom system and inertial sensors. Thus far the saline and first 9 HP-ß-CD infusions
have not been associated with any clinically significant safety issues or changes in laboratory parameters. PostLP headaches and vomiting were observed in both siblings after the first 3 infusions but resolved with a switch
to use of Whitacre spinal needles. Sibling 1 had resolution of choking when eating, decreased seizures,
increased use of language noted in numerous settings and steadier gait by the 4th infusion. Additional safety
data and results of clinical and biomarker outcomes after 6 months of treatment will be presented.
Implementation of the IND protocol has been a process through which treatment timing and dose, safety
monitoring, and outcomes can be explored to inform future phase 2/3 trial design.
17 7. Therapeutic trials for Niemann-Pick Disease, Type C1: 2-Hydroxypropyl-β-Cyclodextrin
Forbes D. Porter and the TRND Team (Nicole Yanjanin1, Aiyi Liu (DESPR)1, Roopa Shankar1, Daniel Ory2, Bill
Pavan3, Charles Vite4, Russell Lonser5, John Heiss5, Steven Walkley6, Chris Austin7, John McKew7, Nuria
Carrillo7, Liz Ottinger7, Juan Marugan7, Pramod Terse7, Xin Xu7, Wei Zeng7, Steven Silber8, Mark Kao8, Marcus
Brewster8, Jon Stocker9, Charles Finn10, Frank Hurley10, Joy Vanderwal10, Patrick Frenchick10, Sandra Morseth10,
Kimberly Lilly10, Carmen Brewer11, Beth Solomon11, Naomi O’Grady11, Kelly King11, Ariane Soldatos5, 12
1NICHD, 2Washington
University School of Medicine, 3NHGRI, 4University of Pennsylvania, 5NINDS, 6Albert
Einstein Collage of Medicine, 7TRND/NCATS, 8Johnson and Johnson, 9SAIC, 10RRD International, 11Clinical
Center, 12NIH
Niemann-Pick Disease, type C1 (NPC1) is an autosomal recessive, lysosomal storage disease characterized by
progressive neurodegeneration. Although miglustat has been approved in multiple countries and is
frequently used off-label in the USA, currently there are no FDA approved therapies for NPC1. Development
of clinical outcome measures is complicated by both the disease rarity and phenotypic heterogeneity. In 2006
we initiated an observational/natural history study to identify potential biomarkers that could be used as tools
to facilitate therapeutic trials, and to identify clinical outcome measures that could ultimately be used to
support labeling a candidate drug for treatment of NPC1. Proteomic and lipidomic studies have identified
multiple biomarkers that are being applied to understand the pathophysiological processes that underlie
NPC1 neurodegeneration, to improve diagnostic and prognostic ability, and to facilitate the development of
safe and effective therapies. These proteomic and lipidomic studies are continuing. The TRND team,
composed of both intramural and extramural scientists, and with significant assistance from Johnson &
Johnson, initiated a clinical trial of intraventricular 2-hydroxypropyl-β-cyclodextrin (HPβCD) in NPC1. This is
an adaptive, dose escalation trial designed to systematically determine a safe and biochemically effective dose
of HPβCD. This trial began in January 2013 and HPβCD was delivered to the lateral ventricle utilizing an
Ommaya reservoir. Although we observed a significant biochemical response, the use of Ommaya reservoirs
to deliver the drug to the lateral ventricle was complicated by P. acnes colonization. As an alternative design,
the clinical protocol was revised to deliver the drug by lumbar intrathecal injection. This trial was initiated in
September of 2013. To date we have enrolled 12 patients and have investigated doses ranging from 50-400 mg.
The safety profile appears to be good although two patients demonstrated grade 1 ototoxicity.
Initial
biomarker data from the trial will be discussed.
18 8. Tracing the Development of Histone Deacetylase Inhibitors as Potential Therapeutic
Agents for Niemann-Pick Type C Disease
Olaf Wiest and Paul Helquist
University of Notre Dame
Our laboratory has a long history of studying histone deacetylase (HDAC) inhibitors. These small molecule
agents were initially developed in the 1990s and became recognized as the first of a now wider class of
compounds known as epigenetic modulators. The HDACs are a family of proteins, of which 18 are known in
humans. One of their functions is to regulate the level of acetylation of histone proteins around which the
strands of DNA are wound in the nucleus of cells. The action of HDACs reduces the level of histone
acetylation, leading to tight binding of DNA and in turn to decreased transcription of DNA and protein
production. Inhibition of HDACs reverses this effect and leads to increased protein production. HDAC
inhibitors have been developed most heavily as treatments for cancer, including some FDA-approved drugs,
but several other applications have also been found for other diseases.i Our work with HDAC inhibitors began
in 1999 in the context of a rare blood disorder. We developed a computational model, which has been widely
adopted in the broader epigenetic research community for the design of new inhibitors,ii,iii and synthesized a
range of selective and pan-HDAC inhibitors.
When we were introduced to the NPC community by the Ara Parseghian Medical Research Foundation in
2005, we began a collaboration with the Maxfield laboratory at Cornell. Our initial efforts focused on the
synthesis of analogues of compounds that had been identified by high-throughput screening as agents
effecting reduction of the abnormally high lysosomal levels of cholesterol seen in NPC disease.iv A spin-off
discovery was the identification of a lysosomal acid lipase inhibitor, now known as Lalistat,v which has been
adopted by several other investigators as a biochemical tool.vi Based upon our prior experience with HDAC
inhibitors as agents that increase protein production, and based upon the knowledge that NPC disease is
associated with insufficient levels of active forms of the NPC1 or NPC2 protein, we put forth the hypothesis
that HDAC inhibitors may restore normal cholesterol trafficking in NPC. In 2009, we supplied the Maxwell
laboratory with a set of HDAC inhibitors having a range of potencies and isoform selectivities. Our hypothesis
was borne out when some of the inhibitors were seen to markedly lower the lysosomal levels of cholesterol in
human NPC fibroblastsvii,viii,ix and led to the filing of a patent for the treatment of lysosomal storage disorders
such as NPC with HDAC inhibitors. We provided a series of HDAC inhibitors to the NPC research community
for further studies, culminating in an application filed with the FDA that has been approved for clinical trials
of an HDAC inhibitor in NPC patients.
i
Wiech, N. L.; Fisher, J. F.; Helquist, P.; Wiest, O. “Inhibition of Histone Deacetylases: A Pharmacological Approach to the Treatment of Non-Cancer Disorders” Curr. Top. Med. Chem.
ii
Wang, D.-F.; Wiest, O.; Helquist, P.; Lan-Hargest, H.; Wiech, N. L. “On the Function of the 14Å Long Internal Cavity of HDLP: Implications for the Design of HDAC Inhibitors”, J. Med.
Chem. 2004, 47, 3409-3417.
Wang, D.-F.; Helquist, P.; Wiech, N. L.; Wiest, O. “Towards Selective HDAC Inhibitor Design: Homology Modeling, Docking Studies, and Molecular Dynamics Simulations of Human
iii
Class I HDACs” J. Med. Chem. 2005, 48, 6936-6947.
Cosner, C. C.; Markiewicz, J. T.; Bourbon, P.; Mariani, C. J.; Wiest, O.; Rujoi, M.; Huang, A.; Maxfield, F. R.; Helquist, P. “Investigation of N-aryl-3-alkylidenepyrrolinones as potential
iv
Niemann-Pick type C disease therapeutics” J. Med. Chem. 2009, 52, 6494-6498.
v
Rosenbaum, A. I.; Cosner, C. C.; Mariani, C. J.; Maxfield, F. R.; Wiest, O.; Helquist, P. “Thiadiazole Carbamates: Potent Inhibitors of Lysosomal Acid Lipase and Potential Niemann-Pick
Type C Disease Therapeutics” J. Med. Chem. 2010, 53, 5281–5289.
vi
Pearson, G. L.; Mellett, N.; Chu, K. Y.; Cantley, J.; Davenport, A.; Bourbon, P.; Cosner, C. C.; Helquist, P.; Meikle, P. J. Biden, T. J. “Lysosomal acid lipase and lipophagy are constitutive
negative regulators of glucose-stimulated insulin secretion from pancreatic beta cells” Diabetologia 2014, 57, 129-139.
Pipalia, N. H.; Cosner, C. C.; Huang, A.; Chatterjee, A.; Bourbon, P.; Farley, N.; Helquist, P.; Wiest, O.; Maxfield, F. R. Proc. Nat. Acad. Soc. 2011, 108, 5620-5625.
vii
viii
Wehrmann, Z. T.; Hulett, T. W.; Huegel, K. L.; Vaughan, K. T.; Wiest, O.; Helquist, P.; Goodson, H. “Quantitative Comparison of the Efficacy of Various Compounds in Lowering
Intracellular Cholesterol Levels in Niemann-Pick Type C Fibroblasts” PLoS ONE 2012, 7(10): e48561.
ix
Helquist, P.; Maxfield, F. R.; Wiech, N. L.; Wiest, O. “Treatment of Niemann-Pick Type C Disease by Histone Deacetylase Inhibitors” Neurotherapeutics 2013, 10, 688–697.
