2001 ANNUAL REPORT
THE J. DAVID GLADSTONE INSTITUTES
University of California, San Francisco
San Francisco General Hospital Medical Center
OF NEUROLOGICAL DISEASE
2001 ANNUAL REPORT
by The J. David Gladstone Institutes
All rights reserved
Editors: Gary Howard and Stephen Ordway
Designers: John C. W. Carroll and John Hull
Photographers: Stephen Gonzales and Christopher Goodfellow
Project Coordinator: Sylvia A. Richmond
THE J. DAVID GLADSTONE INSTITUTES
P.O. Box 419100, San Francisco, CA 94141-9100
Telephone (415) 826-7500 • Facsimile (415) 826-6541
TABLE OF CONTENTS
DESCRIPTION OF THE INSTITUTES.........13
MEMBERS OF THE INSTITUTE.........25
REPORTS FROM THE LABORATORIES.........31
Behavioral Core Laboratory.........95
Gladstone Genomics Core.........103
Lennart Mucke, M.D.
Director’s Report 5
Neurodegenerative disorders impair neuronal function and survival. Interestingly,
rob people of their ability to the pace and severity of neurodegenerative processes
remember, speak, write, ambu- can be modified by endogenous factors secreted from
late, and control their lives. astrocytes and microglia, injury-responsive cells that
These conditions are on the rise closely interact with neurons in the brain and spinal
because people are living cord. Among these modulatory factors are the
longer, and aging strongly apolipoproteins and their receptors, which play impor-
increases the risk of being afflicted by these condi- tant roles in the nervous system in normal develop-
tions. The enormous cost of caring for individuals ment, aging, and disease. Because of its intriguing
with these conditions threatens our health care system. role in Alzheimer’s disease (AD) and other neurolog-
A medical breakthrough is clearly needed, and the ical conditions, apolipoprotein (apo) E has remained a
surest way to such a breakthrough is to determine major research target for several GIND laboratories.
exactly how these diseases result in the dysfunction Other studies have focused on the molecular mecha-
and degeneration of nerve cells. In addition, neurolog- nisms of neural plasticity, which is critical for brain
ical diseases raise a range of fascinating questions that development and adaptations of the nervous system to
are of fundamental scientific interest. While the inves- environmental stimuli and challenges.
tigation of neurological diseases has promoted basic
neuroscientific discoveries for over a century, there Scientific Discoveries
has never been a more promising and exciting conver-
gence of basic and disease-related neuroscience than The laboratory of Dr. Steven Finkbeiner focuses on
now. Investigators at the Gladstone Institute of the pathogenesis of Huntington’s disease. This fatal
Neurological Disease (GIND) have continued to inherited neurodegenerative disorder is associated
unravel the molecular processes that trigger and cul- with increasingly disruptive involuntary movements
minate in neurodegenerative diseases, as well as those and a progressive loss of motor control. It is caused by
that underlie normal functions of the nervous system. abnormal glutamine expansions in the protein hunt-
ingtin, which affect protein folding. Antibodies gener-
Several projects this year have examined the possibili- ated by the Finkbeiner laboratory recognize only dis-
ty that many, if not all, neurodegenerative disorders are ease-causing forms of mutant huntingtin. With these
caused by the abnormal folding or aggregation of pro- antibodies, a specific region was identified in mutant
teins. Although different proteins accumulate in differ- huntingtin that appears to be critical for its pathologi-
ent neurodegenerative disorders, the ways in which cal activities. Interestingly, similar disease-associated
they damage nerve cells appear to overlap. This possi- regions were detected in mutant proteins associated
bility raises hope that it will be feasible to develop with other inherited polyglutamine diseases. To facil-
treatments that can prevent, stall, or even reverse more itate the identification of strategies that can block the
than one of these conditions. Toward this goal, we formation of the disease-associated regions or their
have investigated the conformational states of diverse effects, Dr. Finkbeiner developed a sophisticated
misfolded proteins and the mechanisms by which they robotic microscope for the large-scale analysis of cell
Director’s Report 7
2001 ANNUAL REPORT
cultures. This powerful new device has the potential to the risk of developing AD (E4 > E3 > E2). ApoE4 is
revolutionize the cell biological investigation of neu- the main known inherited risk factor for the most fre-
rodegenerative pathways and other processes. The quent form of AD. This year, lead findings obtained in
Finkbeiner laboratory also studied the molecular cell cultures were extended to transgenic mouse mod-
mechanisms by which the entry of calcium through els and human AD cases, underlining the significance
channels in neuronal membranes affects gene expres- of the original cell-culture data. A much greater accu-
sion and synaptic plasticity. Their findings suggest mulation of apoE fragments was detected in brains of
that intracellular proteins located near the openings of humans and transgenic mice expressing apoE4 than in
the channels interact with the calcium ions, linking those expressing apoE3. Both in cultured neurons and
calcium influx to the activation of specific genes in in transgenic brains, the accumulation of apoE4 frag-
the neuronal nucleus. ments was associated with abnormal phosphorylation
of tau, cytoskeletal derangements, and neurodegener-
The laboratory of Dr. Fen-Biao Gao focuses on the ation. Abnormally phosphorylated tau is the major
genes and molecular pathways that regulate the devel- constituent of neurofibrillary tangles, a pathological
opment and maintenance of neuronal dendrites. hallmark of AD. The differential propensity of apoE
Dendrites are tree-like extensions of neurons that isoforms to be broken down into potentially pathogen-
receive signals and participate in information process- ic fragments (E4 > E3) may relate to their effects on AD
ing and storage. These highly branched structures risk (E4 > E3) and age of onset (E4 < E3). Inhibiting the
account for more than 90% of the surface of some neu- processes that result in intraneuronal fragmentation of
rons. In many neurological disorders, including AD apoE could be of therapeutic benefit, particularly in
and fragile X syndrome, the number of dendritic people with apoE4. Identifying the protease that might
branches and the density of dendritic spines are be involved in apoE cleavage has become a major
altered. However, very little is known about the mech- research target for the Huang laboratory.
anisms that control dendritic branching in vivo. Dr.
Gao developed a method to express mutant proteins The laboratory of Dr. Robert W. Mahley has continued
selectively in individual neurons and to study the con- its cell biological investigation of the mechanisms that
sequences of this manipulation in living fruit flies. This underlie the neuroprotective functions of apoE3 and
new approach has allowed him to demonstrate that the the pathogenic functions of apoE4 in AD and other
dendritic branching patterns of individual neurons are conditions. Previous studies revealed that apoE3 pro-
extremely diverse but well conserved among flies, sug- motes neurite outgrowth, protects the nervous system
gesting that they are of great physiological importance. against diverse injuries, and facilitates neuroregenera-
Some of the mutations Dr. Gao identified in previous tion after trauma. In contrast, apoE4 was not protective
genetic screens turned out to have surprisingly specif- and in many instances even worsened the outcome of
ic effects on the outgrowth of dendrites or axons or neural injuries. This year, studies of apoE isoforms
both. Since neural networks are established through were extended to the interaction of apoE with amyloid
connections between these neuronal structures, these β peptides (Aβ), which accumulate to abnormally high
studies are shedding light on one of the most funda- levels in AD brains. Forms of Aβ with a strong ten-
mental design principles of the nervous system. In the dency to aggregat can be taken up by brain cells
future, they may also help us understand how neural and are known to disrupt lysosomal membranes.
circuits are broken down by neurological diseases and Lysosomes are intracellular vesicles that contain many
how broken circuits might be repaired. protein-degrading enzymes and other highly reactive
molecules. It has been postulated that the destabiliza-
The laboratory of Dr. Yadong Huang has continued its tion of lysosomal membranes may be an important
investigation of intracellular apoE fragmentation and mechanism by which Aβ elicits neuronal degeneration
pathogenic interactions of apoE fragments with the in AD. The Mahley laboratory demonstrated that Aβ-
microtubule-associated protein tau. Human apoE induced lysosomal leakage was strongly enhanced by
exists in three major isoforms that differentially affect apoE4, but not by apoE3, and that the enhanced leak-
8 Director’s Report
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE
age was associated with increased neuronal death. The this analysis was the identification of gene products that
identified effects of apoE isoforms on the destabiliza- showed opposite responses to overexpression of Aβ
tion of lipid membranes (E4 > E3), Aβ-induced lyso- versus Aβ vaccination and were relatively unaffected by
somal leakage (E4 > E3), and associated neuronal human APP or sham vaccination. Some of the identified
death (E4 > E3) may relate closely to their effects on molecules may be effectors of Aβ-induced neurotoxici-
AD risk (E4 > E3) and age of onset (E4 < E3). Notably, ty. Others may mediate beneficial vaccination effects,
pharmacological inhibition of a specific cell death constitute new therapeutic targets, or serve as useful end
mediator completely prevented the potentiation of neu- point measures for vaccination trials.
ronal death by apoE4.
