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  1. 1. 2002 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 2002 ANNUAL REPORT Copyright 2003 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..........4 DESCRIPTION OF THE INSTITUTES..........7 MEMBERS OF THE INSTITUTE..........13 REPORTS FROM THE LABORATORIES..........15 Finkbeiner Laboratory..........16 Gao Laboratory..........20 Huang Laboratory..........23 Mahley Laboratory..........26 Mucke Laboratory..........29 Pitas Laboratory..........33 Weisgraber Laboratory..........36 Behavioral Core Laboratory..........40 Gladstone Genomics Core..........43 EDUCATION AND COMMUNITY OUTREACH..........46 PUBLICATIONS..........47 SEMINARS..........51
  4. 4. DIRECTOR’S REPORT LENNART MUCKE, M.D. Neurodegenerative disorders rob people ating a protein fragment that readily adopts a disease-associated con- of their ability to remember, speak, write, formation. Longitudinal monitoring of neurons in culture with the ambulate, and control their lives. These robotic microscope allowed the investigators to demonstrate that a conditions are on the rise because people protein kinase, Akt, promotes neuronal survival much more potent- are living longer, and aging strongly ly than had been appreciated from initial reports that measured sur- increases the risk of being afflicted by vival at single time points. The potency of Akt makes it an especial- these conditions. The enormous cost of ly attractive therapeutic target to block neurodegeneration. caring for individuals with these condi- The laboratory of Dr. Fen-Biao Gao focuses on the genes and molec- tions threatens our health care system. A ular pathways that regulate the development and maintenance of medical breakthrough is clearly needed, and the surest way to such neuronal dendrites. Dendrites are tree-like extensions of neurons that a breakthrough is to determine exactly how these diseases result in receive signals and participate in information processing and stor- the dysfunction and degeneration of nerve cells. In addition, neuro- age. These structures account for more than 90% of the surface of logical diseases raise a range of fascinating questions that are of fun- some neurons. In many neurological disorders, including damental scientific interest. While the investigation of neurological Alzheimer’s disease (AD) and fragile X syndrome, the number of diseases has promoted basic neuroscientific discoveries for over a dendritic branches is altered. However, little is known about the century, there has never been a more promising and exciting con- mechanisms that control dendritic branching in vivo. Last year, Dr. vergence of basic and disease-related neuroscience than now. Gao’s laboratory selectively mutated genes in individual neurons Investigators at the Gladstone Institute of Neurological Disease and studied the consequences of this manipulation on dendritic (GIND) have continued to examine the possibility that many, if not branching in living fruit flies. This year, the investigators used this all, neurodegenerative disorders are caused by the accumulation of approach to study the pathogenesis of fragile X syndrome, the most proteins that have assumed pathogenic conformations. Although dif- common form of inherited mental retardation in humans. The syn- ferent proteins accumulate in different neurodegenerative disorders, drome is caused by mutations in the fragile X mental retardation 1 the ways in which their neurotoxic assemblies damage nerve cells (fmr1) gene. Their studies revealed that a close homologue of the may overlap. This possibility raises hope that it will be feasible to fmr1 gene is highly expressed in the fruit fly nervous system, where develop treatments that can prevent, stall, or even reverse more than the protein is localized to both dendrites and axons of specific neu- one of these conditions. Other studies have focused on the molecu- rons. Studies are under way to determine whether and how muta- lar mechanisms of neural plasticity, which is critical for the devel- tions in the fly gene alter the function and integrity of these neuronal opment of the nervous system as well as for its adaptations to envi- branches. Dr. Gao and his associates also set up automated behav- ronmental stimuli and for the remodeling of its circuitry after injury. ioral tests to quantify the functional consequences of disrupting gene expression in the fruit fly nervous system. Using such tests, they dis- Scientific Discoveries covered that mutations in the presenilin gene alter the linear loco- The laboratory of Dr. Steven Finkbeiner studies how a genetic muta- motion of fly larvae. Presenilin is a major drug target in AD research tion leads to Huntington’s disease and how the nervous system because it is necessary for the production of the neurotoxic amyloid- adapts to brief experiences by making long-lasting changes in its β peptides (Aβ), which are presumed to cause AD. Dr. Gao’s further structure and function. Huntington’s disease is a fatal inherited neu- analysis of presenilin in the fruit fly may identify ways to inhibit its rodegenerative disorder that is associated with increasingly disrup- AD-promoting activities while preserving its beneficial functions. tive involuntary movements and a progressive loss of motor control. It is caused by abnormal polyglutamine expansions in the protein The laboratory of Dr. Yadong Huang investigates the lipid carrier huntingtin, which affect protein folding. Last year, Dr. Finkbeiner apolipoprotein (apo) E and its relationship to AD. The three major developed a robotic microscope for the large-scale analysis of neu- human isoforms of apoE differentially affect the risk of developing ronal cell cultures expressing different forms of huntingtin or relat- AD (E4 > E3 > E2). Roughly 10 years after the discovery of this ed proteins. This year, he used this powerful new tool to reveal that link, apoE4 remains the main known inherited risk factor for the mutant huntingtin causes a defect in the formation and maintenance most frequent form of AD. Yet, it is still uncertain how exactly it of the branches (neurites) through which brain cells communicate increases the risk and accelerates the onset of this illness. Last year, with each other. These new findings further validate the cell culture Dr. Huang and his collaborators demonstrated that apoE4 has a model Dr. Finkbeiner uses in his studies, since abnormalities in neu- greater proclivity to be broken down into fragments than apoE3, rite extension and regeneration are also early pathological features both in people with AD and in genetically modified (transgenic) of Huntington’s disease. Using an antibody they developed, Dr. mice expressing human apoE in the brain. The accumulation of Finkbeiner and his associates also found that cleavage within a spe- apoE4 fragments was associated with abnormal phosphorylation of cific domain of mutant huntingtin promotes pathogenesis by gener- tau and cytoskeletal derangements. Abnormally phosphorylated tau is the major constituent of neurofibrillary tangles, a pathological 4 Director’s Report
  5. 5. GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE hallmark of AD. This year, Dr. Huang demonstrated that the deficits in our mouse models, they constitute reliable molecular indi- increased fragmentation of apoE is dependent, at least in part, on an cators of clinically relevant deficits and, hence, could improve the intramolecular domain interaction that occurs in apoE4 but not in assessment of novel AD treatments. We also found marked reduc- apoE3. Furthermore, when expressed by themselves in the brains of tions in calcium-dependent proteins in granule cells of humans with transgenic mice, the apoE fragments elicited neurodegenerative AD, although this neuronal population is relatively resistant to AD- alterations in various brain regions, including regions involved in related cell death. This result demonstrates that neuronal populations learning and memory. Inhibitors of the protease presumed to cleave resisting cell death in AD can still be drastically altered at the molec- apoE within neurons might block the adverse effects of apoE4 and ular level. They also suggest that at least some of the neurological thereby benefit the many apoE4 carriers suffering from or at risk for deficits seen in AD may not reflect loss of brain cells. This conclu- developing AD. Therefore, the isolation and further characterization sion has important implications because lost neurons are still hard or of this enzyme is an important goal of ongoing studies. impossible to replace, whereas molecular alterations in surviving The laboratory of Dr. Robert W. Mahley maintains its long-term brain cells may be more readily reversible by pharmacological inter- focus on the mechanisms underlying the pathogenic activities of ventions. We look forward to exploring this possibility further in the apoE4 in AD and other conditions. ApoE3 promotes neurite out- next year. growth, protects the nervous system against diverse injuries, and The laboratory of