19 9. Testing Histone Deacetylase Inhibitors as NPC Therapeutics
Frederick R. Maxfield, Deepti Gadi, Shu Mao, and Nina Pipalia
Weill Cornell Medical College, New York, NY
In collaboration with the laboratories of Drs. Helquist and Wiest (Notre Dame), we
described the use of histone deacetylase inhibitors (HDACi) to reduce the cholesterol
accumulation in human fibroblasts expressing mutant forms of the NPC1 protein (1).
The initial studies were carried out using two human lines. One of these was
homozygous for the NPC1I1061T mutation, and the other was heterozygous with one
NPC1I1061T allele. The HDACi treatment increased the expression of the NPC1 protein,
and this increased protein expression may have been a major reason for the reversal of
the cholesterol accumulation. We have extended the testing of the HDACi to several
other mutations. In collaboration with the Balch laboratory, have found 60 of 80
mutants tested in a model cell system can have stored cholesterol reduced by treatment
with HDACis.
We have now tested several HDACi with selectivity for one of more HDAC enzymes.
This is helping to identify which enzymes are the relevant targets. SAHA (Vorinostat) is
a broad spectrum HDACi, but more selective HDACi may retain the beneficial effects
with fewer off-target effects.
Current plans for animal trials (in collaboration with Drs. Dan Ory, Charles Vite, and
Steve Walkley) as well human trials (in collaboration with Drs. Porter, Patterson, and
Ory) will be discussed.
1. Pipalia, N.H., Cosner, C.C. , Huang, A, Chatterjee, A., Bourbon, P., Farley, N.,
Helquist, P., Wiest, O., and Maxfield, F.R. (2011) Histone deacetylase inhibitor treatment
dramatically reduces cholesterol accumulation in Niemann-Pick Type C1 mutant human
fibroblasts. Proc. Natl. Acad. Sci., USA, 108: 5620-5625.
20 10. Proteostasis Regulators Reprogram Variant NPC1 Folding Environments to
Mitigate Cholesterol Homeostasis in Disease
Kanagaraj Subramanian1, Jason Gestwicki2, Fred Maxfield3 and William E. Balch1, 4
1The
Scripps Research Institute (TSRI), 2Departments of Molecular and Cell Biology and
Chemical Physiology, 4The Skaggs Institute of Chemical Biology, La Jolla, California
92037; University California San Francisco (UCSF) School of Medicine, 2Institute for
Neurodegenerative Disease, San Francisco, California 94158; 3Weill Cornell Medical
College, Department of Biochemistry, Weill Cornell Medical College, New York, NY
10065
Niemann-Pick type C (NPC) disease is caused by the defective folding, trafficking and
function of NPC1 or NPC2 proteins impairing intracellular cholesterol homeostasisresulting in the accumulation of unesterified cholesterol and other lipids in the late
endosomal/lysosomal (LE/LY) compartments. Over 250 disease-causing mutations
residing in different domains trigger a metastable NPC1 fold (referred to as NPC1*)
leading to systemic and neurological disease. A central goal of our efforts is to identify
new therapeutic approaches to correct cholesterol homeostasis by stabilizing the folding
and trafficking of NPC1* through identification of small molecules that impact the
operation of the ‘protein homeostasis’ or ‘proteostasis’ program that manages NPC1*
structure-function relationships. Consistent with this model, we have recently suggested
in collaboration with the laboratory of Dr. Fred Maxfield, a substantial portion of patient
alleles can be rescued by histone deacetylase inhibitors (HDACi) that either indirectly or
directly influence NPC1* function through surface acetylation pathways. In contrast to
surface residues, hydrophobic forces that define core NPC1 structure-function
relationships are managed by heat shock protein (Hsp) 70 (Hsp70) and Hsp90
chaperone/co-chaperone family members. We have identified novel small molecule
proteostasis regulators (PRs) that reprogram the activity of Hsp70 and Hsp90
components to improve NPC1* folding and trafficking, leading to a reduction of
cholesterol in the LE/LY compartments. We anticipate that PRs modulating the
proteostasis environment will provide a strong foundation for development of novel
therapeutics for the treatment of NPC1 disease given the central role of chaperone
biology in defining the structure of a functional fold.
21 11. A murine Niemann-Pick C1 (NPC1) I1061T knockin model recapitulates the
pathological features of the most prevalent human disease allele
Maria Praggastis1, Brett Tortelli1, Jessie Zhang1, Hideji Fujiwara1, Rohini Sidhu1, Anita
Chacko1, Zhouji Chen1, Andrew P. Lieberman2, Cristin Davidson3, Steven U. Walkley3,
Nina H. Pipalia4, Frederick R. Maxfield4, Jean E. Schaffer1, and Daniel S. Ory1
1Washington
University School of Medicine, St. Louis, MO 63110; 2University of
Michigan, Ann Arbor, MI; 3Albert Einstein College of Medicine, NY; 4Weill Cornell
Medical College, New York, NY
Niemann-Pick Type C1 (NPC1) disease is a rare, neurodegenerative cholesterol disorder
characterized by ataxia, motor impairment, and dementia. The most prevalent mutation,
NPC1I1061T, encodes a misfolded protein with a reduced half-life due to ER-associated
degradation. Therapies directed at stabilization of the mutant NPC1 protein reduce
cholesterol storage in fibroblasts, but have not been tested in vivo due to lack of a
suitable animal model. While the prominent features of human NPC1 disease are
modeled in the Npc1-/- mouse, this mouse does not synthesize NPC1 protein and
therefore is not amenable to examining proteostatic therapies. The objective of the
present study was to develop an NPC1I1061T knock-in mouse, in which to test proteostatic
therapies. In comparison to the Npc1-/- mouse, this mouse models a less severe form of
NPC1 disease and displays disease hallmarks, including weight loss, decreased motor
coordination, Purkinje cell death, and lipid storage. The murine NPC1I1061T protein has a
reduced half-life in vivo, consistent with protein misfolding and rapid ER-associated
degradation, and can be stabilized by HDAC inhibition. This novel mouse model
faithfully recapitulates disease caused by the human NPC1I1061T mutation, and provides
the field with a powerful tool for pre-clinical evaluation of proteostatic therapies for
NPC1 disease.
22 12. Inhibition of histone deacetylation in a mouse model of Niemann Pick Type C
disease
Andrew B. Munkacsi1, Natalie Hammond1, and Stephen L. Sturley2
1School
of Biological Sciences and Centre for Biodiscovery, Victoria University of Wellington,
Wellington, New Zealand 6012, 2Department of Pediatrics, Columbia University Medical Center,
New York, NY 10032
Niemann-Pick type C (NPC) disease is a fatal, pediatric neurodegenerative disease arising from
the lysosomal accumulation of cholesterol and sphingolipids. Currently, there is no FDAapproved therapy. In 2011, the Maxfield/Helquist/Wiest laboratories [1] and our group [2]
reported the amelioration of lipid accumulation in NPC patient fibroblasts. This was
accomplished using Vorinostat (suberoylanilide hydroxamic acid; SAHA; Zolinza®), an orallyadministered HDAC inhibitor that is FDA-approved to treat cutaneous T-cell lymphoma.
nmf164
Here we evaluate the onset and progression of NPC disease in Vorinostat-treated Npc1
mice, a missense model of NP-C disease. We demonstrate that in the livers of diseased, treated
animals there is a reduction in the accumulation of free cholesterol and sphingomyelin compared
to vehicle control. Similarly, there were reductions in levels of sphingosine, sphinganine,
nmf164
lactosylceramide, glucosylceramide, and GM1, respectively, in Vorinostat-treated Npc1
compared to untreated controls. We conclude that based on the in vivo efficacy of Vorinostat
with respect to lipid homeostasis in an animal model of NPC disease, that HDAC inhibition
holds great promise for treatment of human NPC disease. To predict the effect of 150 mg/kg
Vorinostat on longevity in this model, we monitored the weight of the animals during this
nmf164
treatment. The Npc1
mice normally cease gaining weight at 8 weeks and plateau at this
weight for 6-7 weeks and then lose weight for 3-4 weeks prior to death [3]. Untreated or 150
nmf164
mg/kg Vorinostat-treated Npc1
animals were not significantly different from each other
and were significantly worse than untreated WT animals. To confirm the vehicle was not toxic,
we monitored weights of untreated mutants and these weights were not different from those of
Vorinostat-treated animals.
Our studies clearly demonstrate the efficacy of Vorinostat in the liver; however it is yet to be
resolved whether this compound penetrates the brain regions affected in NPC disease [4], a result
critical to extending the lifespan of NPC patients. Our results support, but do not prove, the
therapeutic efficacy of HDAC inhibition but suggest that judicious choice of brain penetrant
inhibitors or protocols that result in traversion of the blood brain barrier may be effective.
1.
2.
3.
4.
Pipalia, N.H., et al., Histone deacetylase inhibitor treatment dramatically reduces
cholesterol accumulation in Niemann-Pick type C1 mutant human fibroblasts. Proc Natl
Acad Sci U S A, 2011.