The laboratory of Dr. Robert E. Pitas has followed up
My own laboratory has continued to investigate the on their discovery of a novel brain protein that inter-
molecular pathways that link genetic determinants or acts with apoE. They have begun to characterize the
risk factors of AD to neurodegeneration and cognitive gene that encodes the apoE-binding protein and
decline. In previous studies, we generated transgenic demonstrated that it is expressed in neurons, but not in
mouse models with neuronal expression of mutant other brain cells such as astrocytes. Analyses of data-
human amyloid protein precursors (APP) that cause bases containing information on diverse nucleic acid
overproduction of Aβ and early-onset familial AD in and protein sequences revealed that the apoE-binding
humans. The AD-like pathology that develops in these protein belongs to a family of proteins that have sever-
mice with aging was inhibited or enhanced by glia- al features in common. The existence of a whole fam-
derived factors, such as α1-antichymotrypsin (↑), ily of related proteins and the similarities between fam-
apoE3 (↓), apoE4 (↑), and TGF-β1 (↓), raising hope ily members identified in different species underscore
that AD pathogenesis might be modifiable by diverse the potential physiological importance of the apoE-
therapeutic interventions. It is widely believed that binding protein. This year, research efforts in the Pitas
apoE4 increases AD risk and lowers the age of disease laboratory were extended in an interesting new direc-
onset by increasing the deposition of Aβ into plaques. tion through a collaboration with the Gao laboratory,
However, cognitive decline in AD correlates much bet- which specializes in fruit fly research. The fruit fly sys-
ter with synaptic and cholinergic deficits than with tem will facilitate the rapid molecular manipulation
plaque load. We therefore analyzed these parameters in and physiological characterization of the apoE-binding
mice expressing human Aβ together with apoE3 or protein and its relatives. Good progress has also been
apoE4 in the brain. ApoE3, but not apoE4, delayed Aβ- made toward inactivating the gene that encodes the
dependent synaptic deficits independently of plaque apoE-binding protein in mice, which will help assess
formation. These findings underline the importance of its function in the mammalian brain.
nondeposited, prefibrillar Aβ species and suggest that
the differential effects of apoE isoforms on the risk of The laboratory of Dr. Karl H. Weisgraber has contin-
AD relate to differences in their neuroprotective poten- ued to investigate how amino acid substitutions affect
tial rather than to differences in their effect on plaque the conformation, stability, and biological function of
formation. This year, we also expanded our studies in apoE isoforms. Previous studies revealed that the sin-
two new directions: DNA microarrays and Aβ vaccina- gle amino acid difference between apoE3 and apoE4
tion. While there is evidence that Aβ promotes and has profound effects on the conformation and stabili-
immunization against Aβ inhibits AD-like pathology, ty of these molecules. The biological consequences of
the underlying molecular mechanisms remain to be these biophysical differences are beginning to be
defined. Because at least some of the critical cellular unraveled. This year, the Weisgraber laboratory
and molecular processes may be reflected in changes of proved the biological relevance of an interaction
gene expression, we embarked on a large-scale analysis between different domains within apoE that occurs in
of gene expression in treated and untreated transgenic apoE4 but not in apoE3. Introduction of an apoE4-like
mice expressing human APP and Aβ in the brain. domain interaction into mouse apoE changed the lipid
Among the most interesting preliminary findings from binding profile of apoE in the plasma of the genetically
Director’s Report 9
2001 ANNUAL REPORT
modified mice. In previous studies, the Weisgraber mic environment that is ideal for training in neuro-
laboratory identified small chemical compounds that, science and biomedical research. GIND investigators
based on computer simulations, might be able to dis- have continued to participate actively in the training of
rupt the domain interaction in apoE4. In vitro and cell students and residents from various UCSF departments
culture assays have provided preliminary proof that and interdepartmental programs, including the
some of these compounds can indeed convert apoE4 Departments of Neurology and Physiology, the Neuro-
into a molecule with apoE3-like properties. These science Program, the Biomedical Sciences Program, the
findings bode well for the development of apoE4-tar- Pharmaceutical Sciences and Pharmacogenomics
geted drug treatments to prevent AD in apoE4 carri- Program, and the Medical Scientist Training Program,
ers. Lastly, in vitro studies revealed that apoE4 has a as well as from graduate and undergraduate programs at
greater propensity than apoE3 to assume an unstable UC Berkeley and other institutions. Many members of
conformational state that might promote the degrada- our institute have collaborated this year to make our
tion of apoE4 or its pathogenic interactions with other training environment even more inspiring and reward-
molecules, such as Aβ and tau. The further character- ing for students of all biomedical disciplines. Drs.
ization of this conformational state could shed light on Finkbeiner and Gao deserve particular mention for the
the role of apoE4 in AD and has become an important outstanding efforts they have made in this regard. In
objective of the Weisgraber laboratory. this context, I would also like to highlight the continued
success of our weekly GIND seminar series, which is
The laboratory of Dr. Tony Wyss-Coray has continued organized by Dr. Wyss-Coray. It remains a popular and
to characterize the physiological functions of the stimulating forum for education in disease-related neu-
cytokine transforming growth factor β1 (TGF-β1) in roscience and scientific exchange among members of
the central nervous system. They also investigated the the institutes and colleagues from the greater UCSF
role of inflammation and, in particular, of complement community.
activation in the pathogenesis of AD. TGF-β1 was
shown to promote the activation of microglia and com- GIND investigators have extended their efforts to pro-
plement components in the brains of transgenic mouse mote education and scientific exchange in disease-
models overexpressing human APP. This effect was related neuroscience far beyond the boundaries of our
associated with a decrease in the deposition of Aβ as institute. They have organized and participated in a
amyloid plaques. In contrast, inhibition of complement number of national and international conferences that
activation in the brain increased the plaque load and have advanced our field of research in various ways. I
triggered neuronal degeneration in the mice. These am delighted that their research accomplishments and
findings have important therapeutic implications, as other contributions to the scientific community have
they challenge the widely held belief that activation of not gone unnoticed, as reflected by the honors and
microglia and complement promotes AD. The results awards institute members received this year (see
obtained by the Wyss-Coray laboratory suggest that Outreach section for details).
general inhibition of inflammatory responses in the
brain, including inhibition of complement components, Despite their busy schedules and expanding research
may enhance rather than inhibit the development or efforts, GIND members have continued to devote time
progression of AD. Conversely, the specific enhance- to community outreach. As described in the Outreach
ment of protective inflammatory responses could be of section of this report, these efforts included participa-
therapeutic benefit. tion in activities aimed at educating the public about
AD and neuroscientific research in general. Our syner-
Education, Special Initiatives, gism with the UCSF Memory and Aging Center, the
and Recognition local chapter of the Alzheimer’s Association, and the
Hereditary Disease Foundation has allowed us to main-
The Gladstone Institutes and UCSF provide state-of- tain and expand fruitful links between our research and
the-art research facilities and a highly interactive acade- the patients afflicted by the diseases we study.
10 Director’s Report
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE
The progress we made in 2001 reflects the work of all
I would like to thank the participants of this year’s the researchers at the GIND and of the administrative
Scientific Advisory Board meeting (Drs. Dale E. staff at the Gladstone Institutes. As outlined in this
Bredesen, Buck Institute for Age Research; Eric report, we have advanced our understanding of some
Shooter, Stanford University; Sidney Strickland, of the most devastating diseases known to man. I am
Rockefeller University; and Marc Tessier-Lavigne, confident that this knowledge will contribute to the
UCSF) for their outstanding input. It contributed development of better strategies for their treatment
greatly to our success. and prevention.
Lennart Mucke, M.D.
Director’s Report 11
Description of the
THE J. DAVID GLADSTONE
Richard S. Brawerman
Albert A. Dorman
Richard D. Jones
Robert W. Mahley, M.D., Ph.D.
Chief Financial Officer
Hal Orr, C.M.A.