Dr. Robert E. Pitas has followed up on their dis- facilitates regeneration of the nervous system after trauma. In con- covery of apoE-binding protein (EBP), a novel brain protein that trast, apoE4 is not protective and, in many instances, even worsens belongs to a previously uncharacterized family of proteins with the outcome of neural injuries. This year, Dr. Mahley and his asso- unknown function. The proteins are encoded by four homologous ciates extended their investigation of the most common apoE iso- genes in humans and mice and by one related gene in the fruit fly. forms to the production of Aβ peptides. They examined the effects The association of EBP with apoE3, initially identified by the yeast of apoE3 and apoE4 on the processing of the amyloid precursor two-hybrid approach, has now been confirmed in other biochemical protein (APP) and on Aβ production in cultures of neuronal cells. assays. Dr. Pitas and his associates have also produced evidence that Both apoE isoforms stimulated Aβ production, but apoE4 did so to EBP interacts with the cytoskeleton and have developed polyclonal a significantly greater extent than apoE3. Interestingly, this differ- antibodies that detect both human and mouse EBP. Staining of ential effect of the apoE isoforms was mediated by stimulating cell- mouse brain sections with these antibodies revealed neuron-specific surface APP recycling rather than by altering cellular cholesterol expression of EBP in many regions, including the hippocampus and content or activities of enzymes involved in Aβ production. When dentate gyrus. Ablating the expression of apoE or expressing human the investigators disrupted the intramolecular domain interaction APP and Aβ in neurons altered the expression pattern of EBP in that occurs in apoE4, but not in apoE3, the differential effect of mouse brains, suggesting that the expression of EBP is affected by apoE3 and apoE4 on Aβ production was attenuated, suggesting that factors critically involved in the pathogenesis of AD. Interestingly, this effect involves conformational differences among the apoE in cell cultures, EBP protected neuronal cells against oxidative variants. Since Aβ plays a central role in the pathogenesis of AD, stress. This neuroprotective function of EBP may relate to its inter- the stronger stimulatory effect of apoE4 on Aβ production could action with apoE, which also appears to fulfill neuroprotective func- contribute to the increased AD risk associated with inheritance of tions. Studies are under way to ablate EBP expression in fruit flies this apoE isoform. In addition, apoE4 may increase AD risk through and mice to determine the physiological functions of EBP and the Aβ-independent mechanisms, as demonstrated in collaborative roles it might play in neurological disease. studies with Dr. Huang (see above). The laboratory of Dr. Karl H. Weisgraber investigates the relation- My own laboratory has continued to investigate the molecular path- ship between the three-dimensional structure of different apoE mol- ways that link genetic determinants or risk factors of AD to neu- ecules and their normal and pathological activities. While much is rodegeneration and cognitive decline. We have demonstrated in known about the structure of lipid-free apoE, very little is known transgenic mouse models that human Aβ causes reductions in calci- about apoE’s structure when it is bound to lipids, although apoE um-dependent proteins in granule cells of the dentate gyrus, a neu- probably exists in the body primarily in a lipid-bound (lipidated) ronal population critically involved in learning and memory. Our state. This year, Dr. Weisgraber and his coworkers have made great data suggest that the reductions in calcium-dependent proteins are strides in this area by producing crystals of lipidated apoE whose caused by small soluble neurotoxic Aβ assemblies rather than by the structure can now be investigated by crystallography and other bio- large deposits of Aβ that make up amyloid plaques. Notably, plaque physical approaches. In other studies, the investigators discovered burden remains the most widely used pathological endpoint measure that apoE4 is more likely than apoE3 to assume a particular folding in the preclinical assessment of AD treatments even though it often state (molten globule) that could promote its binding to and translo- does not correlate well with cognitive deficits. Since the molecular cation across membranes. By assuming this folding state, apoE4 alterations we identified correlated extremely well with learning might escape from vesicular compartments within the cell and gain Director’s Report 5
  6. 6. 2002 ANNUAL REPORT access to the cytoskeleton and to enzymes and other molecules in the Klingenstein Fellowship Award in Neuroscience from the cytoplasm. Collaborative studies are under way to determine Klingenstein Foundation, which supports new approaches to neuro- whether this process contributes to the cytoskeletal destabilization scientific research. My own research efforts on AD were recognized and susceptibility to proteolytic cleavage that Drs. Huang and by a Metropolitan Life Foundation Award for Medical Research and Mahley have found to be associated with apoE4. a MERIT award from the National Institute on Aging. Any award reflects the participation of many, and I consider all members of my Education, Special Initiatives, and Recognition laboratory and my collaborators important contributors. I thank them The Gladstone Institutes and UCSF provide state-of-the-art research for their tremendous loyalty and support. We all share these awards facilities and a highly interactive academic environment that is ideal together. for training in neuroscience and biomedical research. GIND investi- gators participate actively in the training of students and residents Dr. Tony Wyss-Coray, a member of the GIND since its inauguration, from various UCSF departments and interdepartmental programs, has been recruited to Stanford University, where he now holds an including the Departments of Neurology and Physiology, the appointment as assistant professor in the Department of Neurology Neuroscience Program, the Biomedical Sciences Program, the and Neurological Sciences. The staff research investigator position Pharmaceutical Sciences and Pharmacogenomics Program, and the he held at the GIND aims to give exceptional young scientists the Medical Scientist Training Program, as well as from graduate and opportunity to focus solely on developing a successful and creative undergraduate programs at UC Berkeley and other institutions. research program before accepting a standard faculty position. Dr. Wyss-Coray made the most of this opportunity and left the institute Several members of our institute have collaborated to make our well published and supported by a substantial amount of independ- training environment even more inspiring and rewarding for stu- ent NIH funding. We wish him and his terrific group the very best in dents of all biomedical disciplines. For example, Dr. Finkbeiner has all their future endeavors and look forward to welcoming the next developed a curriculum for the neurology house staff called the outstanding candidate into the position he vacated. We are equally Neuroscience of Disease Seminar series. He has also served as the excited about the recruitment of a new faculty member to the GIND. neuroscience liaison for UCSF’s Brain Interest Program, which The search for suitable candidates was launched in the fall and will aims to foster interest in the brain and provides an opportunity for be conducted by an eight-member joint UCSF/Gladstone committee. students to learn about different faculty, their careers, and their This faculty recruitment represents the first step toward expanding interests. Also noteworthy in this context is the continued success the GIND as we prepare for the relocation of our research program of our weekly GIND seminar series, which is organized by Dr. Gao. to UCSF’s new Mission Bay campus. These well-attended seminars provide a stimulating forum for edu- cation in disease-related neuroscience and for scientific exchange Gladstone investigators have continued to participate actively in among members of the institutes and colleagues from the greater finalizing the design of the new research facility that will accommo- UCSF community. date all three Gladstone Institutes at Mission Bay. The GIND will occupy the third floor of the new Gladstone building, which is GIND investigators have extended their efforts to promote education scheduled for completion in 2004. The consolidation of our labora- and scientific exchange in disease-related neuroscience far beyond tories, cores, and animal care facilities into one building at the new the boundaries of our institute. They have organized and participat- campus will further enhance our ability to contribute to research and ed in a number of national and international conferences that have training in neurology and neuroscience. advanced our field of research in various ways. Despite their busy schedules