Munkacsi, A.B., et al., An "Exacerbate-reverse" Strategy in Yeast Identifies Histone
Deacetylase Inhibition as a Correction for Cholesterol and Sphingolipid Transport
Defects in Human Niemann-Pick Type C Disease. J Biol Chem, 2011. 286(27): p. 23842-51.
Maue, R.A., et al., A novel mouse model of Niemann-Pick type C disease carrying a
D1005G-Npc1 mutation comparable to commonly observed human mutations. Hum Mol
Genet, 2011. 21(4): p. 730-50.
Hanson, J.E., et al., SAHA enhances synaptic function and plasticity in vitro but has
limited brain availability in vivo and does not impact cognition. PLoS One. 8(7): p.
e69964.
23 13. The Active Phosphorylated Form of FTY720 Is a Histone Deacetylase Inhibitor that
Enhances NPC1 Expression
Sarah Spiegel
VCU School of Medicine, Department of Biochemistry and Molecular Biology, Richmond, VA
It was recently reported that class I histone deacetylase (HDAC) inhibitors can correct cholesterol
storage defects in human NPC1 mutant cells by increasing expression of the low activity mutant
NPC1 protein (1, 2). HDACs remove acetyl groups from histones and play a key role in gene
regulation and HDAC inhibitors have long been used in various brain disorders and are being
investigated as possible treatments for several neurological diseases. We have previously shown
that the sphingolipid metabolite sphingosine-1-phosphate (S1P) is an endogenous inhibitor of
HDAC1/2 (3). Although most of the known actions of S1P, a potent bioactive mediator formed
by sphingosine kinases (SphK1 and SphK2), are mediated by five specific G protein-coupled
receptors, termed S1PR1-5, our work indicates that HDAC1/2 are direct intracellular targets of
S1P produced by nuclear SphK2 and link sphingolipid metabolism to epigenetic regulation of
gene expression (3). Hence, HDACs act as metabolic sensors, converting changes in sphingolipid
metabolism into patterns of gene expression. In recent unpublished studies, we found that the
sphingosine analogue FTY720 (known as fingolimod), a pro-drug that is approved for the
treatment of multiple sclerosis (MS) and sequesters lymphocytes into lymph nodes by
modulating S1PR1 (4), enters the nucleus where it is phosphorylated by SphK2 and that FTY720phosphate, its active form that accumulates there is a potent class I HDAC inhibitor. We also
observed that treatment of mice with FTY720 improved memory impairment independently of its
immunosuppressive actions and S1PR1. Moreover, FTY720 treatment increases expression of
NPC1, NPC2 and lipin 1 in cultured cells and in brains of mice. We are now determining the
mechanisms of action of FTY720. Our goals are to evaluate the therapeutic potential of FTY720 in
NPC1 mutant human fibroblasts and in animal models of NPC disease.
Supported by the Ara Parseghian Medical Research Foundation
Cited Literature
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cholesterol accumulation in Niemann-Pick type C1 mutant human fibroblasts. Proc. Natl. Acad.
Sci. U.S.A. 108,5620-5625.
2. Munkacsi, A. B., Chen, F. W., Brinkman, M. A., Higaki, K., Gutierrez, G. D., Chaudhari, J.,
Layer, J. V.,Tong, A., Bard, M., Boone, C., Ioannou, Y. A., and Sturley, S. L. (2011) An "exacerbatereverse"strategy in yeast identifies histone deacetylase inhibition as a correction for cholesterol
and sphingolipidtransport defects in human Niemann-Pick type C disease. J. Biol. Chem. 286,
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Marmorstein, R., Kordula, T., Milstien, S., and Spiegel, S. (2009) Regulation of histone acetylation
inthe nucleus by sphingosine-1-phosphate. Science 325, 1254-1257.
4. Brinkmann, V., Billich, A., Baumruker, T., Heining, P., Schmouder, R., Francis, G., Aradhye, S.,
and Burtin, P. (2010) Fingolimod (FTY720): discovery and development of an oral drug to treat
multiple sclerosis. Nat. Rev. Drug Discov. 9, 883-897.
24 14. Therapeutic trials for Niemann-Pick Disease, Type C1: Proof of Concept HDAC
Inhibitor Trial
Fred Maxfield1, Daniel Ory2, Forbes Porter3, 4, Paul Helquist5, Olaf Wiest5, Edward
Holson6, Xu Xin4, 7, Marc Patterson8, Mathew Berg5, Cindy Parseghian9, Stephen
Gately10
1Weill
Cornell Medical College, 2Washington University, 3NICHD, 4NIH, 5Notre Dame, 6Broad Institute,
Clinic, 9APMRF, 10TD2
7NCATS, 8Mayo
Niemann-Pick Disease, type C1 (NPC1) is a progressive neurodegenerative disorder due to endolysosomal
accumulation of unesterified cholesterol and lipids. Prior work by the Ory group has shown that if the
p.I1061T mutant NPC1 protein can be protected by from degradation by the cellular quality control systems
in the endoplasmic reticulum, the pI1061T protein can be transported to the endolysosomal compartment
and functions to reduce endolysosomal accumulation of unesterified cholesterol.
More recently the
Maxfield and Sturley groups showed that, in vitro, histone deacetylase inhibitors (HDACi) can reduce the
endolysosomal lipid storage characteristic of NPC1 disease. Over the past year we have been working to
establish a proof of concept clinical trial of HDAC inhibition in NPC1. This proposal was awarded one of
the first U01 grants (Maxfield, Ory, Porter) designed to promote extramural utilization of the NIH Clinical
Center (http://www.nih.gov/news/health/mar2014/nichd-13.htm).
We have now expanded this
intramural/extramural collaboration to include investigators from Notre Dame (Helquist, Wiest), Broad
Institute (Holson) and Mayo Clinic (Patterson). This effort is being supported by Notre Dame College of
Science and the Ara Parseghian Medical Research Foundation. The initial drug to be tested will be
vorinostat. Vorinostat is an HDACi that in vivo can correct the NPC1 cellular phenotype. Vorinostat is
approved by the FDA for the treatment of cutaneous T-cell lymphoma. Since this is a proof of concept trial
and the safety of this drug in NPC subjects is not likely to differ significantly from patients with cutaneous
T-cell lymphoma who have failed alternative chemotherapy, we were able to obtain a waiver of the
requirement for an Investigational New Drug application for the testing of vorinostat in adult subjects with
NPC1. The NICHD Institutional Review Board (IRB) has approved a protocol to test the safety and efficacy
of vorinostat in a cohort of 12 adult NPC1 subjects and we are currently working on IRB approval for a
second site at the Mayo Clinics. This is a phase I proof of concept trial that will focus on safety of HDACi in
NPC1 subjects and determining if HDAC inhibition has a desirable biochemical effect in circulating
peripheral mononuclear cells. Exploratory endpoints will include CSF pharmacokinetics, CSF biomarkers,
and clinical outcome measures.
Nicole M. Yanjanin (NICHD, NIH), Roopa Kanakatti Shankar (NICHD, NIH), Nuria Carrillo (NCATS,
NIH), Forbes D. Porter (NIH, NICHD), and the TRND Team
Niemann-Pick Disease, type C1 (NPC1) is an autosomal recessive, lysosomal storage diseases characterized
by progressive neurodegeneration. Although miglustat has been approved in multiple countries and is
frequently used off-label in the USA, currently there are no FDA labeled therapies for NPC1. Development
of clinical outcome measures is complicated by both the disease rarity and phenotypic heterogeneity. In
2006 we initiated an observational/natural history study to identify potential biomarkers that could be used
as tools to facilitate therapeutic trials, and to identify clinical outcome measures that could ultimately be
used to support labeling a candidate drug for treatment of NPC1. Proteomic and lipidomic studies have
identified multiple biomarkers that are being applied to understand the pathophysiological processes that
underlie NPC1 neurodegeneration, to improve diagnostic and prognostic ability, and to facilitate the
development of safe and effective therapies. These proteomic and lipidomic studies are continuing. In
order to gain insight into factors that contribute to the phenotypic heterogeneity and influence clinical
progression we have initiated an effort to obtain whole exome sequencing data on our cohort of NPC1
patients. This molecular data will be correlated with clinical manifestations. The TRND team, composed of
both intramural and extramural scientists, and with significant assistance from Johnson and Johnson,
initiated a clinical trial of intraventricular 2-hydroxypropyl-β-cyclodextrin (HPβCD) in NPC1. This is an
adaptive, dose escalation trial designed to systematically determine a safe and biochemically effective dose
of HPβCD. This trial was initiated in January of this year and is ongoing. It is our immediate goal to utilize
data from this trial to optimize the design of a larger clinical trial focused on establishing clinical efficacy.