Description of the Institutes
Although autonomous in their areas of specialization,
rimary research efforts at the J. David
Gladstone Institutes focus on three of the most the institutes share a common approach. Each institute
important clinical problems of modern times: is organized around research units consisting of scien-
cardiovascular disease, AIDS, and neurodegenerative tists, postdoctoral researchers, research associates, and
disorders. Cardiovascular disease, the nation’s lead- students. This structure is designed to accommodate
ing killer, claims the lives of over one million small groups of scientists who work together closely
Americans each year. Despite more effective treat- but who also benefit from collegial interactions with
ments, AIDS remains a leading cause of death in the other research groups. Collaborations among staff
United States. Worldwide, more than 40 million peo- members with various areas of expertise create a stim-
ple are living with HIV/AIDS, and more than 21 mil- ulating environment that fortifies the scientific
lion have died as a direct result of HIV infection. lifeblood of the organization.
Alzheimer’s disease, the most recent focus of investi-
gation by Gladstone scientists, is the fourth leading Each institute receives expert input on the progress of
cause of death in adults, affecting four million its science from an advisory board of distinguished sci-
entists. The scientific advisory boards provide a
Americans. The realization of the impact of these dis-
twofold service in reviewing the quality of the research
eases on world health infuses Gladstone scientists
and in advising the president, directors, and trustees.
with a sense of purpose and urgency.
The work of the scientific staff at all three institutes
Gladstone is composed of three institutes, each of
also extends beyond the laboratory to the wider com-
which issues its own annual report. The Gladstone
munity. The mission of the institutes includes the edu-
Institute of Cardiovascular Disease (GICD), which
cation of graduate and medical students, postdoctoral
opened in 1979, focuses on atherosclerosis and its fellows, and visiting scientists; specialized training
complications. In 1992, the Gladstone Institute of for practicing physicians; and educational outreach to
Virology and Immunology (GIVI) was established to the local and extended community.
study HIV, the causative agent of AIDS. The 1993 dis-
covery that apolipoprotein (apo) E—long studied at The J. David Gladstone Institutes are the product of
GICD for its role in heart disease—plays a role in the wisdom and hard work of many individuals. The
Alzheimer’s disease as well led to the establishment first was J. David Gladstone himself, a Los Angeles
of the Gladstone Institute of Neurological Disease real-estate entrepreneur. Others are the trustees. The
(GIND) in 1998. The three institutes are located at the original trustees, all of whom had known or worked
San Francisco General Hospital (SFGH) campus of closely with Mr. Gladstone, were Richard S.
the University of California, San Francisco (UCSF). Brawerman, his attorney and executor of his estate;
While independent, Gladstone is formally affiliated Richard D. Jones, his real-estate attorney; and David
with UCSF, and Gladstone investigators hold univer- Orgell, his cousin and confidant. When Mr. Orgell
sity appointments and participate in many university died in 1987, he was succeeded on the board by Albert
activities, including the teaching and training of grad- A. Dorman, a southern California executive with
uate students. experience in managing large organizations.
2001 ANNUAL REPORT
Gladstone Institute of Cardiovascular Disease of Cardiovascular Disease
Scientific Advisory Board
Close ties already existed between the UCSF School
Göran K. Hansson, M.D., Ph.D. of Medicine and SFGH, the hospital of the City and
Professor of Cardiovascular Research County of San Francisco, when the trustees leased
Center for Molecular Medicine
Karolinska Institute, Karolinska Hospital vacant space from the City in 1977 in which to create
laboratories and offices. The partnership has flour-
Joachim J. A. Herz, M.D. ished. Gladstone scientists collaborate with their col-
Professor of Molecular Genetics leagues at UCSF and SFGH and provide service to
University of Texas Southwestern those organizations as professors and staff physicians.
The mutually beneficial association between the
Aldons J. Lusis, Ph.D. Gladstone, UCSF, and SFGH has created a productive
Professor of Medicine and and supportive environment in which scientists con-
of Microbiology and Molecular Genetics duct basic research while availing themselves of clin-
University of California, Los Angeles
ical and academic opportunities.
Karen Reue, Ph.D.
Research Biologist To choose a director for the developing research facil-
West Los Angeles Veterans Administration ity, the trustees sought guidance from the scientific
Medical Center community. The choice was Robert W. Mahley, M.D.,
Associate Professor of Medicine
University of California, Los Angeles Ph.D. At the time of Mr. Gladstone’s death, he was
just completing his internship. However, by 1979,
Donald M. Small, M.D. when he was appointed director, Dr. Mahley had
Chairman, Department of Biophysics established himself as a leading researcher in the field
Professor of Biophysics, Medicine and
of lipoprotein metabolism and atherosclerosis. He
Boston University School of Medicine came to the Gladstone from the National Institutes of
Health, where he headed the Laboratory of Experi-
Daniel Steinberg, M.D., Ph.D. mental Atherosclerosis. Less than a year after his
Professor Emeritus, Department of Medicine appointment, Dr. Mahley had assembled a staff of 25,
University of California at San Diego
and the new organization, then called the Gladstone
Alan R. Tall, M.D. Foundation Laboratories for Cardiovascu-lar Disease,
Professor of Medicine officially opened on September 1, 1979. Dr. Mahley is
Columbia University College of Physicians professor of pathology and medicine at UCSF and is
and Surgeons a member of the National Academy of Sciences and
the Institute of Medicine.
At the time of Mr. Gladstone’s death in 1971, the By the end of 2001, the research staff of the GICD
southern California real-estate market was just begin- had grown to more than 100 scientists, postdoctoral
ning to flourish. His estate, left almost entirely for fellows, students, and research associates, occupying
medical education and research, was relatively mod- about 48,000 square feet of laboratory and office
est by later standards. However, the trustees recog- space in buildings 9 and 40 on the SFGH campus. In
nized the estate’s potential for growth and, through 21 years of operation, the institute has attained an
their inspired management, increased its worth sever- international reputation for excellence. Its productivi-
alfold within the first decade. From the beginning, the ty is documented in the more than 850 scientific
decisions of the trustees had profound and positive papers published by GICD scientists.
effects on the research organization that evolved.
They continue to manage and enlarge the assets of the Research at the GICD is conducted in five areas and
J. David Gladstone Institutes and to oversee their use. is supported by three core laboratories.
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE
Lipoprotein Biochemistry and Metabolism. A tify unique genetic abnormalities that cause hypercho-
major focus of research in this area is to correlate the lesterolemia and premature myocardial infarction.
structure and function of the apolipoproteins involved Researchers in this unit operate the Lipid Disorders
in cholesterol transport, with particular emphasis on Training Center, which trains medical personnel to
apoE. One of the structural tools that scientists in this manage dyslipidemic patients, and the Lipid Clinic,
unit use is x-ray crystallography to determine the which provides consultation on disease management
three-dimensional structures of proteins. The investi- to SFGH patients and to private, referring physicians.
gators in this unit are Dr. Mahley, Karl H. Weisgraber, This unit also conducts the Turkish Heart Study, which
Ph.D., and Yadong Huang, M.D., Ph.D. investigates cardiovascular risk factors in a developing
nation with a high incidence of heart disease. The
Cell Biology. Studies in this unit examine how the investigators in this unit are Dr. Mahley and Thomas P.
body’s various cells regulate the storage and use of Bersot, M.D., Ph.D.
cholesterol as it relates to the development of athero-
sclerosis. The focus is on the roles of apoB, apoE, and Gladstone Genomics Core. The Genomics Core
class A scavenger receptors in cellular cholesterol assists scientists with the unprecedented research
metabolism and atherogenesis. The investigators in opportunities presented by the decoding of the mouse
this unit are Robert E. Pitas, Ph.D., and Thomas L. and human genomes. Directed by Christopher S.
Innerarity, Ph.D. (retired 3/01). Barker, Ph.D., this laboratory provides state-of-the-art
technologies in the area of functional genomics for
Molecular Biology. Scientists in this unit apply the Gladstone scientists and other investigators at SFGH.
latest DNA techniques to understand the regulation The core focuses on DNA microarray technology,
of genes important in controlling cholesterol, triglyc- including the preparation of custom oligonucleotide
erides, and apolipoprotein production. Studies focus microarrays and customized microarray hybridiza-
on apoE and apoB, which mediate the interaction of tion, array scanning, and data analysis.
lipoproteins with cell-surface receptors. Enzymes
controlling cholesteryl ester and triglyceride produc- Gladstone Transgenics Core. The GICD also main-
tion represent a new area of research. This has led to tains a sophisticated transgenic core facility that is
studies of adipose tissue metabolism and obesity. In heavily used by investigators of all three institutes.
addition, transgenes and homologous recombination The core’s activities are coordinated by John M.
are used to create animal models of human diseases. Taylor, Ph.D.