and expanding research efforts, GIND members have con- In closing, I would like to thank the participants of this year’s tinued to devote time to community outreach. As described in the Scientific Advisory Board meeting, Drs. Dale E. Bredesen (Buck Education and Community Outreach section of this report, these Institute for Age Research), Dennis Selkoe (Harvard Medical efforts included participation in activities aimed at educating the School), and Eric Shooter (Stanford University) for their outstand- public about AD and neuroscientific research in general. Our syner- ing input. It contributed greatly to our success. The progress we gism with the UCSF Memory and Aging Center, the local chapter of made in 2002 reflects the work of all the researchers at the GIND the Alzheimer’s Association, and the Hereditary Disease Foundation and of the administrative staff at the Gladstone Institutes. As out- has allowed us to maintain and expand fruitful links between our lined in this report, we have shed new light on molecular processes research and the patients afflicted by the diseases we study. causing devastating neurological diseases as well as on mechanisms that might be utilized to prevent and cure them. I am delighted that our accomplishments have not gone unnoticed, as reflected by the honors and awards institute members received this year. Dr. Gao received a McKnight Neuroscience of Brain Disorders Award from the McKnight Endowment Fund for Neurosciences, one of six awards to scientists whose research is directed toward finding new ways to diagnose, treat, and cure disor- Lennart Mucke, M.D. ders of the brain and central nervous system. He also received a Director 6 Director’s Report
  7. 7. Description of Trustees Executive Director Richard S. Brawerman Richard Hille THE J. DAVID GLADSTONE Albert A. Dorman Chief Financial Officer Richard D. Jones INSTITUTES Hal Orr, C.M.A. President Robert W. Mahley, M.D., Ph.D. The J. David Gladstone Institutes is the product of the wisdom and P rimary research efforts at the J. David Gladstone Institutes focus on three of the most important clinical problems of mod- hard work of many individuals. The first was J. David Gladstone him- ern times: cardiovascular disease, AIDS, and neurodegenera- self, a Los Angeles real-estate entrepreneur. Others are the trustees. tive disorders. Cardiovascular disease, the nation’s leading killer, The original trustees, all of whom had known or worked closely with claims the lives of over one million Americans each year. Despite Mr. Gladstone, were Richard S. Brawerman, his attorney and execu- more effective treatments, AIDS remains a leading cause of death in tor of his estate; Richard D. Jones, his real-estate attorney; and David the United States. Worldwide, more than 42 million people are living Orgell, his cousin and confidant. When Mr. Orgell died in 1987, he with HIV/AIDS, and more than 21 million have died as a direct result was succeeded on the board by Albert A. Dorman, a southern of HIV infection. Alzheimer’s disease, the most recent focus of inves- California executive with experience in managing large organizations. tigation by Gladstone scientists, robs people of their ability to think, At the time of Mr. Gladstone’s death in 1971, the southern California remember, and control their lives. It currently affects over 4 million real-estate market was just beginning to flourish. His estate, left almost people in the United States alone and is predicted to affect 14 million entirely for medical education and research, was relatively modest by Americans by 2050. The impact of these diseases on world health later standards. However, the trustees recognized the estate’s potential infuses Gladstone scientists with a sense of purpose and urgency. for growth and, through their inspired management, increased its worth Gladstone is composed of three institutes, each of which issues its own severalfold within the first decade. From the beginning, the decisions annual report. The Gladstone Institute of Cardiovascular Disease of the trustees had profound and positive effects on the research organ- (GICD), which opened in 1979, focuses on atherosclerosis and its ization that evolved. They continue to manage and enlarge the assets of complications. In 1992, the Gladstone Institute of Virology and the J. David Gladstone Institutes and to oversee their use. Immunology (GIVI) was established to study HIV, the causative agent Gladstone Institute of Cardiovascular Disease of AIDS. The 1993 discovery that apolipoprotein (apo) E—long stud- Close ties already existed between the UCSF School of Medicine and ied at GICD for its role in heart disease—plays a role in Alzheimer’s SFGH, the hospital of the City and County of San Francisco, when the disease as well led to the establishment of the Gladstone Institute of trustees leased vacant space from the City in 1977 in which to create Neurological Disease (GIND) in 1998. The three institutes are located laboratories and offices. The partnership has flourished. Gladstone at the San Francisco General Hospital (SFGH) campus of the scientists collaborate with their colleagues at UCSF and SFGH and University of California, San Francisco (UCSF), and will soon move provide service to those organizations as professors and staff physi- into a new facility at UCSF’s Mission Bay campus. While independ- cians. The mutually beneficial association between the Gladstone, ent, Gladstone is formally affiliated with UCSF, and Gladstone inves- UCSF, and SFGH has created a productive and supportive environ- tigators hold university appointments and participate in many univer- ment in which scientists conduct basic research while availing them- sity activities, including the teaching and training of graduate students. selves of clinical and academic opportunities. Although autonomous in their areas of specialization, the institutes To choose a director for the developing research facility, the trustees share a common approach. Each institute is organized into research sought guidance from the scientific community. The choice was units consisting of scientists, postdoctoral researchers, research asso- Robert W. Mahley, M.D., Ph.D. At the time of Mr. Gladstone’s death, ciates, and students. This structure is designed to accommodate small he was just completing his internship. However, by 1979, when he groups of scientists who work together closely but who also benefit was appointed director, Dr. Mahley had established himself as a lead- from collegial interactions with other research groups. Collaborations ing researcher in the field of lipoprotein metabolism and atherosclero- among staff members with various areas of expertise create a stimu- sis. He came to the Gladstone from the National Institutes of Health, lating environment that fortifies the scientific lifeblood of the organ- where he headed the Laboratory of Experimental Atherosclerosis. ization. Less than a year after his appointment, Dr. Mahley had assembled a Each institute receives expert input on the progress of its science from staff of 25, and the new organization, then called the Gladstone an advisory board of distinguished scientists. The scientific advisory Foundation Laboratories for Cardiovascular Disease, officially boards provide a twofold service in reviewing the quality of the opened on September 1, 1979. Dr. Mahley is professor of pathology research and in advising the president, directors, and trustees. and medicine at UCSF and is a member of the National Academy of The work of the scientific staff at all three institutes also extends Sciences and the Institute of Medicine. beyond the laboratory to the wider community. The mission of the By the end of 2002, the research staff of the GICD had grown to more institutes includes the education of graduate and medical students, than 100 scientists, postdoctoral fellows, students, and research asso- postdoctoral fellows, and visiting scientists; specialized training for ciates, occupying about 48,000 square feet of laboratory and office practicing physicians; and educational outreach to the local and space in buildings 9 and 40 on the SFGH campus. In 22 years of oper- extended community. ation, the institute has attained an international reputation for excel- Description 7