25 15. rHSP70: A novel therapeutic opportunity for lysosomal storage diseases
Thomas Kirkegaard Jensen1, 4, James Gray2, Ole Dines Olsen1, 4, Svetlana Drndarski3,
Linda Ingemann1, Signe Humle Jørgensen1, Ian Williams2, Kerri-Lee Wallom2, David A
Priestman2, David Begley3, Marja Jäättelä4, Frances M. Platt2
1Orphazyme
ApS, Copenhagen, Denmark, 2Department of Pharmacology, Oxford
University, Oxford, UK, 3Institute of Pharmaceutical Science, King’s College London,
London, UK, 4Department of Cell Death and Metabolism, Danish Cancer Society
Research Center, Copenhagen, DK [Abstract #15, p. 26]
The heat shock response is an ancient biological defense mechanism employing the
power of a series of stress-inducible proteins – the heat shock proteins (HSPs). The HSPs
are very well described cytoprotective agents and their ability to confer resistance to a
wide array of pathological stress insults is of fundamental importance in keeping the
physiological homeostasis in balance.
Together with the heat shock response, the lysosomes with their arsenal of catabolic
enzymes necessary for the efficient re-utilization of macromolecules constitute another
crucial part in the regulation of cellular metabolic homeostasis.
Through an understanding of the intricate interplay between these two important
homeostatic response systems: the lysosomal system and the heat shock response, and
by focusing on the cytoprotective and lysosome-enhancing effects of the heat shock
response, Orphazyme develops a platform of novel therapies for rare, orphan diseases
centered on lysosome-related diseases and diseases particularly responsive to heat shock
response therapy.
Orphazymes lead program, Orph-001, is a recombinant version of human HSP70 being
developed for the treatment of lysosomal storage diseases. HSP70 has been shown to be
effective in correcting the primary pathology of a number of lysosomal storage disorders
at the cellular level and recent data from animal models of LSDs have corroborated these
findings (unpublished data), prompting the ongoing clinical development of rHSP70.
26 16. Rational drug discovery using patient-derived human-induced pluripotent stem
cell models of Niemann Pick type C1
Lawrence S.B. Goldstein, PhD and Paulina Ordonez-Naranjo, MD
University of California San Diego
Successful development of effective therapeutic interventions for Niemann-Pick type C1
(NPC1) will require a deeper understanding of mechanisms of disease initiation and
progression. We have used reprogramming technology to develop sets of NPC1 and
control human induced pluripotent stem cell (hIPSC) lines, and we systematically
generate patient-specific pure neuronal cultures using a standard differentiation
protocol. Our expanded mechanistic studies have provided crucial insights into the dual
role of autophagy in NPC1 neurons and the implications of manipulating this pathway.
We found that cholesterol starvation, such as that caused by lysosomal sequestration of
cholesterol, can induce autophagy, and that autophagy functions as a back-up pathway
that releases and distributes trapped cholesterol, albeit at lower efficiency than the
primary NPC1-dependent pathway. This autophagy-dependent backup cholesterol
distribution pathway is sufficient to support neuronal viability in the short term, but
appears to generate fragmented mitochondria in the long-term as a consequence of
persistent activation. Further mechanistic studies lead us to identify a potential new
transporter with significant homology to NPC1 that mediates autophagy- dependent
cholesterol efflux from the lysosomal compartment. Our work defines the hypothetical
profile of a novel useful drug for NPC1 patients. Such a drug should rescue aberrant
mitophagy without substantially disturbing bulk autophagy, which promotes viability
of NPC1 neurons. We are now actively testing compounds that have been shown, or
predicted to be, protective of mitochondrial function focusing on PKA activators and on
specific inhibitors of the mitochondrial fission machinery. The profile of the agent we are
seeking is one that rescues mitochondrial fragmentation without reducing bulk
autophagy, as this pathway is likely to be essential for normal long-term neuronal
viability as the backup system that distributes accumulated cholesterol in NPC1
neurons.
27 17. Reactions of a CNS neuron to impaired intracellular cholesterol transport
Valérie Demais1, Nicole Ungerer2, Martine Perraut2, Frank W. Pfrieger2
1Plateforme
Imagerie in Vitro, CNRS UPS 3156, 67084 Strasbourg, France,
of Cellular and Integrative Neurosciences, CNRS UPR 3212, University of
Strasbourg, 67084 Strasbourg, France
2Institute
Although neurodegeneration is the main culprit of Niemann-Pick type C disease, it is
still largely unknown, why specific neurons are highly vulnerable to dysfunction or loss
of NPC1 or NPC2. We are studying the impact of NPC1 deficiency on retinal ganglion
cells (RGCs), the neuron of the mammalian central nervous system that connects the
retina to the rest of the brain. Based on our previous studies (Mauch et al., 2001; Nieweg
et al., 2009) we hypothesized that RGCs depend on the NPC1-mediated import of
cholesterol via endocytotic uptake of lipoproteins. Indeed, we observed in RGCs of
NPC1-deficient mice an age-dependent accumulation of ultrastructurally defined
inclusions (Claudepierre et al., 2010) similar to those observed in other types of CNS
neurons, notably Purkinje cells. To further characterize these inclusions and their impact
on cellular metabolism, we use an advanced in vitro preparation, where RGCs are
purified from retinae of postnatal rats and cultured in the absence of serum and glial
cells (Barres et al., 1988; Meyer-Franke et al., 1995) and thus under full control of the
neuronal cholesterol content. As a first approach to block intracellular cholesterol
transport, we treated RGCs with U18666A at 0.5 µg/ml for 48 hours and observed
reliably the induction of filipin-positive puncta in neuronal somata. Notably, this effect
occured in the absence of glial lipoproteins indicating the accumulation of cell-intrinsic
cholesterol, and it was reversed within 24 hours by cyclodextrin in a dose-dependent
manner. The U18-induced filipin-positive puncta were costained with cholera toxin B
suggesting the concomitant accumulation of GM1, and with antibodies against CD63
and LAMP1 indicating their endosomal-lysosomal origin. Modified fixation and staining
methods for electron microscopy revealed that U18 treatment induces multilamellar
inclusions, which contain large amounts of cholesterol. Correlative light and electron
microscopy (CLEM) provided a first proof that U18-induced filipin-positive puncta
observed by fluorescence microscopy correspond to inclusions. Our quantitative
ultrastructural analysis revealed specific U18-induced changes in the endosomallysosomal system, but no effects on the autophagic pathway. Together, our results reveal
how neurons react to a disturbance of intracellular cholesterol trafficking and identify
the ultrastructural correlate of cholesterol accumulation.
Supported by the Niemann-Pick Selbsthilfegruppe Deutschland e.V.
28 18. Heat shock protein 27 protects vulnerable neurons in NPC1 deficiency
Chan Chung, Matthew J. Elrick, Brittany Dixon and Andrew P. Lieberman
From the Department of Pathology, University of Michigan Medical School, Ann Arbor,
Michigan 48109, USA
Many progressive neurological diseases are characterized by the selective vulnerability
of certain neuronal populations. Identification of the mechanisms underlying this
phenomenon is an important problem that will improve our understanding of the
neurodegenerative process and offer therapeutic targets for these devastating disorders.
Purkinje cell degeneration in an anterior-to-posterior gradient is a common feature of
many cerebellar disorders, including Niemann-Pick type C disease (NPC), a lysosomal
storage disorder characterized by childhood onset of multiple progressive neurologic
deficits. Here, we describe an approach to identify candidate genes underlying selective
vulnerability of Purkinje cells in anterior cerebellar lobules using data freely available in
the Allen Brain Atlas. This approach provides candidate neuroprotective genes and
candidate susceptibility genes. Furthermore, we demonstrate that one of candidate
neuroprotective genes, HSP27, promotes neuronal survival in an in vitro model of NPC
disease, through a mechanism that likely involves inhibition of apoptosis. Additionally,
we show that HSP27 overexpression in vivo slows the progression of motor impairment
and diminishes cerebellar Purkinje cell loss in posterior lobules. These results highlight
the novel use of bioinformatic tools to uncover pathways leading to neuronal protection
in neurodegenerative disorders.
* Chan Chung and Matthew Elrick contributed equally to this work
29 19. Regulation of Cholesterol Homeostasis with GEX1A: A Potential Lead for
Niemann-Pick Type C Disease
Jarred R.E. Pickering, Eve A. Granatosky, Michael J. Ahlers, D. Cole Stevens, and
Richard E. Taylor
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame,
IN, 46556, USA
Recent studies have shown that histone deacetylase (HDAC) inhibitors are effective in
correcting the cholesterol storage defects characteristic of Niemann-Pick Type C (NPC)
disease in human NPC1 mutant fibroblasts. Our efforts to identify a novel treatment
option for NPC disease are focused on the Streptomyces-derived polyketide GEX1A,
which increases gene expression similar to known HDAC inhibitor trichostatin A. We
have observed that GEX1A is capable of facilitating cholesterol trafficking in NPC1 cells,
however GEX1A does not affect histone acetylation, and likely acts through a novel
mechanism. Based on these findings, we have developed a multidisciplinary approach
to access GEX1A, as well as synthetic, semisynthetic and bioengineered analogues, in
order to investigate their potential as therapeutics for NPC disease. Here we present our
efforts to develop a synthetic route to GEX1A and analogues, our work in engineering
mutant strains of Streptomyces chromofuscus capable of generating GEX1A analogues,
and our current progress towards evaluating GEX1A in both NPC1 and NPC2 mutant
fibroblasts.