The investigators in this unit are John M. Taylor,
Ph.D., Stephen G. Young, M.D., and Robert V. Gladstone Microscopy Core. The Microscopy Core,
Farese, Jr., M.D. under the direction of David A. Sanan, Ph.D., pro-
vides expertise, instrumentation, service, and training
Vascular Biology. This research aims to elucidate how for the generation and capture of research data in the
monocytes/macrophages are attracted to sites of ather- form of microscopic images and for the quantitation,
osclerotic lesion formation and to delineate the role of analysis, and interpretation of those images to all
platelets in forming the occlusive thrombus that leads three institutes.
to myocardial infarction. Another research goal is to
elucidate cell-signaling pathways that can be used to Gladstone Institute
confer proliferative advantages to genetically modi- of Virology and Immunology
fied cells. The investigators in this unit are Israel F.
Charo, M.D., Ph.D., and Bruce R. Conklin, M.D. The GIVI resulted from the convergence of several
factors. On the forefront of the battle against AIDS
Clinical Molecular Genetics. Patient studies and since the beginning of the pandemic, SFGH is widely
national and international population screening pro- recognized as one of the world’s leading clinical
jects conducted by Gladstone researchers aim to iden- research centers for the study of HIV disease. The
2001 ANNUAL REPORT
Warner C. Greene, M.D., Ph.D., an internationally
Gladstone Institute of Virology recognized immunologist and virologist, officially
and Immunology took the helm in September 1991. Before coming to
Scientific Advisory Board Gladstone, Dr. Greene was professor of medicine and
investigator in the Howard Hughes Medical Institute
Elizabeth H. Blackburn, Ph.D.
Professor of Microbiology at Duke University Medical Center. Currently, Dr.
and Immunology Greene is also a professor of medicine and of micro-
University of California, San Francisco biology and immunology at UCSF, co-director of the
UCSF Center for AIDS Research, and a member of
Robert C. Gallo, M.D.
Director, Institute of Human Virology the executive committees of the UCSF AIDS
Professor of Medicine Research Institute and UCSF Biomedical Sciences
and of Microbiology and Immunology Graduate Program.
University of Maryland at Baltimore
Edward W. Holmes, M.D. Formally dedicated on April 19, 1993, the GIVI occu-
Vice Chancellor for Health Sciences pies 27,000 square feet of space on the top two floors
Dean, School of Medicine of SFGH’s building 3. Studies at the GIVI are con-
University of California at San Diego ducted under the direction of an outstanding group of
Stanley J. Korsmeyer, M.D. physician-scientists in six state-of-the-art laboratories
Sidney Farber Professor of Pathology and three supporting laboratories.
and Professor of Medicine
Harvard Medical School Laboratory of Molecular Immunology. The
Director, Program in Molecular Oncology
Department of Cancer Immunology Laboratory of Molecular Immunology studies the
and AIDS mechanisms by which proteins within the immune
Dana-Farber Cancer Institute cells harboring HIV may act to trigger the growth of
the virus and how the virus’s own proteins subse-
Joseph R. Nevins, Ph.D.
James B. Duke Professor and Chairman quently amplify its replication and pathogenic effects
Department of Genetics in primary T cells and macrophages. This work, head-
Duke University Medical Center ed by the director of the institute, Dr. Warner C.
Greene, specifically focuses on the HIV proteins Vpr
Robin A. Weiss, Ph.D.
Professor of Viral Oncology and Nef and select host factors, including the NF-
Wohl Virion Centre κB/Rel family of transcription factors.
Windeyer Institute of Medical Sciences
University College London Laboratory of Molecular Evolution. The
Laboratory of Molecular Evolution focuses on evolu-
tion and its implications for medicine and epidemiol-
State of California provided funding to build an AIDS ogy. Genetic variations in host susceptibility and in
research center at SFGH under the auspices of the microbial replication capacity, virulence, and drug
UCSF School of Medicine. Additional funds were susceptibility typically determine who develops dis-
needed to finish and equip the center and to undertake ease and who remains healthy. The laboratory exam-
the research. The success of the established relation- ines several consequences of molecular evolution,
ship of UCSF and the City with the Gladstone formed including HIV-1 drug resistance, selection pressures
the foundation for a unique agreement by which the bearing on HIV-1 populations during transmission
Gladstone would lease the center, establish the and in tissues, and nonpathogenic simian immunode-
research program, and manage the ongoing studies. ficiency virus infection in natural host species. This
laboratory is directed by Robert M. Grant, M.D.,
Gladstone and UCSF were able to attract an outstand- M.P.H., an assistant investigator in GIVI and assistant
ing physician-scientist to direct the new institute. professor of medicine at UCSF.
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE
Laboratory of Receptor Biology. The Laboratory of for analysis. The laboratory is directed by Martin
Receptor Biology investigates the molecular basis of Bigos, M.S., a staff research scientist in GIVI.
transmembrane signaling by cell-surface receptors on
hematopoietic cells and the interplay between lym- Gladstone-UCSF Laboratory of Clinical Virology.
photropic viruses and normal intracellular pathways of The Gladstone-UCSF Laboratory of Clinical Virology,
signal transduction. The laboratory is headed by Mark directed by Dr. Grant, provides key virological testing
A. Goldsmith, M.D., Ph.D., an associate investigator in support of HIV-related clinical research projects at
in GIVI and associate professor of medicine at UCSF. UCSF. Established in collaboration with the UCSF
AIDS Research Institute, this laboratory evaluates
Laboratory of Viral Pathogenesis. The Laboratory patients who are failing combination antiviral therapy,
of Viral Pathogenesis focuses on the pathogenic studies HIV replication in the central nervous system,
mechanisms of HIV in vivo, with the specific intent of and investigates mechanisms of primary HIV infection
finding better ways to prevent or suppress HIV- and sexual transmission. This laboratory is also devel-
induced disease. The work falls into two areas: effects oping state-of-the-art assays for genotypic and pheno-
of HIV on the central hematopoietic system and trans- typic drug resistance and assessment of viral loads
mission of HIV across mucosal and placental barriers. using ultrasensitive techniques to further enhance clin-
Research in this laboratory is directed by Joseph M. ical AIDS research at SFGH and UCSF.
McCune, M.D., Ph.D., a senior investigator in GIVI
and professor of medicine at UCSF. Antiviral Drug Research Division. The Antiviral
Drug Research Division evaluates potential new
Laboratory of Molecular Virology. The Laboratory antiviral drugs. Using a novel animal model system,
of Molecular Virology studies how HIV transcription the SCID-hu mouse, the group is developing new
is controlled by host chromatin structure and by viral methods for drug evaluation and extending its work to
proteins such as HIV Tat. More recently, this labora- the field of viral pathogenesis. The laboratory is
tory has also investigated the molecular basis for HIV- directed by Cheryl A. Stoddart, Ph.D., a staff research
induced T-cell death, focusing on the role of apopto- scientist in GIVI.
sis. This laboratory is directed by Eric M. Verdin,
M.D., a senior investigator in GIVI and professor of Gladstone Institute
medicine at UCSF. of Neurological Disease
Laboratory of Cellular Immunology. The The GIND resulted from the natural expansion of
Laboratory of Cellular Immunology investigates highly successful research programs. Its predecessor,
innate and adaptive cellular immune responses the Gladstone Molecular Neurobiology Program, was
against HIV and simian immunodeficiency virus at created in 1996 as a joint venture of the Gladstone
mucosal and systemic sites. The work focuses on Institutes and the UCSF Department of Neurology.
understanding host immune/pathogen interactions Lennart Mucke, M.D., recruited to head the new pro-
that might be manipulated by vaccination or thera- gram, brought with him a group of researchers with
peutic drugs. The laboratory is headed by Douglas F. expertise in diverse areas of disease-related neuro-
Nixon, M.D., Ph.D., an associate investigator in GIVI science. With its establishment, neuroscientists in the
and associate professor of medicine at UCSF. new program could leverage Gladstone’s wealth of
experience with apoE by applying it to the burgeoning
Flow Cytometry Core Laboratory. The Flow field of neurodegenerative diseases. These efforts were
Cytometry Core Laboratory is dedicated to providing complemented with research on amyloid proteins,
cutting-edge techniques in fluorescence-based cell which play a seminal role in Alzheimer’s disease.
sorting and analysis to Gladstone and UCSF scien-
tists. This laboratory operates both a Becton- Significant findings were rapidly made in a broad
Dickinson FACS Vantage for sorting and a FACScan range of research areas, including molecular biology,
2001 ANNUAL REPORT
Massachusetts General Hospital, and Harvard
Gladstone Institute of Neurological Disease Medical School. He came to Gladstone from The
Scientific Advisory Board Scripps Research Institute to expand Gladstone’s
research efforts in disease-oriented neuroscience in
Dale E. Bredesen, M.D. the context of the Molecular Neurobiology Program
President and CEO and then to direct the new institute. Dr. Mucke is also
Buck Institute for Age Research
Professor of Neurology the Joseph B. Martin Distinguished Professor of
University of California, San Francisco Neuroscience at UCSF.