  8. 8. 2002 ANNUAL REPORT are John M. Taylor, Ph.D., Stephen G. Young, M.D., and Robert V. Gladstone Institute of Cardiovascular Disease Farese, Jr., M.D. Vascular Biology. This research aims to elucidate how mono- Scientific Advisory Board cytes/macrophages are attracted to sites of atherosclerotic lesion Göran K. Hansson, M.D., Ph.D. formation and to delineate the role of platelets in forming the occlu- Professor of Cardiovascular Research sive thrombus that leads to myocardial infarction. Another research Center for Molecular Medicine goal is to elucidate cell-signaling pathways that can be used to confer Karolinska Institute, Karolinska Hospital proliferative advantages to genetically modified cells. The investiga- tors in this unit are Israel F. Charo, M.D., Ph.D., and Bruce R. Joachim J. A. Herz, M.D. Professor of Molecular Genetics Conklin, M.D. University of Texas Southwestern Medical Center Clinical Molecular Genetics. Patient studies and national and inter- national population screening projects conducted by Gladstone Aldons J. Lusis, Ph.D. Professor of Medicine researchers aim to identify unique genetic abnormalities that cause and of Microbiology and Molecular Genetics hypercholesterolemia and premature myocardial infarction. University of California, Los Angeles Researchers in this unit operate the Lipid Disorders Training Center, which trains medical personnel to manage dyslipidemic patients, Karen Reue, Ph.D. and the Lipid Clinic, which provides consultation on disease man- Research Biologist West Los Angeles Veterans Administration Medical Center agement to SFGH patients and to private, referring physicians. This Associate Professor of Medicine unit also conducts the Turkish Heart Study, which investigates car- University of California, Los Angeles diovascular risk factors in a developing nation with a high incidence of heart disease. The investigators in this unit are Dr. Mahley and Donald M. Small, M.D. Thomas P. Bersot, M.D., Ph.D. Chairman, Department of Biophysics Professor of Biophysics, Medicine and Biochemistry Gladstone Genomics Core. The Genomics Core assists scientists Boston University School of Medicine with the unprecedented research opportunities presented by the decoding of the mouse and human genomes. Directed by Daniel Steinberg, M.D., Ph.D. Christopher S. Barker, Ph.D., this laboratory provides state-of-the- Professor Emeritus, Department of Medicine University of California at San Diego art technologies in the area of functional genomics for Gladstone scientists and other investigators at SFGH. The core focuses on Alan R. Tall, M.D. DNA microarray technology, including the preparation of custom Professor of Medicine oligonucleotide microarrays and customized microarray hybridiza- Columbia University College of Physicians and Surgeons tion, array scanning, and data analysis. Gladstone Transgenic Core. The GICD also maintains a sophisti- cated core facility for the generation of transgenic mice that is lence. Its productivity is documented in the more than 900 scientific heavily used by investigators of all three institutes. The core’s papers published by GICD scientists. activities are coordinated by Dr. Taylor. Research at the GICD is conducted in five areas and is supported by Gladstone Microscopy Core. The Microscopy Core, under the three core laboratories. direction of Dr. Young, provides expertise, instrumentation, service, Lipoprotein Biochemistry and Metabolism. A major focus of and training for the generation and capture of research data in the research in this area is to correlate the structure and function of the form of microscopic images and for the quantitation, analysis, and apolipoproteins involved in cholesterol transport, with particular interpretation of those images to all three institutes. emphasis on apoE. One of the structural tools that scientists in this Gladstone Institute of Virology and Immunology unit use is x-ray crystallography to determine the three-dimensional structures of proteins. The investigators in this unit are Dr. Mahley The GIVI resulted from the convergence of several factors. On the and Karl H. Weisgraber, Ph.D. forefront of the battle against AIDS since the beginning of the pan- demic, SFGH is widely recognized as one of the world’s leading Cell Biology. Studies in this unit examine how the body’s various clinical research centers for the study of HIV disease. The State of cells regulate the storage and use of cholesterol as it relates to the California provided funding to build an AIDS research center at development of atherosclerosis. The focus is on the roles of apoB, SFGH under the auspices of the UCSF School of Medicine. apoE, and class A scavenger receptors in cellular cholesterol metab- Additional funds were needed to finish and equip the center and to olism and atherogenesis. The investigators in this unit are Robert E. undertake the research. The success of the established relationship Pitas, Ph.D., and Yadong Huang, M.D., Ph.D. of UCSF and the City with the Gladstone formed the foundation for Molecular Biology. Scientists in this unit apply the latest DNA tech- a unique agreement by which the Gladstone would lease the center, niques to understand the regulation of genes important in controlling establish the research program, and manage the ongoing studies. cholesterol, triglycerides, and apolipoprotein production. Studies Gladstone and UCSF were able to attract an outstanding physician- focus on apoE and apoB, which mediate the interaction of lipopro- scientist to direct the new institute. Warner C. Greene, M.D., Ph.D., teins with cell-surface receptors. Enzymes controlling cholesteryl an internationally recognized immunologist and virologist, official- ester and triglyceride production represent a new area of research. ly took the helm in September 1991. Before coming to Gladstone, This has led to studies of adipose tissue metabolism and obesity. In Dr. Greene was professor of medicine and investigator in the addition, transgenes and homologous recombination are used to cre- Howard Hughes Medical Institute at Duke University Medical ate animal models of human diseases. The investigators in this unit Center. Currently, Dr. Greene is also a professor of medicine and of 8 Description
  9. 9. GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE microbiology and immunology at UCSF, co-director of the UCSF Center for AIDS Research, and a member of the executive commit- Gladstone Institute of Virology and Immunology tees of the UCSF AIDS Research Institute and UCSF Biomedical Sciences Graduate Program. Scientific Advisory Board Formally dedicated on April 19, 1993, the GIVI occupies 27,000 Elizabeth H. Blackburn, Ph.D. square feet of space on the top two floors of SFGH’s building 3. Professor of Biochemistry and Biophysics Studies at the GIVI are conducted under the direction of an out- University of California, San Francisco standing group of physician-scientists in five state-of-the-art labora- tories and three supporting laboratories. Robert C. Gallo, M.D. Director, Institute of Human Virology Laboratory of Molecular Immunology. The Laboratory of Professor of Medicine, Microbiology and Immunology Molecular Immunology studies the mechanisms by which proteins University of Maryland at Baltimore within the immune cells harboring HIV may act to trigger the growth of the virus and how the virus’s own proteins subsequently amplify its Beatrice H. Hahn, M.D. Professor of Medicine and Microbiology replication and pathogenic effects in primary T cells and macrophages. University of Alabama at Birmingham This work, headed by the director of the institute, Dr. Greene, specifi- cally focuses on the HIV proteins Vpr and Nef and select host factors, Edward W. Holmes, M.D. including the NF-κB/Rel family of transcription factors. Vice Chancellor for Health Sciences Dean, School of Medicine Laboratory of Molecular Evolution. The Laboratory of Molecular University of California, San Diego Evolution focuses on evolution and its implications for medicine and epidemiology. Genetic variations in host susceptibility and in micro- Stanley J. Korsmeyer, M.D. bial replication capacity, virulence, and drug susceptibility typically Sidney Farber Professor of Pathology and Professor of Medicine determine who develops disease and who remains healthy. The lab- Harvard Medical School oratory examines several consequences of molecular evolution, Director, Program in Molecular Oncology including HIV-1 drug resistance, selection pressures bearing on Department of Cancer Immunology and AIDS HIV-1 populations during transmission and in tissues, and nonpath- Dana-Farber Cancer Institute ogenic simian immunodeficiency virus infection in natural host Joseph R. Nevins, Ph.D. species. This laboratory is directed by Robert M. Grant, M.D., James B. Duke Professor and Chairman M.P.H., an assistant investigator in GIVI and assistant professor of Department of Genetics medicine at UCSF. Duke University Medical Center Laboratory of Viral Pathogenesis. The Laboratory of Viral Thomas A. Waldmann, M.D. Pathogenesis focuses on the pathogenic mechanisms of HIV in vivo, Chief, Metabolism Branch with the specific intent of finding better ways to prevent or suppress National Cancer Institute HIV-induced disease. The work falls into two areas: effects of HIV National Institutes of Health on the central hematopoietic system and transmission of HIV across mucosal and placental barriers. Research in this laboratory is direct- Robin A. Weiss, Ph.D. Professor of Viral Oncology ed by Joseph M. McCune, M.D., Ph.D., a senior investigator in GIVI Wohl Virion Centre and professor of medicine at UCSF. Windeyer Institute of Medical Sciences Laboratory of Molecular Virology. The Laboratory of Molecular University College London Virology studies how HIV transcription is controlled by host chro- matin structure and by viral