30 20. Proteastatic rescue of a high fraction of human NPC1 mutations by HDAC
inhibitors
Shu Mao, Nina H. Pipalia, and Frederick R. Maxfield
Department of Biochemistry, Weill Cornell Medical College, Cornell University
95% of Niemann–Pick disease type C (NPC) is caused by mutations in NPC1, a
transmembrane protein, that blocks efflux of cholesterol from late endosomes and
lysosomes. There are over 200 mutations observed in NPC1 patients, and most studies
have been done with NPC1I1061T mutation found in about 20% of NPC1 patients.
Previous studies have found that histone deacetylase inhibitors (HDACi), such as SAHA
and LBH589, are able to correct the NPC1 phenotype in human fibroblast cells with an
NPC1 I1061T mutation. The effects of drugs on NPC1 mutations other than NPC1
I1061T are still unclear. In order to examine the effectiveness of HDACi treatment on
hundreds of different NPC1 mutations simultaneously, a high throughput screen has
been designed using an NPC1-null human osteosarcoma cell line (U2OS_shNPC1).
U2OS_shNPC1 cells were transfected with a bicistronic vector expressing GFP and
mutants of NPC1 (pMIEG3-NPC1) by reverse transfection. Using this system, we tested
the effect of SAHA or LBH589 on 81 different NPC1 mutations. After HDACi treatment,
a high fraction of NPC1 mutant proteins were found to be effective in reducing
cholesterol accumulation. This suggests that HDACi therapy might be effective for a
large majority of NPC1 patients. We previously reported that HDACi treatment
increases the expression of NPC1I1061T protein in fibroblast cells. We also found that
treatment of NPC1 mutant cells with Vorinostat led to increased delivery of the NPC1
protein to LE/Ly and increased stability of the mutant NPC1 protein. These indicate that
HDACis might be involved in proteostasis of the mutant protein.
31 21. Cholesterol lowering effect of c-Abl inhibitors, a new therapeutic tool for Niemann-Pick C
disease
Contreras P.1,2,3, González-Zuñiga M.1,2, Dulcey A4., Marugan J4., Alvarez A.R.1,2, Zanlungo S.3
1Cell
Signaling Laboratory. Biological Sciences Faculty, Pontificia Universidad Católica de Chile,
Santiago, Chile, 2CARE-Chile-UC, 3School of Medicine, Pontificia Universidad Católica de Chile,
Santiago, Chile, 4 NCGC, National Institutes of Health, Rockville, USA.
NPC research heavily focuses on therapies that help to improve two central hallmarks: i)
decrease the neuronal damage and ii) restore intracellular cholesterol homeostasis.
Regarding the first point, our laboratory has shown that the tyrosine kinase c-Abl plays a key role
in the neurodegenerative process observed in NPC. The c-Abl specific inhibitor, Imatinib,
prevented apoptosis in the cerebellum, improved locomotor behavior and increased survival
rates of Npc1-/- mice. We have also shown that Imatinib prevented neuronal damage induced by
oxidative stress through inhibition of the c-Abl/p73 pro-apoptotic pathway.
In relation to the second point, cyclodextrin (CD) and histone deacetylase inhibitors (HDACi)
have emerged as possible cholesterol lowering drugs for NPC treatment. Unexpectedly and
interestingly, our results show that Imatinib reduces cholesterol accumulation in both Hepa 1-6
and neuronal HT22 U188666A (U18)- treated cells. Although Imatinib specifically inhibits c-Abl
and is an approved drug by the FDA, it has a low penetrance of the blood-brain barrier (BBB).
Our principal aim is to analyze the effect of GNF-2, a new brain-penetrating c-Abl inhibitor, in
NPC animal models, testing its possible effects on decreasing cholesterol accumulation and
cellular damage in key organs such as the cerebellum and brain and thus opening the possibility
of finding a new therapeutic strategy for treating NPC disease. We are also interested in
exploring the mechanisms involved in the cholesterol-lowering effect of c-Abl inhibition in NPC
models.
Our laboratory in collaboration with the NCGC-NIH group has found that GNF-2, an allosteric
inhibitor of c-Abl, with better BBB penetrance, reduces cholesterol accumulation in HT22 U18treated cells. Moreover, the same effect was observed using GNF-2 in a fibroblast NPC1 genetic
model. Furthermore, our results show that GNF-2 has a beneficial effect in the Npc1-/- mice
improving motor coordination and the gain of weight. We are starting to explore the possible
mechanisms involved in the decrease in cholesterol accumulation mediated by c-Abl inhibition in
NPC cells studying the modulation of histone deacetylases (HDACs) and the upregulation of the
authophagy flux.
Our data strongly suggest that c-Abl could be modulating cholesterol homeostasis in NPC cells
and accordingly this kinase becomes a key therapeutic target due to its participation in the two
central hallmarks sought for NPC treatment.
Supported by Fondecyt 1120512 (AAR) and 1110310 (SZ)
Fondef: D10I1077
CARE-Chile-UC PFB 12/2007
32 22. Hydrophobic amine inhibition of cholesterol trafficking can be reversed by
cathepsin B inhibitors
Joslyn Mills, Jerry Faust, and Laura Liscum
Program in Cellular and Molecular Physiology, Sackler School of Graduate Biomedical
Sciences, Tufts University School of Medicine, Boston, MA
Our lab previously showed that cultured cells treated with hydrophobic amines exhibit
defective cholesterol trafficking. Specifically, free cholesterol accumulates in lysosomes
of hydrophobic amine-treated cells, and LDL-stimulation of cholesterol esterification
and suppression of cholesterol homeostatic genes are reduced. The mechanism by
which hydrophobic amines cause impaired cholesterol transport is unknown.
Tricyclic antidepressants, e.g. imipramine and desipramine, are one class of
hydrophobic amines that cause defective cholesterol trafficking. Recently, we have
observed that CHO cells treated with the irreversible cathepsin B inhibitor, CA-074Me,
show no imipramine inhibition of LDL-stimulated cholesterol esterification.
Furthermore, imipramine-induced accumulation of cholesterol in lysosomes was
prevented when CA-074Me was added to the culture media.
Leupeptin, a reversible cathepsin B inhibitor, also restored cholesterol trafficking to
imipramine-treated CHO cells; however, pepstatin A, which inhibits cathepsin D, did
not.
These data lead us to hypothesize that tricyclic antidepressants induce the cathepsin Bmediated degradation of lysosomal protein(s) that are required for efficient cholesterol
transport through the lysosome and to the endoplasmic reticulum.
This abstract is written for public dissemination.
33 23. Cerebellar neurobiology of Niemann-Pick type C1 disease
Ian M. Williams, Celine Cluzeau, Christopher A. Wassif and Forbes D. Porter
The lysosomal storage disorder Niemann-Pick type C1 (NPC1) is a progressive
neurodegenerative disease caused by mutations in the Npc1 gene, which codes for a
lysosomal transmembrane protein of unknown function. Npc1 defects lead to lysosomal
lipid accumulation, impairments in trafficking and dysregulated lysosomal calcium
homeostasis. Symptoms include ataxia and tremor due to gradual yet near-complete loss
of cerebellar Purkinje cell neurons in an anterior to posterior gradient. Interestingly,
Purkinje cells in the lobule X region do not die in either NPC1 patients or the Npc1-/mouse model. NPC1 is typically considered a cholesterol storage disorder, but a number
of other cellular phenotypes initiate prior to murine Purkinje cell cholesterol
accumulation becoming apparent, for example (in order of appearance) axonal
dystrophy of Purkinje cells, followed by microgliotic then astrogliotic onset.
Npc1-/- and littermate control mice cerebella were studied immunohistochemically to
ascertain whether lobule X Purkinje cells are immune to the NPC1-defect or merely
resistant to a certain aspect of pathology. Reduced levels of axonal pathology, microglial
activation and astroglial recruitment were observed in lobule X Purkinje cells compared
to others from midstage disease onwards. Crucially, when looking at early-stage disease,
the level of Purkinje axon pathology and microgliosis was equally present in lobule X
and disease-susceptible posterior regions, and yet these pathologies did not appear to
worsen from then on in Lobule X, with little development in neuronal or gliotic
pathology.
This suggests pathology initiates in Lobule X Purkinje cells, but after the 4-week stage
disease progression is relatively static. Discovering what protective factor(s) is/are
unique to Lobule X Purkinje cells may well prove invaluable in finding novel ways to
manipulate the NPC1 disease pathway.
34 24. Assessing Cholesterol Metabolism, Storage and Transport in Live Cells and C.
elegans by SRS Imaging of Phenyl-Diyne Cholesterol
Hyeon Jeong Lee1, 2, Wandi Zhang3, Yang Yang3, Delong Zhang3, Bin Liu4, Eric L.