Gerald D. Fischbach, M.D. The GIND was formally dedicated on September 11,
Dean of Faculty of Medicine
and for Health Sciences 1998. Its laboratories are housed in buildings 1, 9, and
Harold and Margaret Hatch Professor 40 of the SFGH campus. Studies at the GIND are con-
Columbia University ducted in eight state-of-the-art laboratories and a
behavioral core laboratory. The research focuses on
Dennis J. Selkoe, M.D.
Co-Director, Center for Neurologic Diseases six major areas relating to neurodegenerative disor-
Brigham and Women’s Hospital ders, cognitive function, and brain inflammation as
Professor of Neurology and Neuroscience outlined below.
Eric M. Shooter, Ph.D. Physiological and Pathophysiological Roles of
Professor of Neurobiology Amyloidogenic Proteins in the Brain. While amy-
Stanford University loidogenic molecules, such as the amyloid β protein
precursor and α-synuclein, may facilitate learning and
Sidney Strickland, Ph.D.
Dean of Educational Affairs memory, they can be broken down into peptides or
Professor of Neurobiology and Genetics altered in their conformation to form neurotoxic
Rockefeller University aggregates in cells and tissues. Understanding how
these toxic proteins form and act could facilitate the
Marc Tessier-Lavigne, Ph.D.
Professor of Anatomy design of better treatments for Alzheimer’s disease
and of Biochemistry and Biophysics and other neurodegenerative disorders. Defining the
University of California, San Francisco normal function of the amyloidogenic precursor mol-
ecules is of fundamental neuroscientific interest.
Investigators involved in research on this topic are Dr.
cell biology, physical structure, signal transduction, Mucke, Tony Wyss-Coray, Ph.D., Robert W. Mahley,
experimental pathology, and behavioral neurobiology. M.D., Ph.D., Yadong Huang, M.D., Ph.D., and Karl
In 1998, the trustees expanded the program to create H. Weisgraber, Ph.D.
the GIND. Its goal is to increase understanding of the
molecular pathogenesis of neurological deficits Role of ApoE in Neurodegeneration and Cognitive
resulting from neurodegenerative disorders such as Impairment. The apoE4 allele is the main known
Alzheimer’s disease, from cerebrovascular disease, or genetic risk factor for the most common form of
from HIV infection. Productive synergies exist with Alzheimer’s disease and for poor neurological out-
scientists studying cardiovascular disease and AIDS come after head injury and cardiac bypass surgery.
in the other institutes. Defining the effects of the three main human apoE
isoforms (E2, E3, and E4) on the structure and func-
As was the case with the two other institutes, tion of the brain should provide crucial insights into
Gladstone and UCSF were able to attract an out- the contribution of the apoE4 variant to neurological
standing physician-scientist to head the new institute. disease. Characterizing how changes in the x-ray
Dr. Mucke was educated at the Max-Planck-Institute crystallographic three-dimensional structure of apoE
for Biophysical Chemistry in Germany, the affect its activity may result in the development of
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE
novel apoE-targeted drug treatments for Alzheimer’s decline in diverse diseases. Investigators involved in
disease and other neurological conditions. research on this topic include Drs. Finkbeiner, Mucke,
Investigators involved in research on this topic are and Fen-Biao Gao, Ph.D.
Drs. Mahley, Weisgraber, Huang, Mucke, and Robert
E. Pitas, Ph.D. Neurobiological Function of Glial Cells and Their
Role in Neurological Disease. Glial cells are special-
Huntingtin and Other Polyglutamine-Repeat ized brain cells that support the health and function of
Proteins. Huntington’s disease, the most common neurons. In response to brain injuries, these cells pro-
inherited neurodegenerative disorder, is caused by an duce a large number of molecules that participate in
abnormally long stretch of the amino acid glutamine inflammatory and immune responses. While acute
within the protein called huntingtin. Abnormal polyg- glial responses may help prevent neuronal damage
lutamine stretches within other proteins are responsi- and facilitate the removal of toxic amyloid proteins,
ble for several other midlife neurodegenerative disor- abnormal activation of these cells could contribute to
ders. Determining how abnormal polyglutamine neurological disease. Genetic and pharmacological
stretches cause neurons to die may make it possible to strategies are used to characterize the beneficial and
develop specific therapies for these disorders. It may detrimental roles of glial cells in cerebral amyloido-
also reveal general mechanisms of neurodegeneration sis, neurodegeneration, and HIV-associated dementia.
that are relevant to other neurological diseases. This Investigators involved in research on this topic
topic is a major focus of Steven M. Finkbeiner, Ph.D. include Drs. Wyss-Coray, Mucke, and Pitas.
Neural Plasticity. Plasticity is a property of the ner- Behavioral Core Laboratory. Because many of our
vous system that enables it to undergo long-lasting, mouse models are designed to simulate aspects of
sometimes permanent adaptive responses to brief human diseases resulting in memory deficits, behav-
stimuli. Plasticity is believed to be important for ioral disturbances, or movement disorders, the
establishing precise patterns of synaptic connections detailed behavioral characterization of these models
during early neuronal development and for learning plays an important role in the assessment of their clin-
and memory in adults. Disturbances in plasticity and ical relevance. Behavioral alterations in transgenic
synaptic function could contribute significantly to models can shed light on the central nervous system
memory disorders characteristic of many neurodegen- effects of diverse molecules and are used to assess
erative diseases, such as Alzheimer’s disease and novel therapeutic strategies at the preclinical level.
Huntington’s disease. An understanding of the molec- Established by Jacob Raber, Ph.D., and Dr. Mucke,
ular mechanisms that regulate the formation, activity, this core is now engaged in collaborative studies with
degeneration, and regeneration of synapses and neu- investigators at all three Gladstone Institutes, as well
ronal dendrites could form the basis for therapeutic as with scientists in various UCSF departments and
strategies to prevent memory loss and cognitive other institutions.
New Gladstone Laboratories at the UCSF Mission Bay Campus
Most recently, the trustees—Richard S. Brawerman,
he vision of Gladstone’s new research build-
ing at Mission Bay is becoming clearer as the Albert A. Dorman, and Richard D. Jones—completed
planners make good progress in hammering the bond financing necessary to fund the project. They
out the details. This vision represents Gladstone’s worked hard to secure the highest ratings and the low-
optimistic and progressive spirit for growth over the est interest rate possible for this 30+ year investment.
next 10 years. The interest rate of the $145 million loan is the lowest
secured in the past 20 years.
15A 15B 16A 16B 17A 17B
Proposed 25A 25B
Location of the Gladstone building at the new UCSF Mission Bay campus. The new laboratories will be located across from building
24A/B, the initial UCSF building, currently under construction as part of Phase I, and in close proximity to the Student/Community
Center to be located in building 21A.
Mission Bay 23
2001 ANNUAL REPORT
The architects have also been hard at work. They have grow from the current 265 employees to more than
prepared a preliminary blueprint of the six-floor build- 500. This will help Gladstone achieve its goal of being
ing. The first floor will hold the offices for administra- home to 27–30 investigators by 2010. Gladstone will
tion and building operations, a 150-person lecture hall, also enjoy the synergy that comes from having all
four seminar rooms, and an eating facility. The second three institutes, the core laboratories, and central facil-
floor will be left as shell space, which can be leased out ities integrated in a single research building.
and/or developed into laboratories in the future. Floors
three, four, and five will house the offices and labora- Scheduled for completion in 2004, the Gladstone
tories of the three institutes, while the animal quarters building will be located at Owens and 16th Streets at
will be located on the sixth floor. The architects are UCSF’s new 43-acre Mission Bay campus. When
currently working with the design team—composed of completed, this campus will host about 9000 scien-
Robert Mahley, Lennart Mucke, Warner Greene, Karl tists and support staff and will house many of UCSF’s
Weisgraber, Robert Pitas, Israel Charo, Debbie Addad, basic biomedical research programs. Initial research
Todd Sklar, and designers—to refine the plans for the programs include structural and chemical biology,
laboratories and the building’s layout. The planners molecular and cell biology, neuroscience, develop-
have also benefited significantly from the input of mental biology, and genetics. The new building
other Gladstone scientists and staff members. promises to strengthen Gladstone’s excellent rela-
tionship with UCSF and provide an opportunity for
With about 200,000 square feet of space, the new increasingly productive collaborations with universi-
building will provide sufficient room for Gladstone to ty colleagues.
