2001 ANNUAL REPORT




                      GLADSTONE
                    INSTITUTE OF
                   NEUROLOGICAL
  ...
GLADSTONE INSTITUTE
            OF NEUROLOGICAL DISEASE
2001 ANNUAL REPORT




Copyright 2002
by The J. David Gladstone In...
TABLE OF CONTENTS



DIRECTOR’S REPORT.........5

DESCRIPTION OF THE INSTITUTES.........13

MEMBERS OF THE INSTITUTE.........
DIRECTOR’S REPORT



Lennart Mucke, M.D.




                      Director’s Report 5
Director’s Report



                        Neurodegenerative disorders         impair neuronal function and survival. In...
2001 ANNUAL REPORT




cultures. This powerful new device has the potential to      the risk of developing AD (E4 > E3 > E...
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE




age was associated with increased neuronal death. The       this analysis ...
2001 ANNUAL REPORT




modified mice. In previous studies, the Weisgraber            mic environment that is ideal for tra...
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE




Acknowledgements
                                                        T...
Description of the
THE J. DAVID GLADSTONE
INSTITUTES

Trustees
Richard S. Brawerman
Albert A. Dorman
Richard D. Jones


Pr...
Description of the Institutes



                                                           Although autonomous in their a...
2001 ANNUAL REPORT




                                                          Gladstone Institute
Gladstone Institute o...
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE




Lipoprotein Biochemistry and Metabolism. A                  tify unique ge...
2001 ANNUAL REPORT




                                                         Warner C. Greene, M.D., Ph.D., an internat...
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE




Laboratory of Receptor Biology. The Laboratory of         for analysis. Th...
2001 ANNUAL REPORT




                                                          Massachusetts General Hospital, and Harva...
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE




novel apoE-targeted drug treatments for Alzheimer’s       decline in diver...
New Gladstone Laboratories at the UCSF Mission Bay Campus



                                                             ...
2001 ANNUAL REPORT




The architects have also been hard at work. They have            grow from the current 265 employee...
MEMBERS OF THE INSTITUTE




              Members of the Institute 25
Members of the Gladstone Institute of Neurological Disease



Director                                   Staff Research In...
2001 ANNUAL REPORT




Postdoctoral Fellows            Senior Research Associates
Montserrat Arrasate, Ph.D.      Maureen ...
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE




Administrative Staff
of the J. David Gladstone Institutes


President     ...
2001 ANNUAL REPORT




Intellectual Property/Technology Transfer   Receptionist
Joan V. Bruland, J.D., Officer            ...
REPORTS FROM
THE LABORATORIES


   Finkbeiner Laboratory.........33


   Gao Laboratory.........41


   Huang Laboratory.....
FINKBEINER LABORATORY


Assistant Investigator
Steven M. Finkbeiner, M.D., Ph.D.


Postdoctoral Fellows
Montserrat Arrasat...
Molecular Mechanisms of Plasticity and Neurodegeneration



Steven M. Finkbeiner, M.D., Ph.D.




                        ...
2001 ANNUAL REPORT




Figure 1. The robotic microscope facilitates high-throughput cell       formed 15, 39, 48, 72, 96, ...
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE




Molecular Mechanisms                                       subtype of glut...
2001 ANNUAL REPORT




NMDA receptor itself, might play a role in coupling        The fact that deletions in the carboxyl ...
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE




demonstrated that huntingtin is an Akt substrate. We     mutant huntingtin...
GAO LABORATORY


Assistant Investigator
Fen-Biao Gao, Ph.D.


Postdoctoral Fellows
Sarah Goulding, Ph.D.
Wen-Jun Li, Ph.D....
Molecular Mechanisms of Dendritic Morphogenesis
and Their Involvement in Neurological Diseases


Fen-Biao Gao, Ph.D.




 ...
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GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE

  1. 1. 2001 ANNUAL REPORT GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE THE J. DAVID GLADSTONE INSTITUTES University of California, San Francisco San Francisco General Hospital Medical Center
  2. 2. GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE 2001 ANNUAL REPORT Copyright 2002 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
  3. 3. TABLE OF CONTENTS DIRECTOR’S REPORT.........5 DESCRIPTION OF THE INSTITUTES.........13 MEMBERS OF THE INSTITUTE.........25 REPORTS FROM THE LABORATORIES.........31 Finkbeiner Laboratory.........33 Gao Laboratory.........41 Huang Laboratory.........47 Mahley Laboratory.........55 Mucke Laboratory.........63 Pitas Laboratory.........73 Weisgraber Laboratory.........79 Wyss-Coray Laboratory.........87 Behavioral Core Laboratory.........95 Gladstone Genomics Core.........103 OUTREACH.........109 PUBLICATIONS.........115 SEMINARS.........123
  4. 4. DIRECTOR’S REPORT Lennart Mucke, M.D. Director’s Report 5
  5. 5. Director’s Report 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
  6. 6. 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
  7. 7. 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
  8. 8. 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
  9. 9. GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE Acknowledgements 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 Director’s Report 11
  10. 10. Description of the THE J. DAVID GLADSTONE INSTITUTES Trustees Richard S. Brawerman Albert A. Dorman Richard D. Jones President Robert W. Mahley, M.D., Ph.D. Executive Director Richard Hille Chief Financial Officer Hal Orr, C.M.A. Description 13
  11. 11. Description of the Institutes Although autonomous in their areas of specialization, P 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. Description 15
  12. 12. 2001 ANNUAL REPORT Gladstone Institute 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. Medical Center 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 Biochemistry 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. 16 Description
  13. 13. 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 Description 17
  14. 14. 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. 18 Description
  15. 15. 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, Description 19
  16. 16. 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. Harvard University 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 20 Description
  17. 17. 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. Description 21
  18. 18. New Gladstone Laboratories at the UCSF Mission Bay Campus Most recently, the trustees—Richard S. Brawerman, T 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. The Commons 15A 15B 16A 16B 17A 17B 16B 17A The Courts 18A 18B 20A 20B 19A 19B The Plaza 21A O 23A w The Green en 21B 23B s St G nst re la itu I ds t et to 3rd Street ne es Pa ruc 24C St rk tur in e 24A/B g Proposed 25A 25B Biotech Labs 16th Street 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
  19. 19. 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
  20. 20. MEMBERS OF THE INSTITUTE Members of the Institute 25
  21. 21. 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 of Neuroscience 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 and Neurology Research Scientists 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
  22. 22. 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 Lab Aide John A. Gray 28 Members of the Institute
  23. 23. GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE Administrative Staff 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
  24. 24. 2001 ANNUAL REPORT Intellectual Property/Technology Transfer Receptionist Joan V. Bruland, J.D., Officer Hope S. Williams Erin Madden 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 Operations Deborah S. Addad, Officer Facilities Vincent J. McGovern, M.S., Manager David R. Bourassa Randy A. Damron George R. Leeds Roger A. Shore Purchasing P. J. Spangenberg, Manager Tyler G. Campos Judy H. Cho P. Sidney Oduah Alberto L. Reynoso Benjamin V. Young 30 Members of the Institute
  25. 25. REPORTS FROM THE LABORATORIES Finkbeiner Laboratory.........33 Gao Laboratory.........41 Huang Laboratory.........47 Mahley Laboratory.........55 Mucke Laboratory.........63 Pitas Laboratory.........73 Weisgraber Laboratory.........79 Wyss-Coray Laboratory.........87 Behavioral Core Laboratory.........95 Gladstone Genomics Core.........103 Reports from the Laboratories 31
  26. 26. FINKBEINER LABORATORY Assistant Investigator Steven M. Finkbeiner, M.D., Ph.D. Postdoctoral Fellows Montserrat Arrasate, Ph.D. John Bradley, Ph.D. Students Sarah Carter Patrick Chang Ammon Corl Jennifer Fu Siddhartha Mitra Hélène Rangone Vikram Rao Research Associates Elizabeth Brooks Jessica Curtis Shyamal Kapadia Administrative Assistant Nannette Nemenzo Reports from the Laboratories 33
  27. 27. Molecular Mechanisms of Plasticity and Neurodegeneration Steven M. Finkbeiner, M.D., Ph.D. neurodegeneration, cell-specific death, and formation O 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
  28. 28. 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
  29. 29. 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
  30. 30. 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
  31. 31. 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. Selected References 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 57:394–401. (http://stke.sciencemag.org/cgi/content/ full/OC_sigtrans;2001/94/pe1). 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
  32. 32. GAO LABORATORY Assistant Investigator Fen-Biao Gao, Ph.D. Postdoctoral Fellows Sarah Goulding, Ph.D. Wen-Jun Li, Ph.D. Students Richard Han Linda Ngo Neal T. Sweeney Research Associates Nhue L. Do Yu-Hua Zheng Administrative Assistant Marlette A. Marasigan Reports from the Laboratories 41
  33. 33. Molecular Mechanisms of Dendritic Morphogenesis and Their Involvement in Neurological Diseases Fen-Biao Gao, Ph.D. two-dimensional plane. The dendritic branching pat- S 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

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