Barker5, Kimberly K. Buhman6, Lyudmila V. Slipchenko3, Mingji Dai3, Ji-Xin Cheng1, 3, 7
1Interdisciplinary
Life Science Program, Purdue University, West Lafayette, Indiana,
USA, 2Department of Comparative Pathobiology, Purdue University, West Lafayette, IN
47907, 3Department of Chemistry, Purdue University, West Lafayette, Indiana, USA,
4National Key Laboratory of Science and Technology on Tunable Laser, Harbin Institute
of Technology, Harbin 150080, China, 5Medicinal Chemistry and Molecular
Pharmacology, Purdue University, West Lafayette, IN 47907, 6Department of Nutrition
Science, Purdue University, West Lafayette, IN 47906, 7Weldon School of Biomedical
Engineering, Purdue University, West Lafayette, IN 47907
We report an in vivo cholesterol imaging method using rationally synthesized phenyldiyne cholesterol (PhDY-Chol) and stimulated Raman scattering (SRS) microscope. The
phenyl-diyne group is biologically inert and provides a Raman scattering cross section
that is 122 times larger than the endogenous C=O stretching mode. SRS microscopy
offers an imaging speed that is faster than spontaneous Raman microscopy by three
orders of magnitude, and a detection sensitivity of 31 µM PhDY-Chol, corresponding to
~1,800 molecules in the excitation volume. Inside living CHO cells, PhDY-Chol closely
mimics the behavior of cholesterol, including membrane incorporation and
esterification. In a cellular model of Niemann-Pick type C disease, PhDY-Chol reflects
the accumulation of cholesterol in lysosomes more selectively than filipin staining. SRS
imaging of PhDY-Chol-fed live C. elegans identifies previously unnoticed cholesterol
storage compartments. Together, our work demonstrates an enabling platform for
mechanistic study of cholesterol storage and trafficking in living cells and vital
organisms.
35 25. Site directed mutagenesis of NPC2 reveals sterol transfer and LBPA-interaction
domains
Leslie McCauliff and Judith Storch
Department of Nutritional Sciences and Rutgers Center for Lipid Research, Rutgers
University, NJ, USA
The cholesterol storage disorder Niemann-Pick type C (NPC) disease is caused by
mutations in either of two lysosomal proteins, NPC1 or NPC2. NPC2 is a 16kDa soluble
protein that binds cholesterol. Previous work showed that NPC2 can rapidly transport
cholesterol to/from vesicles via direct interaction with membranes. Site-directed
mutagenesis studies suggest that the NPC2 surface may have two membrane interacting
domains necessary for its cholesterol transport properties. Membrane interaction assays
demonstrate that NPC2 promotes vesicle-vesicle contacts, supporting the hypothesis
that the protein contains at least two membrane interaction domains on its surface.
Sterol transfer assays also demonstrate that lysobisphosphatidic acid (LBPA), found
uniquely in late endosomes/lysosomes (LE/LY), dramatically enhances cholesterol
transfer rates by wt NPC2, suggesting a relationship between NPC2 and LBPA in LE/LY
cholesterol transport. Moreover, we found that LBPA dramatically enhances the rate at
which the NPC2-mediated vesicle-vesicle interactions occur. In the current studies we
have identified two residues on the surface of NPC2 that are particularly sensitive to
LBPA; several mutations causing deficient sterol transfer rates to zwitterionic egg
phosphatidylcholine (EPC) vesicles exhibit sterol transfer rates identical to WT NPC2
when LBPA is incorporated into the membranes, whereas two mutations on the surface
of NPC2 generate proteins that are entirely insensitive to LBPA. Furthermore, NPC2
mutants whose sterol transfer rates were enhanced by LBPA were found to bind LBPA
similar to WT protein, while the two mutants with transfer rates unaffected by LBPA
exhibit decreased binding to the phospholipid. Thus, it is likely that LBPA aids in
NPC2-mediated LE/LY cholesterol transport by directly interacting with a specific
domain on the surface of the protein. We hypothesize that the relationship between
NPC2 and LBPA in cholesterol transport may involve the formation of membrane
contact sites within the LE/LY, facilitating rapid cholesterol egress from the interior
lamellae of the compartment.
36 26. Phosphatidic acid and ubiquitin catabolism modify intracellular lipid
accumulation and neurodegeneration in Niemann Pick type C disease.
Andrew B. Munkacsi1, Lauren Csaki2, Katsumi Higaki4, Giselle Domínguez-Gutiérrez3,
Robin B. Chan5, Bowen Zhou5, Manjari Vasanthan1, Noor Alsarrage1, Namal Coorey1,
David N. Brindley6, Daniel Finley7, Aaron D. Gitler8, Gilbert Di Paolo5, Karen Reue2, and
Stephen L. Sturley5, 9
1School
of Biological Sciences and Centre for Biodiscovery, Victoria University of Wellington, Wellington,
New Zealand 6012, 2Department of Human Genetics, University of California at Los Angeles, Los Angeles,
CA 90095, 3Institute of Human Nutrition, Columbia University Medical Center, New York, NY 10032,
4Division of Functional Genomics, Research Center for Bioscience and Technology, Tottori University,
Japan, 5Department of Pathology and Cell Biology, Columbia University Medical Center, New York, NY
10032, 6Department of Biochemistry, University of Alberta Edmonton, Alberta T6G 2H7 Canada,
7Department of Cell Biology, Harvard Medical School, Boston, MA 02115, 8Department of Genetics, Stanford
University School of Medicine, Stanford, CA 94305, 9Department of Pediatrics, Columbia University Medical
Center, New York, NY 10032
School of Biological Sciences and Centre for Biodiscovery1, Victoria University of Wellington, Wellington,
New Zealand 6012. Department of Human Genetics2, University of California at Los Angeles, Los Angeles,
CA 90095. Institute of Human Nutrition4, Columbia University Medical Center, New York, NY 10032.
Division of Functional Genomics4, Research Center for Bioscience and Technology, Tottori University,
Japan, Department of Pathology and Cell Biology5, Columbia University Medical Center, New York, NY
10032, Department of Biochemistry6, University of Alberta Edmonton, Alberta T6G 2H7 Canada,
Department of Cell Biology7, Harvard Medical School, Boston, MA 02115. Department of Genetics8, Stanford
University School of Medicine, Stanford, CA 94305. Department of Pediatrics9, Columbia University Medical
Center, New York, NY 10032.
The mutations that cause subcellular sterol and sphingolipid accumulation associated with NP-C disease are
known, however mutant alleles do not always correlate with disease severity, either between or sometimes
even within afflicted families. This provokes the hypothesis that extrinsic factors (environmental and/or
genetic) may impact disease outcome. The genes defective in NP-C disease are conserved across 2 billion
years of evolution to the extent they are functionally interchangeable between yeast and humans [1].
Curiously, yeast strains that lack the NPC pathway are aphenotypic; they are indistinguishable from control
strains. This compelling observation led us to embark on a search for modifier pathways that confer
“normality” to these yeast strains with the hypothesis that these pathways would represent treatment
options for this devastating disease [2]. We identified 11 genes that exacerbated lethality of the yeast model
in the presence of myriocin, a pharmacologic modifier of sphingolipid metabolism. We extrapolated these
findings to demonstrate that deletion or knockdown of the mammalian orthologs of UBP6, a deubiquitinase
that activates microautophagy, and PAH1, a phosphatidic acid hydrolase, exacerbate disease severity in
rodent and human systems. We present here the continuation of these studies and will pursue a unifying
hypothesis regarding the impact of these modifiers on NP-C disease. Our results advance the molecular
understanding of eukaryotic sphingolipid metabolism, particularly with regard to NP-C disease severity
and consequently offer promising therapeutic intervention points to reverse NP-C disease.
1.
2.
Malathi, K., et al., Mutagenesis of the putative sterol-sensing domain of yeast Niemann Pick Crelated protein reveals a primordial role in subcellular sphingolipid distribution. J Cell Biol, 2004.
164(4): p. 547-56.
Munkacsi, A.B., et al., An "Exacerbate-reverse" Strategy in Yeast Identifies Histone Deacetylase
Inhibition as a Correction for Cholesterol and Sphingolipid Transport Defects in Human NiemannPick Type C Disease. J Biol Chem, 2011. 286(27): p. 23842-51.
37 27. Determination of the Allelic Frequency in Niemann-Pick Type C by Analysis of
Massively Parallel Sequencing Data Sets
Christopher A. Wassif, Joanna L. Cross, James Iben, Forbes D. Porter
Program in Developmental Endocrinology and Genetics, Eunice Kennedy Shriver
National Institute of Child Health and Human Development, National Institutes of
Health, Department of Health and Human Services
We sought to determine the pathogenic carrier frequency of NPC1 and NPC2 utilizing
data amassed from 4 independent massively parallel sequencing projects: NHLBI GO
Exome Sequencing Project (ESP), 1000 Genomes Project, ClinSeq®, and a database from
a NIH inter-institute collaboration on Autism.