Courtesy of NBBJ Architects
24 Mission Bay
MEMBERS OF THE INSTITUTE
Members of the Institute 25
Members of the Gladstone Institute of Neurological Disease
Director Staff Research Investigators
Lennart Mucke, M.D. Yadong Huang, M.D., Ph.D.
Joseph B. Martin Distinguished Professor Assistant Professor of Pathology
Tony Wyss-Coray, Ph.D.
Investigators Assistant Professor of Neurology
Steven M. Finkbeiner, M.D., Ph.D.
Assistant Professor of Neurology Staff Research Scientists
and Physiology Christopher S. Barker, Ph.D.
Fen-Biao Gao, Ph.D. Jacob Raber, Ph.D.
Assistant Professor of Physiology Assistant Professor of Neurology
Robert W. Mahley, M.D., Ph.D. Manuel J. Buttini, Ph.D.
Professor of Pathology and Medicine Robert L. Raffaï, Ph.D.
Robert E. Pitas, Ph.D. Visiting Scientists
Professor of Pathology Andrea Barczak
Marion Buckwalter, M.D., Ph.D.
Karl H. Weisgraber, Ph.D. Toru Kawamura, Ph.D.
Professor of Pathology Yvonne Kew
Xiao Xu, M.D., Ph.D.
Shirley Zhu, Ph.D.
Members of the Institute 27
2001 ANNUAL REPORT
Postdoctoral Fellows Senior Research Associates
Montserrat Arrasate, Ph.D. Maureen E. Balestra
John Bradley, Ph.D. Walter J. Brecht
Jeannie Chin, Ph.D. Zhong-Sheng Ji, Ph.D.
Luke A. Esposito, Ph.D. Yvonne M. Newhouse
Christian Essrich, Ph.D. Gui-Qiu Yu, M.S.
Paul C. R. Hopkins, Ph.D.
Amy Hsiu-Ti Lin, Ph.D. Research Associates
Jorge J. Palop-Esteban, Ph.D. Thomas C. Brionne
Juan Santiago-Garcia, Ph.D. Elizabeth S. Brooks, M.S.
Kimberly Scearce-Levie, Ph.D. Anita Chow
Ina Tesseur, Ph.D. Jacob Corn
Zheng Wang, Ph.D. Jessica J. Curtis
Shiming Ye, Ph.D. Nhue Do
Sarah E. Goulding, Ph.D.
Students Kristina Hanspers, M.S.
Duane M. Allen Yanxia Hao, M.D.
Gerold Bongers Faith M. Harris
Sarah Carter Shyamal G. Kapadia
Patrick Chang Lisa N. Kekonius
Ammon Corl Anthony D. LeFevour
Jennifer Fu Wenjun Li, Ph.D.
Richard J. Han Xiao Qin Liu, M.D.
Sharon E. Haynes Rene D. Miranda
Jason Held Lauren Mondshein
Marian L. Logrip Hilda C. Ordanza
Siddhartha Mitra Jon-Paul Pepper
Linda Ngo Michelle E. Rohde
Ryan P. Owen Nathan G. Salomonis
Amina A. Qutub Kristina P. Shockley
Hélène Rangone Richard M. Stewart
Vikram Rao Fenrong Yan
Neal T. Sweeney Yuhua Zheng
John A. Gray
28 Members of the Institute
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE
of the J. David Gladstone Institutes
President Human Resources
Robert W. Mahley, M.D., Ph.D. Migdalia Martinez, M.S., Officer
John R. LeViathan, M.A., Manager
Administrative Assistants Wendy M. Foster
Catharine H. Evans Anthony R. Gomez
Karina G. Fantillo Chad E. Popham
Mariena D. Gardner Alyssa S. Uchimura
Marlette A. Marasigan
Kelley S. Nelson Information Services and Communications
Nannette I. Nemenzo Reginald L. Drakeford, Sr., Officer
September C. Plumlee Susan H. Dan
Emily K. O’Keeffe Iris Newsum, M.B.A., M.S.
Aileen C. Santos Sylvia A. Richmond
Bethany J. Taylor Teresa R. Roberts
Executive Assistants Editorial
Brian Auerbach Gary C. Howard, Ph.D.
Denise Murray McPherson Stephen B. Ordway
Sylvia A. Richmond
Graphics and Photography
Finance and Accounting John C. W. Carroll, Manager
Marc E. Minardi, M. Div., Officer Stephen Gonzales
Richard S. Melenchuk, M.S., Manager Christopher A. Goodfellow
Emilia M. Herrera John R. Hull, M.F.A.
Kai Yun W. Sun
Kenneth J. Weiner Information Services
Jon W. Kilcrease, Manager
Grants and Contracts Theodore E. Doke
Rex F. Jones, Ph.D., Officer Matthew L. Lyon
Frank T. Chargualaf, M.B.A. Joseph R. Solanoy
Lynne M. Coulson
Marga R. Guillén, M.P.A. Public Affairs
Marya Pezzano Laura Lane, M.S., Manager
Martin C. Rios
Michael S. Whitman
Yvonne L. Young
Members of the Institute 29
2001 ANNUAL REPORT
Intellectual Property/Technology Transfer Receptionist
Joan V. Bruland, J.D., Officer Hope S. Williams
Anne Scott, M.A., M.S. Student Assistants
Shannon P. Chi
Office of the President April D. Hughes
Susan H. Dan Christina N. Luna
Teresa R. Roberts Luvy C. Vanegas
Deborah S. Addad, Officer
Vincent J. McGovern, M.S., Manager
David R. Bourassa
Randy A. Damron
George R. Leeds
Roger A. Shore
P. J. Spangenberg, Manager
Tyler G. Campos
Judy H. Cho
P. Sidney Oduah
Alberto L. Reynoso
Benjamin V. Young
30 Members of the Institute
Behavioral Core Laboratory.........95
Gladstone Genomics Core.........103
Reports from the Laboratories 31
Steven M. Finkbeiner, M.D., Ph.D.
Montserrat Arrasate, Ph.D.
John Bradley, Ph.D.
Reports from the Laboratories 33
Molecular Mechanisms of Plasticity and Neurodegeneration
Steven M. Finkbeiner, M.D., Ph.D.
neurodegeneration, cell-specific death, and formation
ur laboratory is interested in two biological
questions. First, how does the nervous system of abnormal deposits of aggregated huntingtin known
adapt to brief experiences with long-lasting as intranuclear inclusions.
changes in its structure and function? The molecular
mechanisms that underlie this process, collectively However, this immunocytochemical approach has
known as plasticity, are important for the proper significant limitations. First, it is relatively insensi-
development of the nervous system and for forming tive. Only a fraction of the neurons that undergo neu-
memories. We are especially interested in the role that rodegeneration are caught—some have not begun to
new gene expression plays in coupling transient neu- degenerate at the time the cells are fixed, whereas oth-
ronal activity to long-term changes in synaptic func- ers have degenerated so completely that they are dif-
tion. The second question that we are pursuing is how ficult to identify or are absent altogether. Second,
a genetic mutation leads to an adult-onset progressive manual scoring is extremely time consuming.
neurodegenerative disease. We have focused on Because the assay relies on a microscopic assessment
understanding the molecular mechanisms that medi- of morphology, it is inherently user-dependent.
ate the inherited neurological disorder Huntington’s Criteria may not be applied uniformly, and if neurons
disease (HD). degenerate by multiple effector pathways, the range
of morphological changes may not be fully encom-
A Robotic Microscope passed in a single definition. Third, the static nature of
immunocytochemistry limits its potential for address-
The molecular mechanisms that mediate gene ing mechanistic questions, for unraveling the
expression–dependent synaptic plasticity and muta- sequence of events that are responsible for the process
tion-dependent neurodegeneration have at least one under study, and for determining whether an observed
feature in common. Significant time must elapse change is a cause or an effect.