proteins such as HIV Tat. More recent- ly, this laboratory has also investigated the molecular basis for HIV- Gladstone-UCSF Laboratory of Clinical Virology. The induced T-cell death, focusing on the role of apoptosis. This labora- Gladstone-UCSF Laboratory of Clinical Virology, directed by Dr. tory is directed by Eric M. Verdin, M.D., a senior investigator in Grant, provides key virological testing in support of HIV-related GIVI and professor of medicine at UCSF. clinical research projects at UCSF. Established in collaboration with Laboratory of Cellular Immunology. The Laboratory of Cellular the UCSF AIDS Research Institute, this laboratory evaluates Immunology investigates innate and adaptive cellular immune patients who are failing combination antiviral therapy, studies HIV responses against HIV and simian immunodeficiency virus at replication in the central nervous system, and investigates mecha- mucosal and systemic sites. The work focuses on understanding host nisms of primary HIV infection and sexual transmission. This labo- immune/pathogen interactions that might be manipulated by vacci- ratory is also developing state-of-the-art assays for genotypic and nation or therapeutic drugs. The laboratory is headed by Douglas F. phenotypic drug resistance and assessment of viral loads using ultra- Nixon, M.D., Ph.D., an associate investigator in GIVI and associate sensitive techniques to further enhance clinical AIDS research at professor of medicine at UCSF. SFGH and UCSF. Flow Cytometry Core Laboratory. The Flow Cytometry Core Antiviral Drug Research Division. The Antiviral Drug Research Laboratory is dedicated to providing cutting-edge techniques in Division evaluates potential new antiviral drugs. Using an animal fluorescence-based cell sorting and analysis to Gladstone and model system, the SCID-hu mouse, the group is developing new UCSF scientists. This laboratory operates both a Becton-Dickinson methods for drug evaluation and extending its work to the field of FACS Vantage and FACS DiVa for sorting and a FACScan for viral pathogenesis. The laboratory is directed by Cheryl A. Stoddart, analysis. The laboratory is directed by Martin Bigos, M.S., a staff Ph.D., a staff research scientist in GIVI. research scientist in GIVI. Description 9
  10. 10. 2002 ANNUAL REPORT Gladstone Institute of Neurological Disease The GIND resulted from the natural expansion of highly successful Gladstone Institute of Neurological Disease research programs. Its predecessor, the Gladstone Molecular Scientific Advisory Board Neurobiology Program, was created in 1996 as a joint venture of the Gladstone Institutes and the UCSF Department of Neurology. Lennart Dale E. Bredesen, M.D. Mucke, M.D., recruited to head the new program, brought with him a President and CEO group of researchers with expertise in diverse areas of disease-related Buck Institute for Age Research neuroscience. With its establishment, neuroscientists in the new pro- Professor of Neurology University of California, San Francisco gram could leverage Gladstone’s wealth of experience with apoE by applying it to the field of neurodegenerative disease research. These Gerald D. Fischbach, M.D. efforts were complemented with research on amyloid proteins, which Dean of Faculty of Medicine and for Health Sciences play a seminal role in Alzheimer’s disease. Harold and Margaret Hatch Professor Columbia University Significant findings were rapidly made in a broad range of research areas, including molecular biology, cell biology, physical structure, Dennis J. Selkoe, M.D. signal transduction, experimental pathology, and behavioral neurobi- Co-Director, Center for Neurologic Diseases ology. In 1998, the trustees expanded the program to create the GIND. Brigham and Women’s Hospital Its goal is to advance the understanding of the nervous system to the Professor of Neurology and Neuroscience Harvard University point where rational strategies can be developed for the treatment and prevention of Alzheimer’s disease, cerebrovascular disease, and other Eric M. Shooter, Ph.D. major neurological conditions. Productive synergies exist with scien- Professor of Neurobiology tists studying cardiovascular disease and AIDS in the other institutes. Stanford University As was the case with the two other institutes, Gladstone and UCSF Sidney Strickland, Ph.D. were able to attract an outstanding physician-scientist to direct the Dean of Educational Affairs new institute. Dr. Mucke was educated at the Max-Planck-Institute for Professor of Neurobiology and Genetics Biophysical Chemistry in Germany, the Massachusetts General Rockefeller University Hospital, and Harvard Medical School. He came to Gladstone from Marc Tessier-Lavigne, Ph.D. The Scripps Research Institute to first expand Gladstone’s research Susan B. Ford Professor of Humanities and Sciences efforts in disease-oriented neuroscience in the context of the Professor of Biological Sciences Molecular Neurobiology Program and then to direct the new institute. Stanford University Dr. Mucke teaches neurology and neuroscience at UCSF, where he is the first holder of the Joseph B. Martin Distinguished Professorship in Neuroscience. treatments for Alzheimer’s disease and other neurological conditions. The GIND was formally dedicated on September 11, 1998. Its labora- Investigators involved in research on this topic are Drs. Mahley, tories are housed in buildings 1, 9, and 40 of the SFGH campus. Weisgraber, Huang, Mucke, and Robert E. Pitas, Ph.D. Studies at the GIND are conducted in eight state-of-the-art laborato- Huntingtin and Other Polyglutamine-Repeat Proteins. ries and a behavioral core laboratory. The research focuses on six Huntington’s disease, the most common inherited neurodegenerative major areas relating to neurodegenerative disorders, cognitive func- disorder, is caused by an abnormally long stretch of the amino acid tion, and brain inflammation as outlined below. glutamine within the protein called huntingtin. Abnormal polygluta- Physiological and Pathophysiological Roles of Amyloidogenic mine stretches within other proteins are responsible for several other Proteins in the Brain. While amyloidogenic molecules, such as the midlife neurodegenerative disorders. Determining how abnormal amyloid β protein precursor and α-synuclein, may normally facilitate polyglutamine stretches cause neurons to die may make it possible learning and memory, they can be broken down into peptides or to develop specific therapies for these disorders. It may also reveal altered in their conformation to form neurotoxic aggregates in cells general mechanisms of neurodegeneration that are relevant to other and tissues. Understanding how these toxic proteins form and act neurological diseases. This topic is a major focus of Steven M. could facilitate the design of better treatments for Alzheimer’s disease Finkbeiner, Ph.D. and other neurodegenerative disorders. Defining the normal function Neural Plasticity. Plasticity is a property of the nervous system that of the amyloidogenic precursor molecules is of fundamental neurosci- enables it to undergo long-lasting, sometimes permanent adaptive entific interest. Investigators involved in research on this topic are Dr. responses to brief stimuli. Plasticity is believed to be important for Mucke, Robert W. Mahley, M.D., Ph.D., Yadong Huang, M.D., Ph.D., establishing precise patterns of synaptic connections during early neu- and Karl H. Weisgraber, Ph.D. ronal development and for learning and memory in adults. Role of ApoE in Neurodegeneration and Cognitive Impairment. Disturbances in plasticity and synaptic function could contribute sig- The apoE4 allele is the main known genetic risk factor for the most nificantly to memory disorders characteristic of many neurodegenera- common form of Alzheimer’s disease and for poor neurological out- tive diseases, such as Alzheimer’s disease and Huntington’s disease. come after head injury. Defining the effects of the three main human An understanding of the molecular mechanisms that regulate the for- apoE isoforms (E2, E3, and E4) on the structure and function of the mation, activity, degeneration, and regeneration of synapses and neu- brain should provide crucial insights into the contribution of the ronal dendrites could form the basis for therapeutic strategies to pre- apoE4 variant to neurological disease. Characterizing how changes in vent memory loss and cognitive decline in diverse diseases. the x-ray crystallographic three-dimensional structure of apoE affect Investigators involved in research on this topic include Drs. its activity may result in the development of novel apoE-targeted drug Finkbeiner, Mucke, and Fen-Biao Gao, Ph.D. 10 Description