In total we evaluated 17,754
chromosomes against the human reference sequence for NPC1 and NPC2 and found
16,455 and 271 nonsynonymous sequence variants in the 2 genes respectively. Further
analysis of the 16,455 variants found in NPC1 revealed 147 distinct changes; 129 coding
single nucleotide base variants, 9 splice mutations, and 9 insertions or deletions.
Analysis of NPC2 discovered 14 distinct changes; 12 coding single nucleotide base
variants, and 2 splice mutations. In order to determine the rate of pathogenic alleles we
conducted a literature search and queried HGMD® and found 25 of 147 distinct variants
published in NPC1, additionally we noted 2 additional mutations in the NPC patient
cohort, and 5 of 14 distinct variants published in NPC2. We further performed in silico
analysis on all the coding single nucleotide base variants using Polyphen-2, which
provided a predictive assignment of “benign”, “possibly damaging”, or “probably
damaging”. Combining those variants that have been published as disease causing with
variants that pholyphen predicted to be possibly damaging or probably damaging
totaled 112 pathogenic alleles or a pathogenic carrier rate of 0.63% (112/17,754) for
NPC1. Based on this carrier frequency we are able to predict a NPC1 disease incidence
of 1/100,776 conceptions. This estimate is well in line with published reports. Similarly
for NPC2 we surmised 58 pathogenic alleles or a pathogenic carrier rate of 0.33%
(58/17,754). Based on this carrier frequency we are able to predict a NPC2 disease
incidence of 1/367,242 conceptions.
38 28. Regulation of NPC1-mediated Cholesterol export by the lysosome glycocalyx
Maika Deffieu, Jian Li and Suzanne R. Pfeffer
Department of Biochemistry, Stanford University School of Medicine
Stanford, CA 94305
NPC1 protein is needed to transport low density lipoprotein-derived cholesterol from the lumen
of lysosomes into the cell. The protein has 13 transmembrane domains, three large lumenal
domains, and a cytoplasmic tail. NPC1’s lumenally oriented, N-terminal domain binds
cholesterol and has been proposed to receive cholesterol from NPC2 protein as part of this
process. We have shown that the second lumenal domain of NPC1 binds directly to cholesterolbearing NPC2 protein; disease-causing NPC1 mutations decrease NPC2 binding, suggesting that
NPC2 binding is needed for NPC1 function in humans.
Why do cells need NPC1 and NPC2 proteins? Our working model is that a glycocalyx comprises
a barrier to protect the lysosome membrane from lysosomal degradative enzymes, and cells need
NPC1 protein to transfer LDL-derived cholesterol across this glycocalyx. A prediction of this
model is that cells will be less dependent upon NPC1 protein if their glycocalyx is thinner. Our
recent data strongly support this model.
LAMP1 and LAMP2 proteins represent the major glycoproteins of the lysosome membrane.
These proteins are highly glycosylated on both Asp (N-linked) and Ser/Thr residues (O-linked),
and their oligosaccharides contribute to the structure of the lysosomal glycocalyx. Initial
experiments to test the role of the glycocalyx in driving cholesterol accumulation indicated that
NPC1-deficient CHO cells containing shorter, Man-5 N-linked oligosaccharides accumulate less
cholesterol in lysosomes than NPC1-deficient, wild type CHO cells. Cell sorting of antibody
stained cells showed that bis-monoacylglycerol levels (that generally track lysosomal cholesterol
levels) were significantly higher in NPC1-depleted HeLa cells or NPC1-deficient CHO cells,
compared with the same cells treated with an inhibitor of O-linked glycosylation. Lysosomespecific filipin content, determined by quantitative immunofluorescence microscopy, confirmed a
significant decrease in lysosome cholesterol upon O-linked glycosylation inhibition. Direct
biochemical measurement of cholesterol showed that lysosomes purified from NPC1-deficient
human fibroblasts contained at least 30% more cholesterol that those obtained from cells in which
O-linked glycosylation was blocked. As an independent means to modify protein Oglycosylation, we used siRNA to target GCNT1, a key enzyme in the pathway of mucin-type Oglycan synthesis. Similar to our findings with O-GalNAc addition inhibitors, this treatment led
to a ~25% decrease in lysosomal filipin staining in NPC1-deficient human fibroblasts, two days
after GCNT1 enzyme depletion. Finally, increased CD68 reflects the presence of elevated
oxidized LDL in lysosomes; CD68 levels decreased in NPC1-deficient cells after addition of the
O-linked glycosylation inhibitor.
These experiments strongly support a model in which the glycocalyx interferes with the ability of
cholesterol to access the limiting lysosome membrane; NPC1 functions to bypass the glycocalyx.
Our findings suggest that O-linked glycosylation inhibitors may be beneficial for the treatment of
NPC disease in the future. It is very possible that miglustat (N-butyl-deoxynojirimycin), an
imino sugar, influences the composition of the glycocalyx, a prospect that we hope to test
directly.
39 29. Niemann-Pick C Type 1 With Severe Pulmonary Manifestations
Orna Staretz-Chacham1, Micha Aviram2, Eliyahu Hershkovitz3
1Soroka
University Medical Center, Ben-Gurion University of the Negev, Beer-Sheva,
Israel, Israel, 2Soroka University Medical Center, Ben-Gurion University of the Negev,
Beer-Sheva, Israel, Israel, 3Soroka University Medical Center, Ben-Gurion University of
the Negev, Beer-Sheva, Israel, Israel
Niemann- Pick disease type C (NPC) is a lysosomal storage disorder, caused due to
abnormalities of intracellular transport of endocytosed cholesterol. The hallmark of NPC
is abnormal cholesterol esterification. Estimated prevalence of NPC is 1: 120,000-150,000.
There are four phenotypic types of NPC, and two genes are involved in NPC: NPC1
which accounts for approximately 90% of cases, localized to the late endosomal
membrane and involved in cholesterol trafficking and NPC2. Lung involvement has
been described in only a few patients with NPC, mostly NPC2. We describe here a series
of 12 patients, with NPC1, seven of them with pulmonary manifestations .
Patients and Methods
Data was collected from patients' files diagnosed with NPC by mutation analysis in our
clinical center.
Results
All patients were homozygous to mutation R404Q, and presented during the first year
of life, the majority of them immediately after birth. Seven of the patients had
pulmonary manifestations, including recurrent episodes of asthma exacerbations, and
also mixed with restrictive lung disease .
Conclusion
Pulmonary involvement in NPC1 is underestimated, as an obstructive or mixed lung
disease that can lead to respiratory insufficiency due to accumulation of cholesterol,
abnormal surfactant and abnormal signaling. The involved proteins: NPC1- a large
transmembrenal protein and NPC2- a soluble lysosomal protein. Therefore, blocking the
pathway of alveolar type II cells, whether in NPC1 protein or NPC2, would lead to
disruption of cholesterol trafficking and as a result to accumulation of cholesterol with
pulmonary manifestation as well as the well-recognized neurological signs.
40 30. Effect of TFEB activation on NPC phenotype
Hiroko Nagase, Joslyn Mills and Laura Liscum
Program in Cellular and Molecular Physiology, Sackler School of Graduate Biomedical
Sciences, Tufts University, 136 Harrison Avenue, Boston, MA 02111
Niemann Pick type C disease (NPC) is a lysosomal storage disorder characterized by the
accumulation of cholesterol and glycosphingolipids. NPC disease primarily affects the
liver and the brain, but the two organs have different responses to the lipid storage. In
the liver, the cholesterol storage deprives hepatocytes of needed cholesterol, but the cells
compensate by increasing de novo cholesterol synthesis to meet the organ’s demand.
Clearing hepatocytes of the storage material may represent a therapeutic approach for
NPC liver. In contrast, cells in the brain fail to increase cholesterol synthesis to supply
needed cholesterol [1]. The brain’s pathogensis is thought to occur as a result of reduced
cholesterol in cellular membranes. Therefore, a therapeutic approach for the NPC brain
must distribute the stored cholesterol to the needed membranes.
Recently, it was reported that overexpression of transcription factor EB (TFEB) promotes
lysosomal exocytosis and clearance of pathogenic material in other lysosomal storage
diseases, including Pompe, Batten diseases, and Multiple Sulfatase Deficiency [2]. This
led us to question whether TFEB activation in NPC cells will clear cholesterol
accumulation in lysosomes, but also result in cholesterol redistribution within the cells
thereby normalizing cholesterol homeostasis. Our hypothesis is that TFEB activation in
NPC cells will reduce its stored cholesterol. This may be due to efflux of contents into
the extracellular space, which would be therapeutic for the NPC liver. However, it may
also lead to redistribution of stored cholesterol which would be therapeutic for NPC
neurons and glia.
We induced TFEB activation in NPC cells using Torin1, an mTORC1 inhibitor [3,4].
Torin1 treatment led to TFEB translocation to the nucleus and downstream changes in
gene expression. Torin1 treatment also led to a reduction in NPC lysosomal cholesterol
storage as measured by filipin fluorescence microscopy. However, we did not detect
efflux of stored cholesterol or differences in cellular cholesterol content upon Torin1
treatment, which suggested that cholesterol was being redistributed within the cells.