between the initiating process (e.g., transient synap-
tic activity or the inheritance of a genetic mutation) In the last year, we have developed a new platform for
and the final outcome (lasting changes in synaptic studying mechanisms of plasticity and neurodegener-
function or neurodegeneration). That these processes ation. Central to this effort is a device we call a robot-
occur over a significant time span has complicated ic microscope. The core of the device is an inverted
our efforts to study them. microscope body equipped with special optics to
make automated image collection and analysis feasi-
We have developed a cellular model of HD by intro- ble. Computer-controlled motors move the stage, the
ducing versions of the causative gene into neurons focus knob, multiple fluorescence filters, and shutters.
grown from regions of the brain that are most vulner- We programmed the instrument to automatically
able in people. We fix the neurons days after intro- focus itself by brief 20-millisecond pulses of light and
ducing the gene and then use immunocytochemistry fast Fourier transform analysis. Once focused, the
to assay for neurodegeneration. The model recapitu- computer collects fluorescence images of different
lates three key features of HD: mutation-dependent wavelengths and then precisely moves the stage to an
Reports from the Laboratories 35
2001 ANNUAL REPORT
Figure 1. The robotic microscope facilitates high-throughput cell formed 15, 39, 48, 72, 96, and 136 hours after transfection with GFP.
biology. Striatal neurons have been cultured in multiwell dishes and After the experiment was complete, a field was chosen from the
transfected with the reporter gene, green fluorescent protein (GFP). stack of images collected on the first day and then the correspond-
The microscope has been programmed to scan the whole dish, col- ing image was chosen from the stacks collected on the subsequent
lecting stacks of images representing nonoverlapping fields from five days. The arrows point to individual neurons whose fate can be
each well of the entire dish. The plate of neurons is returned to the followed over the course of the six days. The arrowheads point to a
incubator for a period of time and then remounted on the micro- neuron that survives the experiment; the arrows point to a neuron
scope for repeated imaging. In this experiment, imaging was per- that dies between the third and fourth days.
adjacent but nonoverlapping field. These steps are fected neurons from a single 24-well plate in about
repeated until selected fields of each well of a multi- 15 minutes. In our previous assay, this would have
well plate are automatically imaged. We have written taken us nearly 6 weeks. Third, the criteria for scor-
programs that automatically analyze the stacks of ing are defined explicitly in the automated analysis
images and quantify features of interest. program. This removes a component of user bias,
ensures that each neuron is quantified similarly, and
This platform has many new capabilities. For the makes it possible to communicate the exact criteria
first time, we have the ability to follow the fates of to other labs. Fourth, the assay is much more sensi-
specific neurons over any time interval. We can tive than our previous assay. By collecting images
image a particular living neuron or field of living within an hour of transfection and periodically
neurons, return the tissue-culture plate to the incu- thereafter, we can accurately measure the initial
bator, and at any time mount the plate back on the transfected population and precisely follow the rate
microscope and quickly find the same neuron and of neurodegeneration. This method accounts for all
collect another image (Figure 1). This is a key capa- the cells and has the capability to detect neuron-
bility for elucidating mechanisms of plasticity and specific patterns of degeneration. The system has
neurodegeneration that develop over days to weeks. many other capabilities and is useful for a wide
Second, the system is very fast, making possible variety of different applications that would benefit
experiments that were previously unfeasible. For from high-throughput analysis, including our own
example, in one typical experiment, we collected studies on gene expression–dependent mechanisms
images and counted approximately 300,000 trans- of synaptic plasticity.
36 Reports from the Laboratories
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE
Molecular Mechanisms subtype of glutamate receptor, plays an essential role in
of Synaptic Plasticity initiating activity-dependent plasticity at many synaps-
es. Our laboratory is interested in understanding how
Although new gene transcription and protein expres- calcium signals are encoded into the activity of specific
sion are believed to play a critical role in the process signal transduction pathways, how the activity of these
of learning and memory formation, delineating that pathways regulates the transcription of certain gene tar-
role poses a number of difficult cell biological ques- gets that are important for synaptic plasticity, and how
tions. The pattern of synaptic activity determines newly transcribed or translated genes regulate the spe-
whether the strength of a synapse grows, diminishes, cific subsets of synapses whose strength has been ini-
or remains unchanged. How are these distinct patterns tially and transiently changed.
of synaptic activity related to the gene expression pro-
grams that regulate synaptic strength? If the influx of In the last year, we have especially focused on how
extracellular calcium is crucial for initiating the bio- calcium influx through the NMDA receptor regulates
chemical processes that strengthen or weaken a neuronal gene expression. Our previous work had
synapse as well as the processes that control activity- suggested that calcium influx through the NMDA
dependent gene transcription, how does calcium spec- receptor couples to signal transduction pathways near
ify these distinct responses? the cytoplasmic mouth (within ~1 µm of the plasma
membrane) to control gene expression in the nucleus.
Although multiple neuronal calcium channels are sensi- Thus, accessory proteins near the cytoplasmic mouth
tive to activity, one, the N-methyl D-asparate (NMDA) of the channel, possibly directly associated with the
Figure 2. (A) The domain structure of the cytoplasmic tail of wild-
type NR1 subunit (NR1A), a normal splice variant (NR1C), and a
deletion mutant (NR1∆). M4 designates a portion of the fourth trans-
membrane-spanning region, and C0, C1, and C2 are the names
given to cassette domains of the carboxyl terminus. The numbers
along the top correspond to the amino acid positions of the bound-
aries, counting from the amino terminus. (B) NMDA receptor–medi-
ated gene expression reconstituted in NR1–/– neurons. NR1–/– and
NR1+/– neurons were transfected with a reporter gene (CaRE-
luciferase) and stimulated to activate NMDA receptors (100 µM,
NMDA) or L-type voltage-sensitive calcium channels (L-VSCCs) (55
mM, KCl). NR1+/– neurons responded to all stimuli (left set of bars);
similar responses were seen with NR1+/+ (wild-type) neurons (not
shown). NR1–/– neurons responded normally to depolarization but
not to NMDA (middle set of bars) until the NR1A subunit was
restored by transfection (pCMV-NR1A, right set of bars) (n = 5, *p <
0.005). (C) Variants of the NR1 subunit flux Ca2+ but differ in their
ability to regulate gene expression. NR1–/– neurons were transfected
with NR1A, NR1C, or NR1∆ and the transfection marker GFP-NLS.
Ca2+ responses to NMDA (100 µM, arrow) measured by imaging
Fura-2, were comparable. Scale bars: time (tim) = 20 seconds; [Ca2+]
= 5% ∆F/F for NR1A and NR1C and 4% for NR1∆. ∆F/F is propor-
tional to intracellular Ca2+ concentration. It is calculated by measur-
ing the average fluorescence intensity before and after stimulation,
subtracting the former from the latter, and then dividing the result by
the fluorescence intensity before stimulation. (D) NR1–/– neurons
were transfected with NR1A, NR1C, or NR1∆, the reporter gene
CaRE-luciferase, and the control gene Ren-luciferase. NMDA recep-
tors reconstituted with NR1C or NR1∆ mediated significantly less
NMDA-stimulated CaRE-dependent gene expression than NR1A (n
= 5, *p < 0.005). Similarly transfected neurons mediated compara-
ble responses to L-VSCC stimulation, indicating that the effect of the
NR1 subunit is specific to the ability of the reconstituted NMDA
receptor to regulate gene expression. Control fold induction = 1.