  11. 11. GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE Neurobiological Function of Glial Cells and Their Role in Behavioral Core Laboratory. Because many of our mouse models Neurological Disease. Glial cells are specialized brain cells that sup- are designed to simulate aspects of human diseases resulting in mem- port the health and function of neurons. In response to brain injuries, ory deficits, behavioral disturbances, or movement disorders, the these cells produce a large number of molecules that participate in detailed behavioral characterization of these models plays an important inflammatory and immune responses. While acute glial responses may role in the assessment of their clinical relevance. Behavioral alterations help prevent neuronal damage and facilitate the removal of toxic amy- in transgenic models can shed light on the central nervous system loid proteins, abnormal activation of these cells could contribute to effects of diverse molecules and are used to assess novel therapeutic neurological disease. Genetic and pharmacological strategies are used strategies at the preclinical level. This core is engaged in collaborative to characterize the beneficial and detrimental roles of glial cells in studies with investigators at all three Gladstone Institutes, as well as cerebral amyloidosis, neurodegeneration, and HIV-associated demen- with scientists in various UCSF departments and other institutions. tia. Investigators involved in this research include Drs. Mucke and Pitas. New Gladstone Laboratories at the UCSF Mission Bay Campus C onstruction of Gladstone’s new facility at Mission Bay is on schedule. In late 2004, all three institutes will move to a new six-story building, containing about 190,000 square feet of space, adjacent to UCSF’s new basic science campus. The move is a milestone for Gladstone. “This decision was an investment in the future,” said Gladstone president Robert W. Mahley. “The move will enhance our ability to fulfill our mission of contributing to the health and well-being of all people.” The programming and schematic design phases for the facility have been completed, and the design development phase is proceeding on schedule. The San Francisco Redevelopment Agency has approved the schematic design. Utilities, lighting, trees, and sidewalks at the Model showing Gladstone’s location at Mission Bay. building site have been installed. Preparation of the site has begun, and the steel for the building has been purchased. of downtown San Francisco and the bay. One main entrance will face In conjunction with NBBJ Architects of San Francisco, Todd Sklar, the entrance to UCSF’s campus, and the other will open onto a land- Gladstone’s project development and management consultant, has scaped plaza. directed a team of more than 20 firms through a rigorous planning The new building will greatly enhance the research environment for and review process. The review process has included significant Gladstone scientists. Having all three institutes under one roof will input from the institute directors, investigators, and others. foster a sense of unity. Larger and more open laboratories will facil- The first floor will hold the administrative offices and building oper- itate communication and collaborations among the researchers. The ations, a 150-person lecture hall, and four seminar rooms. The sec- additional space will allow Gladstone to increase its staff of scientists ond floor will be left as shell space for future expansion or may be and administrators from about 300 currently to more than 500 with- developed and leased out temporarily. The third, fourth, and fifth in the next 7 years. floors will house the offices and laboratories of the three institutes. The move will also strengthen the interactions between Gladstone The plans provide for a pleasant work environment. The building’s and UCSF. The Gladstone building will be directly across the street long and relatively narrow footprint will maximize the amount of from UCSF’s new basic science campus. “We foresee a constant natural light in the offices and laboratories. Equipment areas will be exchange of ideas that will benefit researchers at both institutions,” located in the center of each floor. Stairwells at the ends of the build- said Dr. Mahley. “There will be a synergy that will promote the ing will have large windows to take advantage of the beautiful views progress of research and the advancement of our basic science.” Description 11
  12. 12. 2002 ANNUAL REPORT Michael Sechman Associates * Top: artist’s rendering of the new Gladstone laboratories at Mission Bay. Bottom: aerial view showing the location of UCSF’s new campus within the larger Mission Bay redevelopment area. Asterisk indicates approximate location of Gladstone. 12 Mission Bay
  13. 13. Members of the Gladstone Institute of Neurological Disease Director Jukka Puoliväli, Ph.D. Laboratory Associate Lennart Mucke, M.D. Juan Santiago-Garcia, Ph.D. John A. Gray Joseph B. Martin Distinguished Professor Miki Tamura, Ph.D. of Neuroscience Kanyan Xu, Ph.D. Administrative Staff Qin Xu, Ph.D. of the J. David Gladstone Institutes Investigators Shi-Ming Ye, Ph.D. Steven Finkbeiner, M.D., Ph.D. President Students Assistant Professor of Neurology Robert W. Mahley, M.D., Ph.D. and Physiology Brigitte Bogert Sarah Carter Vice President Fen-Biao Gao, Ph.D. Patrick Chang for Administrative Affairs Assistant Professor of Neurology Ruomeng Dong Jennifer Hsu Daniel M. Oshiro, M.S. and Physiology Emily Lesch Peter Li Administrative Assistants Robert W. Mahley, M.D., Ph.D. Professor of Pathology and Medicine Catherine Massaro Catharine H. Evans Siddhartha Mitra Karina G. Fantillo Robert E. Pitas, Ph.D. Sean Pintchovski Mariena D. Gardner Professor of Pathology Vikram Rao Leslie T. Manuntag Kimmy Su Marlette A. Marasigan Karl H. Weisgraber, Ph.D. Neal T. Sweeney Kelley S. Nelson Professor of Pathology Amy Tang Jennifer L. Polizzotto Brigitte Watkins Emily K. O’Keeffe Staff Research Investigator Senior Research Associates Executive Assistants Yadong Huang, M.D., Ph.D. Assistant Professor of Pathology Kay S. Arnold Brian Auerbach Maureen E. Balestra Denise Murray McPherson Staff Research Scientist Walter J. Brecht Sylvia A. Richmond Zhong-Sheng Ji, Ph.D. Christopher S. Barker, Ph.D. Yvonne M. Newhouse Communications Gui-Qiu Yu, M.S. Daniel M. Oshiro, M.S., Officer Research Scientists Susan H. Dan Robert L. Raffaï, Ph.D. Research Associates Sylvia A. Richmond Kimberly A. Scearce-Levie, Ph.D. Elizabeth S. Brooks, M.S. Teresa R. Roberts Blanca Cabezas Visiting Scientists Kenneth Cheung Editorial Andrea J. Barczak Nhue L. Do Gary C. Howard, Ph.D. Liming Dong, Ph.D. Kristina Hanspers, M.S. Stephen B. Ordway Chandi Griffin, M.S. Yanxia Hao, M.S. Graphics and Photography Dionysos Slaga Gregor Hare John C.W. Carroll, Manager Tony Wyss-Coray, Ph.D. Faith M. Harris Stephen Gonzales Brian Jones Christopher A. Goodfellow Postdoctoral Fellows Shyamal G. Kapadia John R. Hull, M.F.A. Lorenzo Arnaboldi, Ph.D. Lisa N. Kekonius Montserrat Arrasate, Ph.D. Alan Lee Public Affairs John Bradley, Ph.D. Sam Loeb Laura Lane, M.S., Manager Shengjun Chang, Ph.D. Maya Mathew Irene Cheng, Ph.D. Rene D. Miranda Finance and Accounting Jeannie Chin, Ph.D. Hilda C. Ordanza Marc E. Minardi, M.A., Officer Luke A. Esposito, Ph.D. David S. Peterson Richard S. Melenchuk, M.S., Manager Christian Essrich, Ph.D. Belma Sadikovic Emilia M. Franco Danny M. Hatters, Ph.D. Kristina P. Shockley Marga R. Guillén, M.P.A. Paul C.R. Hopkins, Ph.D. Richard M. Stewart Kai Yun W. Sun Wenjun Li, Ph.D. Maryam A. Tabar Kenneth J. Weiner Jorge J. Palop, Ph.D. Chunyao Xia Clare A. Peters-Libeu, Ph.D. Fengrong Yan Members of the Institutet 13
  14. 14. 2002 ANNUAL REPORT Grants and Contracts Intellectual Property/ Purchasing Rex F. Jones, Ph.D., Officer Technology Transfer P.J. Spangenburg, Manager Frank T. Chargualaf, M.B.A. Joan V. Bruland, J.D., Officer Tyler G. Campos Lynne M. Coulson Erin Madden P. Sidney Oduah Martin C. Rios Anne Scott, M.S. Alberto L. Reynoso Yvonne L. Young Benjamin V. Young Office of the President Human Resources Receptionist Susan H. Dan Migdalia Martinez, M.S., Officer Teresa R. Roberts Hope S. Williams John R. LeViathan, M.A., Manager Wendy M. Foster Operations Student Assistants Chad E. Popham Deborah S. Addad, Officer Shannon P. Chi Alyssa S. Uchimura Christina N. Luna Facilities Information Services Vincent J. McGovern, M.S., Manager Reginald L. Drakeford, Sr., Officer David R. Bourassa Jon W. Kilcrease, Manager Randy A. Damron Matthew L. Lyon Roger A. Shore Sarah K. Mays Joseph R. Solanoy Waldo Yee 14 Members of the Institute
  15. 15. REPORTS FROM THE LABORATORIES Finkbeiner Laboratory.........16 Gao Laboratory.........20 Huang Laboratory.........23 Mahley Laboratory.........26 Mucke Laboratory.........29 Pitas Laboratory.........33 Weisgraber Laboratory.........36 Behavioral Core Laboratory.........40 Gladstone Genomics Core.........43 Reports from the Laboratories 15