Thus, we are testing the effect of Torin1 on all cholesterol trafficking pathways.
1. Peake, K.B., and J.E. Vance. (2012) Normalization of cholesterol homeostasis by 2hydroxypropyl-b-cyclodextrin in neurons and glia from Niemann-Pick C1 (NPC1)deficient mice. J Biol Chem, 287:9290-9298.
2. Medina, D.L., A. Fraldi, et al. (2011) Transcriptional activation of lysosomal
exocytosis promotes cellular clearance. Dev Cell, 21:421-430.
3. Thoreen, C.C., S.A. Kang, et al. (2009) An ATP-competitive mammalian target of
rapamycin inhibitor reveals rapamycin-resistant functions of mTORC1. J Biol Chem,
284:8023-8032.
4. Settembre, C., R. Zoncu, et al. (2012) A lysosome-to-nucleus signaling mechanism
senses and regulates the lysosome via mTOR and TFEB. EMBO J, 31:1095-1108.
41 31. Plasma signature of neurological disease in the monogenetic disorder Niemann
Pick Type C
Md. Suhail Alam1, 2, Michelle Getz1, 2, Sue Yi1, 2, Forbes Dennis Porter3, Nicole Farhat3,
Tamar Pounardjian4, Elizabeth M Berry-Kravis4, Jeffrey Kurkewich1, 2, Innocent
Safeukui1, 2 and Kasturi Haldar1, 2
1Center
for Rare and Neglected Diseases, 2Department of Biological Sciences University
of Notre Dame, IN 46556, USA; 3National Institute of Child Health and Human
Development, National Institutes of Health; 4Rush University Medical Center, 1725 West
Harrison Street, Chicago, IL 60612.
Neurodegenerative diseases like Niemann Pick Type C disease are associated with
inflammation in the brain and other organs. Inflammatory proteins are frequently a
source of biomarkers in many diseases: however inflammatory products of cerebral
disease are not easily detected in blood. Inflammation in multiple organs and
heterogeneity in disease present additional challenges in distinguishing the extent to
which a blood-based marker reflects disease in brain or other afflicted organs. Murine
models of Niemann Pick Type C (NPC) present aggressive forms of cerebral and liver
inflammatory disease. Microarray analyses previously revealed age-dependent changes
in innate immunity transcripts in the mouse brain. We have now validated four putative
secretory inflammatory markers that are also elevated in mouse liver. We include
limited, but first time analysis of human NPC liver and cerebellum. Further we utilized
2-hydroxypropyl-beta-cyclodextrin (HPβCD; an emerging therapeutic) administered
intraperitoneally in mice, which abrogates inflammatory pathology in the liver but has
limited effect on the brain. By analyzing the corresponding effects on inflammatory
plasma proteins, we identified cathepsin S as a lead indicator of liver disease. In contrast
lysozyme was a marker of both brain and liver disease.
Our data suggest that dual analysis of levels of the inflammatory markers lysozyme and
cathepsin S may enable detection of multiple distinct states of neurodegeneration in
plasma. We are validating these markers to separately track neurological disease from
liver disease in NPC patient plasma obtained from natural history studies and in
response to emerging therapeutics.
42 Attendees
Last Name
Alam
Alvarez Rojas
Balch
Balmert
Berry-Kravis
Calhoun
Chung
Cluzeau
Collins
Cologna
Crawford
Csimma
Davidson
Dixon
Gafni
Goodson
Granatosky
Green
Haldar
Healy
Helquist
Hill
Imrie
Jacoby
Kiefer
Kirkegaard
Koujaian
Lee
Li
Liang
Liscum
Liu
Mao
Maxfield
McCauliff
Milla
Mills
Morris
O'Keefe
O'Neill
First Name
Suhail
Alejandra
William
Mary
Elizabeth
Barbara
Chan
Celine
Chris
Stephanie
Gregory
Cristina
Cristin
Brittany
Juliette
Holly
Eve
James
Kasturi
Aileen
Paul
Nadine
Jacqueline
Jonathan
Luke
Thomas
Harry
Hyeon
Jeong
Jian
Guosheng
Laura
Gang
Shu
Fred
Leslie
Luis
Joslyn
Jill
Joanne
Matthew
Ordonez
Ory
Parseghian
Perlstein
Pfeffer
Pfrieger
Pipalia
Porter
Paulina
Daniel
Cindy
Ethan
Suzanne
Frank
Nina H.
Forbes D.
Quandt
Quandt
Reid
Gene
Karen
Alison
Institution
University of Notre Dame
P. Universidad Catolica de Chile
Scripps Research Institute
University of Notre Dame
University of Michigan Medical School
NICHD/NIH
Purdue University
National Institutes of Health
University of Notre Dame
Cydan
Albert Einstein College of Medicine
Andrew Lieberman Lab
BioMarin Pharmaceutical
University of Notre Dame
University of Notre Dame
International Niemann-Pick Disease Alliance
University of Notre Dame
Cydan Development, Inc.
University of Notre Dame
Director, National Niemann-Pick Disease Foundation
NP-C Parent
University of Notre Dame
Orphazyme
Purdue University
Stanford University
UT Southwestern Medical Center
University of Notre Dame
Weill Cornell Medical College
Weill Cornell Medical College
Rutgers University
Stanford University
Tufts University
Rush University
Department of Chemistry and Biochemistry - University of Notre
Dame
UCSD
Washington University School of Medicine
Ara Parseghian Medical Research Foundation
Perlstein Lab
Stanford University School of Medicine
CNRS UPR 3212 INCI
Weill Cornell Medical College
Program in Developmental Endocrinology and Genetics, NICHD,
NIH, DHHS
NNPDF
National Niemann-Pick Disease Foundation
Actelion
Email Address
[email protected]bio.puc.cl
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
Rizk
Schultz
Scott
Sedgwick
Spiegel
Staretz
Chacham
Storch
Strattan
Strattan
Sturley
Subramanian
Taylor
Thompson
Tripicchio
Vaughan
Vite
Wassif
Wiest
Williams
Wood
Wood
Zanlungo
Shahir
Mark
Matthew
Alanna
Sarah
Orna
University of Notre Dame
University of Michigan
Stanford University
University of Notre Dame
Virginia Commonwealth University
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
Judith
Rick
Nancy
Stephen
Kanagaraj
Richard
David H
Kristen
Kevin
Charles
Christopher
Olaf
Ian
Aletha
Andy
Silvana
Rutgers University
CTDInc
TFBU
Columbia University Medical Center
The Scripps Research Institute
University of Notre Dame
Purdue University
University of Notre Dame
University of Notre Dame
University of PA
NIH\NICHD
University of Notre Dame
NIH / NICHD
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
Pontificia Universidad Catolica de Chile (UC)
NP-C Research - Community Resources/Information
OGT-918 (Zavesca or Miglustat) Trial Information
Contact: Marc Patterson, Mayo Clinic, Rochester, MN
[email protected]
NPC Natural History Study and proposed Drug Trial
Cell lines, serum, plasma and CSF from over 50 well-characterized NP-C1 patients
Contacts: Forbes “Denny” Porter and Nicole Yanjanin, National Institutes of Health, Maryland
[email protected]; [email protected]
NPC Cat Colony at UPenn
Contacts: Charles Vite, University of Pennsylvania School of Veterinary Medicine
[email protected]
NPC-2 Reagents (gene-targeted mice and antibodies)
Contact: Peter Lobel, Rutgers, NJ
[email protected]
NPC Tissue
Contact: Melissa Larkins Davis, University of Maryland Brain and Tissue Bank for Developmental Disorders.
Director: H. Ronald Zielke, Ph.D.
[email protected]; BTBank.org
Drug Design and Development Services
Contact: Richard Taylor, Interim Director, Warren Family Center for Drug Discovery and Development
[email protected]
NPC Imaging Studies
Contacts: Ted Trouard and Robert Erickson, University of Arizona
[email protected]; [email protected]
NPC Diagnostics: Sequencing and Filipin Testing
Contact: Peter Bauer, University of Tuebingen, Germany
[email protected]
BC-Theta – High Quality Cholesterol-binding Reagent
Contact: Yoshiko Iwashita, Faculty of Pharmacy Iwaki Meisei University
[email protected]
NPC Mouse Core to test emerging therapies
Contacts: Michelle Getz and Kasturi Haldar
Dept. Biological Sciences, Center for Rare and Neglected Diseases, University of Notre Dame
[email protected]; [email protected]
45
Save the Date
The 2015 Michael, Marcia, and Christa Parseghian
Conference for Niemann-Pick Type C Research will
be held at the University of Notre Dame (South
Bend, IN) with guest rooms available at the new
Morris Inn on campus!
Thursday, June 11th – Saturday, June 13th
Abstract Submission Deadline: March 27th, 2015
As the date draws near, information will be posted on
http://niemannpick.nd.edu/conference
In the meantime, feel free to contact Jenna Rangel (574-631-6456 or
[email protected]) with questions.
46