Reports from the Laboratories 37
2001 ANNUAL REPORT
NMDA receptor itself, might play a role in coupling The fact that deletions in the carboxyl terminus of
calcium influx to nuclear gene expression. The idea NR1 can dissociate bulk cytoplasmic calcium
that channel-associated proteins could play a role in responses from the ability of the NMDA receptor to
long-range signal transduction emanating from the regulate adaptive gene expression suggests two
NMDA receptor could explain how a pleiotropic sec- important conclusions. First, a critical component of
ond messenger, the calcium ion, could nonetheless calcium-dependent signal transduction specificity
send a “channel-specific” signal. may be conferred by the molecules that the calcium
ion engages near its site of entry. Second, some of the
To explore this hypothesis further, we have developed long-range signals that a calcium channel sends may
a reconstitution system that enables us to determine be determined by the repertoire of proteins located
how channel-associated proteins couple calcium influx near its cytoplasmic mouth. Future work will focus on
through the NMDA receptor to new gene transcription. refining our understanding of the sequences within
The NMDA receptor is believed to be a heteromeric NR1 and associated proteins that are required to cou-
channel, possibly a tetramer composed of two protein ple calcium influx to gene transcription and on ana-
subunits encoded by the NR1 gene and two subunits lyzing the promoters of NMDA-induced genes to
encoded by members of the NR2 gene family. Without determine the extent to which a channel-specific sig-
the NR1 gene product, no functional NMDA receptor nal is perceived by the nucleus.
is synthesized, and animals that lack the NR1 gene fail
to live beyond their first postnatal day. Molecular Mechanisms of HD
If we stimulate cultured neurons from NR1–/– mice with From initial work with our cellular model of HD, we
NMDA, they fail to show calcium or gene expression had found that the addition of caspase inhibitors, the
responses. However, when we transfect NR1–/– neurons cotransfection of the anti-apoptosis gene BClXL, or
with an expression plasmid that encodes NR1, we res- the addition of extracellular factors such as brain-
cue both responses (Figure 2A–C). Since the binding derived neurotrophic factor, ciliary neurotrophic factor,
site for NMDA is on the NR2 subunit and channels that or insulin growth factor 1 (IGF-1) delayed or sup-
lack the NR1 subunit fail to function, the rescued pressed neurodegeneration in response to mutant
responses must be mediated by reconstituted het- huntingtin. Surprisingly, the manipulations had different
eromeric NMDA receptors that have incorporated the effects on the abnormal deposition of mutant hunt-
transfected NR1 subunit. Thus, the system has given us ingtin within neurons to form intranuclear inclusions.
control of the cytoplasmic tail of the NR1 subunit with- IGF-1 suppressed the fraction of neurons with visible
in every functional NMDA receptor. inclusion bodies, whereas all the other manipulations
doubled or tripled the fraction of neurons with
To test whether the cytoplasmic tail of the NMDA intranuclear inclusions. In the last year, we and our
receptor or the proteins that associate with it are collaborators have focused on the signal transduction
important for NMDA-dependent gene expression, we mechanisms by which IGF-1 suppresses degeneration
compared the abilities of carboxyl-terminal truncation and inclusion body formation by mutant huntingtin.
mutants of NR1 to mediate NMDA-induced calcium
and gene expression responses in our reconstitution We have discovered that the pro-survival kinase, Akt,
system (Figure 2A). We found that specific deletions is responsible for many of the effects of IGF-1 in our
in the carboxyl terminus of the NMDA receptor did cellular model of HD. IGF-1 activates Akt in the stri-
not measurably affect the ability of the channel to pro- atal neurons that we study, and active forms of Akt
duce cytoplasmic calcium increases, as measured introduced into these neurons recapitulate the effects
with the calcium indicator Fura-2 (Figure 2C). of IGF-1 on huntingtin-induced neurodegeneration
However, some of these same deletions substantially and inclusion body formation. Surprisingly, we have
diminished the ability of the NMDA receptor to acti- discovered a highly conserved Akt phosphorylation
vate neuronal gene transcription (Figure 2D). site within the huntingtin protein itself and have
38 Reports from the Laboratories
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE
demonstrated that huntingtin is an Akt substrate. We mutant huntingtin nontoxic. This is the first evidence
have generated antibodies that specifically recognize that huntingtin is a phosphoprotein and that its abili-
the Akt-phosphorylated form of huntingtin and have ty to induce neurodegeneration can be regulated by
found that IGF-1 stimulation leads to huntingtin phosphorylation. These results also suggest that hunt-
phosphorylation in neurons. Blocking phosphoryla- ingtin may be an in vivo target of Akt. Finally, since
tion by site-directed mutation (S421A) renders Akt activity can be regulated by small-molecule
mutant huntingtin partly resistant to the protective drugs, these findings suggest that Akt may be an
properties of IGF-1. Conversely, mutation of this site attractive therapeutic target for HD and other neu-
to mimic tonic phosphorylation (S421D) renders rodegenerative disorders.
Saudou F, Finkbeiner S, Devys D, Greenberg, ME Finkbeiner S (2001) Disease-associated polyglutamine
(1998) Huntingtin acts in the nucleus to induce apop- expansions as protein epitopes involved in neurode-
tosis but death does not correlate with the formation generation. Soc. Neurosci. 27 (Part 2):674.5 (abstract).
of intranuclear inclusions. Cell 95:55–66.
Finkbeiner S (2001) New roles for introns: Sites of
Finkbeiner S (2000) Calcium regulation of the brain- combinatorial regulation of Ca2+- and cyclic AMP-
derived neurotrophic factor gene. Cell. Mol. Life Sci. dependent gene transcription. Science’s STKE
Bradley J, Curtis J, Finkbeiner S (2001) The C-termi-
nus of the NMDA receptor NR1 subunit is required
for NMDA-dependent gene expression. Soc.
Neurosci. 27 (Part 2):2451 (abstract).
Reports from the Laboratories 39
Fen-Biao Gao, Ph.D.
Sarah Goulding, Ph.D.
Wen-Jun Li, Ph.D.
Neal T. Sweeney
Nhue L. Do
Marlette A. Marasigan
Reports from the Laboratories 41
Molecular Mechanisms of Dendritic Morphogenesis
and Their Involvement in Neurological Diseases
Fen-Biao Gao, Ph.D.
two-dimensional plane. The dendritic branching pat-
ignaling between neurons requires specialized
subcellular structures, including axons and den- tern of the MD neurons is fairly invariant from
drites. Dendrites can be highly branched and embryo to embryo, suggesting that a genetic pro-
may account for more than 90% of the postsynaptic gram controls dendritic morphogenesis. Using this
surface of some neurons. Only recently have den- assay system, we have begun to identify the molec-
drites been appreciated as having much more active ular components of the genetic program, including
roles in neuronal function. In addition, the number of the flamingo and sequoia genes.
dendritic branches and dendritic spines is altered in
many neurological disorders, including Alzheimer’s GFP Labeling of Single Neurons
disease and fragile X syndrome. Despite the impor- in Living Drosophila Larvae
tance of dendrites in neuronal function and dysfunc-
tion, the molecular mechanisms underlying dendritic To study how neuronal morphogenesis is controlled
morphogenesis in vivo remain elusive. by intracellular factors during development, our labo-
ratory uses the mosaic analysis with a repressible
Using Drosophila as a Model System marker technique to visualize single wildtype and
to Study Dendrite Development mutant PNS neurons in living Drosophila larvae.
Briefly, with the UAS-GAL4-targeted expression sys-
Systematic genetic analysis in Drosophila offers a tem, all of the larval neurons are labeled by mCD8-
powerful approach to unravel complex biological GFP. When GAL80, a yeast repressor that can bind to
processes. To understand the molecular mechanisms GAL4, is ubiquitously expressed, mCD8-GFP expres-
that control dendritic morphogenesis during devel- sion is suppressed in all neurons. When FLP recombi-
opment and in neurological disorders, our laboratory nase–mediated recombination occurs in precursor
uses the Drosophila peripheral nervous system cells, one daughter cell loses GAL80 and is labeled by
(PNS) as a genetic model system. Each abdominal GFP. Using this approach, we could label different
hemisegment contains only 44 PNS sensory neu- individual MD neurons with GFP in living
rons, which can be grouped into dorsal, lateral, and Drosophila larvae.
ventral clusters. In the dorsal cluster, there are six
mutiple dendritic (MD) neurons, four external sen- Morphological Diversity
sory (ES) neurons, one bipolar dendritic (BD) neu- of PNS Sensory Neurons
ron, and one internal sensory neuron. Using the
UAS-GAL4 system to express green fluorescent We obtained images of the dendritic branching pat-
protein (GFP) in a cell type–specific manner, we can terns of each subtype of PNS neuron in the dorsal
directly visualize dendritic morphogenesis of dorsal cluster, as well as images of other PNS sensory neu-
MD neurons in living Drosophila embryos and lar- rons in the lateral and ventral clusters. Our genetic
vae and follow their growth, branching, and remod- studies mainly focused on the dorsal cluster.
eling in real time. These neurons elaborate their den- Therefore, only the development of dendritic fields of
drites just underneath the epidermal cell layer in a dorsal cluster MD neurons is described here in detail.
Reports from the Laboratories 43