  16. 16. FINKBEINER LABORATORY Assistant Investigator Students Research Associates Steven Finkbeiner, M.D., Ph.D. Sarah Carter Elizabeth Brooks Patrick Chang Kenneth Cheung Postdoctoral Fellows Jennifer Hsu Shyamal Kapadia Montserrat Arrasate, Ph.D. Peter Li John Bradley, Ph.D. Siddhartha Mitra Administrative Assistant Sean Pintchovski Kelley Nelson Vikram Rao Brigitte Watkins Molecular Mechanisms of Plasticity and Neurodegeneration Steven Finkbeiner, M.D., Ph.D. eration in HD by manually examining immunocytochemically O ur laboratory is interested in two biological questions. First, how does the nervous system adapt to brief experiences by stained neurons transfected with wildtype or disease-associated making long-lasting changes in its structure and function? forms of huntingtin. This approach was painstaking and relatively The molecular mechanisms that underlie this process, collectively insensitive. With the microscope, we can observe neurons as they known as plasticity, are important for the proper development of the begin to express transfected huntingtin and then monitor them over nervous system and for forming memories. We are especially inter- time, observing their survival, degeneration, and eventual death. ested in the role of new gene expression in coupling transient neu- The new approach is 100-fold faster than our old assay, more sen- ronal activity to long-term changes in synaptic function. The second sitive, and less susceptible to user bias. question is how a genetic mutation leads to an adult-onset progres- We also designed the microscope and the controlling computer sive neurodegenerative disease. We focus on Huntington’s disease programs to be able to return to the same field of neurons or the (HD), the most common inherited neurodegenerative disorder in the same neuron after the tissue-culture plate has been removed from United States. Unlike Alzheimer’s disease and Parkinson’s disease, the microscope stage. In fact, although the high-throughput capa- the cause of HD is known. HD is caused by a mutation that leads to bilities of the microscope are very useful, the ability to follow cells an abnormal polyglutamine expansion in the huntingtin protein. longitudinally may be the key capability that helps us unravel Since HD shares some common pathological features with cause and effect mechanisms. By observing a neuron and the adap- Parkinson’s disease and Alzheimer’s disease, we hope that, by tive or maladaptive changes that it undergoes over time, we can understanding HD, we will understand the molecular mechanisms of reconstruct dynamic relationships that we lose by examining a sin- neurodegeneration more broadly. gle snapshot. For example, longitudinal analysis of huntingtin- Applications of Robotic Microscopy transfected neurons revealed a polyglutamine-expansion-depend- Is it a cause or an effect? That question is often difficult to address ent defect in dendrite and axon formation (Figure 1A). Initially, experimentally but it must be answered to establish biological or neurons transfected with either wildtype or mutant huntingtin pathological mechanisms and to identify appropriate therapeutic extended neurites similarly; however, neurons transfected with targets. The difficulty lies in understanding the true relationship mutant huntingtin eventually failed to extend neurites and then between the outcome of a long-term dynamic process and a partic- retracted them before the neuron died. Dendritic dystrophy and ular subsidiary change. The conundrum is especially common in axon regeneration are early pathological features of HD and so our studies of pathogenesis in which multiple changes occur in paral- ability to recapitulate another feature of HD with our simple neu- lel. Some changes are a direct consequence of an inciting event and ronal model adds to the validity of the model. We expect that lon- mediate morbidity, others are beneficial coping responses, and still gitudinal analysis will be useful to trace pathogenic pathways others are neither helpful nor harmful. The interrelatedness of backwards through time to elucidate critical inciting events. many molecular and cellular processes can also make it challeng- In the past year, we discovered that the ability to monitor the same ing to isolate one process and to manipulate it to evaluate its role. neuron or population of neurons over time permits us to use pow- Last year, we described our invention of a computer-controlled erful statistical tools that are commonly used in clinical studies, robotic microscope that performs automated imaging and analysis such as Kaplan-Meier analysis and Cox proportional hazards of living or fixed cells. The capabilities of the microscope have regression analysis. In collaboration with UCSF biostatistician Dr. fundamentally altered the way we perform many of our experi- Marc Segal, we have begun to adapt these tools to analyze data sets ments. For example, we used to study mechanisms of neurodegen- generated by the robotic microscope. These tools are powerful for 16 Reports from the Laboratories
  17. 17. GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE GFP fluorescence within individual neurons, serving as a surrogate for Akt expression within that neuron, could predict which neurons survived. We found that the levels of GFP in neurons cotransfect- ed with empty vector did not predict survival, whereas the levels of GFP in neurons cotransfected with Akt were highly predictive (Figure 1C and D). The observation is important because it demon- strates our ability to measure a variable in a living neuron that can predict its fate. The approach is especially well suited to studying neurodegenera- tive diseases. In HD, a mutation increases the risk to all neurons of initiating the biochemical processes that lead to death. However, stochastic processes appear to play a major role in determining when a particular neuron is affected and eventually dies. Because these processes may take time to unfold, specific changes may be visible in only a small fraction of neurons at one point in time. Thus, the significance and even the detection of some changes may not be recognized with conventional methods. On the other hand, the ability to track the fate of individual neurons with the robotic microscope and apply these statistical tools makes it possible to Figure 1. Applications of robotic microscopy. (A) Mutant huntingtin (Htt) induces identify highly significant and predictive relationships even if the neurite retraction. Striatal neurons were transfected with amino-terminal hunt- changes are transient or affect a small fraction of neurons at a time. ingtin fragments fused to GFP and containing either a normal (Htt-25Q-GFP) or Another reason the approach could be useful to study pathogenesis disease-associated (Htt-103Q-GFP) stretch of polyglutamines. After automated imaging approximately daily, corresponding microscope fields were examined to is its potential for identifying therapeutic targets. The approach reconstruct the pattern of neurite extension of individual neurons over the should help us distinguish factors that predict survival or dysfunc- course of the experiment. (B) Akt potently protects neurons. Cultured cortical tion and thereby identify therapeutic targets before we fully under- neurons were transfected with GFP together with either a control vector or an stand the molecular pathways that link them. active form of the Akt kinase. Transfected neurons were quantified by automat- ed imaging and analysis for 10 minutes each day, resulting in over 31,000 obser- Molecular Mechanisms of Synaptic Plasticity vations. Neurons transfected with Akt survived significantly better than those transfected with control vector. The extraordinary statistical significance It is widely held that new gene transcription must occur in neurons achieved here reflects the large number of observations and the power of for long-term memories to form. Our laboratory is interested in a Kaplan-Meier analysis. (C, D) The level of Akt expression predicts neuronal number of questions concerning the role of new gene expression in longevity. The expression of the GFP marker directly varies with the expression plasticity. What synaptic signals trigger new gene transcription? of cotransfected Akt. Therefore, we used the level of GFP fluorescence as a sur- What are the gene targets of these signals and how do the encoded rogate for Akt expression in living neurons. Expression of Akt (GFP) in a partic- ular neuron predicted how long it would survive (C). However, the expression of proteins promote synaptic plasticity? How do genes transcribed in GFP did not predict longevity in neurons transfected with empty vector (D). the nucleus strengthen only a subset of synapses made by a neuron Censored data were removed from both Akt and control graphs for clarity. that are directly involved in forming a particular memory? Previous work from our laboratory and others has identified Ca2+ influx and neurotrophins as two synaptic signals that influence two reasons. First, Kaplan-Meier analysis is extraordinarily sensi- synaptic plasticity and potently regulate neuronal gene transcription. tive, in part because it can detect important differences that emerge Ca2+ influx through either of two ion channels, the N-methyl-D- or accrue over the course of an experiment. We used this approach aspartate (NMDA) receptor or the L-type voltage-sensitive Ca2+ to evaluate the ability of a protein kinase, Akt, to promote neuronal channel (L-VSCC), appears to be particularly effective in regulating survival (Figure 1B). Longitudinal monitoring and Kaplan-Meier neuronal gene transcription. Neurotrophins are a family of peptides analysis showed that Akt promotes neuronal survival much more that are essential for the normal growth, development, and differen- potently than had been appreciated from initial reports that meas- tiation of neuronal subpopulations in the central and peripheral nerv- ured survival at single time points. The newly found potency of ous systems. One neurotrophin, brain-derived neurotrophic factor Akt makes it an especially attractive therapeutic target. (BDNF), has also been shown to regulate synaptic plasticity and Perhaps the most important reason that statistical tools such as Cox learning and memory during adulthood. proportional hazards regression analysis are so powerful is that Earlier, we had shown that Ca2+ influx through the L-VSCC direct- they enable us to identify and quantify variables that have predic- ly induces BDNF gene transcription. We hypothesized that BDNF tive value. This method provides a way to use longitudinal data and Ca2+ influx signals might cooperate more broadly to regulate (the appearance of identified changes within a particular neuron neuronal gene transcription and synaptic plasticity. The hypothesis and the knowledge of that neuron’s fate) to identify whether and to attracted us because it offered a potential biochemical mechanism to what extent a change that appears on one day predicts a particular transduce rapidly varying electrical activity (through the production fate for that neuron on another day. We have applied this method of transient Ca2+ influx signals) into longer-lasting biochemical sig- of analysis to neurons cotransfected with the gene for green fluo- nals (BDNF signaling). BDNF-induced signaling cascades generate rescent protein (GFP) and either an active form of Akt or a control very long-lasting adaptive responses, such as those envisioned for plasmid. Previously, we showed that the expression level of GFP learning and memory. correlated with the expression level of Akt in individual neurons To test this hypothesis, we introduced reporter genes into cortical even though the overall expression of these two proteins varied neurons and examined the responses to Ca2+ influx and BDNF. To from neuron to neuron. Therefore, we asked whether the amount of Reports from the Laboratories 17
  18. 18. 2002 ANNUAL REPORT our surprise, we discovered that the efficacy with which BDNF elicits neuronal gene expression is highly dependent on the level of L-VSCC activity. For example, modest plasma membrane depolar- ization significantly potentiated the ability of BDNF to induce expression of the immediate-early gene c-fos (Figure 2A). Conversely, application of any one of three structurally distinct and specific L-VSCC antagonists significantly reduced BDNF-induced c-fos expression. In contrast, application of both NMDA and BDNF led to additive gene expression responses, and coapplication of an NMDA receptor antagonist had no effect on responses to BDNF. These results suggest that Ca2+ influx signals synergistically regulate BDNF responses in a channel-specific fashion. Ca2+ influx through L-VSCCs could potentiate BDNF signals at any site from the plasma membrane (where BDNF initiates a signal) to the nucleus (where the c-fos promoter is located). The c-fos pro- moter contains two DNA elements that can each mediate responses to either Ca2+ influx or growth factor signals. One element is the Ca2+ and cyclic AMP response element (CaRE) and the other is the serum response element (SRE). We tested whether luciferase reporter genes containing only CaREs or SREs could mediate syn- ergistic responses to BDNF and L-VSCC activation. We found that only the SRE or Elk-1, one of the transcription factors that binds the SRE, showed clear evidence of synergistic activation. These results suggest that Ca2+ influx through L-VSCCs potentiates BDNF- induced gene expression in a response element–selective fashion. Since BDNF induces the phosphorylation and activation of Elk-1 through the Ras pathway, Ca2+ influx might potentiate BDNF responses by upregulating this pathway. We examined the effects of L-VSCC activation and BDNF on extracellular signal–regulated kinase (ERK), a downstream component of the Ras pathway and the major Elk-1 kinase. We found that L-VSCC activation and BDNF had additive effects on ERK activation (Figure 2B). These results suggest that the site of synergy is in the nucleus, downstream of ERK but upstream of transcription initiation. Recently, a family of factors called histone deacetylases (HDACs) has been identified. These factors regulate gene expression through effects on specific transcription factors and on chromatin structure. Deacetylation of specific histones suppresses gene expression by making the associated DNA less available to the transcription machinery. Importantly, Ca2+ influx can promote the translocation of two HDAC family members from the nucleus to the cytoplasm, thereby removing their suppressive effects. We found that pharma- Figure 2. Synaptic signals can cooperate to synergistically regulate neuronal gene cological agents known to inhibit a subset of HDACs (e.g., tricho- transcription. (A) Cortical neurons were left unstimulated or were stimulated with statin A or tripoxin) do not by themselves induce expression of a c- either modest membrane depolarization (KCl, 10 mM), BDNF (10 ng/ml), or both. Transcription of the immediate-early gene c-fos was evaluated by reverse-tran- fos-luciferase reporter gene in neurons. However, when added scriptase polymerase chain reaction. Costimulation with KCl and BDNF had a syn- together with BDNF, HDAC inhibitors significantly potentiate neu- ergistic effect on c-fos induction (the shaded portion of the bar corresponds to the ronal gene expression, similar to L-VSCC activation (Figure 2C). amount of induction that exceeds an additive response). (B) Costimulation with KCl (10 mM) and BDNF (10 ng/ml, 15 minutes) did not produce synergistic phospho- In summary, we have elucidated a new signaling pathway in neu- rylation and activation of ERK or CREB, two factors that critically determine the rons by which Ca2+ influx through L-VSCCs cooperates with activity of the c-fos promoter. Thus, the site of synergy is downstream of ERK and BDNF to regulate gene expression. We hypothesize that synergy is probably in the nucleus. The additional lane of BDNF (10 ng/ml, 45 minutes) arises from the integration of two pathways: BDNF signals that stimulation is a later time point meant to show that the failure to observe synergis- tic ERK and CREB phosphorylation earlier was not due to signal saturation. (C) activate Elk-1 bound to the SRE and L-VSCCs signals to HDACs Synergy may depend on Ca2+ influx–mediated inhibition of HDACs. Cortical neu- that derepress the chromatin structure of the SRE-containing pro- rons were transfected with a luciferase reporter gene under the control of the c-fos moter. promoter (fos-luc). The addition of the HDAC inhibitor trichostatin A (TSA, 400 nM) had almost no effect alone compared with untreated neurons. BDNF modestly Molecular Mechanisms of Huntington’s Disease induced c-fos-luciferase; however, application of TSA and BDNF synergistically Abnormal deposits of huntingtin known as intranuclear inclusions induced neuronal gene expression. Ca2+ influx induces the nuclear export of at least two HDACs and could indicate that synergy arises from the spatial coinci- are a hallmark of HD. Although our previous work showed that dence of two signals at the promoter: Ca2+-mediated HDAC inhibition and BDNF- inclusion formation was not required for huntingtin-induced degen- induced activation of critical nuclear enhancer transcription factors (e.g., Elk-1). eration, nuclear localization was nonetheless important. Reduction 18 Reports from the Laboratories
  19. 19. GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE in the nuclear accumulation of huntingtin eliminated degeneration polyglutamine expansions, we discovered that, in the absence of the in our HD neuronal model, and since we reported this finding, sev- adjacent domain, the polyglutamine expansion appeared to be more eral laboratories have reproduced the observation in other cell mod- likely to adopt a conformation recognized by our antibody and less els and in transgenic and knock-in mice. likely to aggregate. This result is interesting because cleavage of hunt- The factors that govern the subcellular localization of huntingtin are ingtin at a site within the domain that we have defined has been pro- unknown. In fact, although huntingtin was discovered almost a posed to play a pathogenic role. Our findings suggest that cleavage decade ago, its normal function remains a mystery. Therefore, we promotes pathogenesis by generating a fragment of huntingtin that can performed a large-scale screen of the huntingtin protein (3144 more readily adopt a disease-associated conformation. In the coming amino acids) for sequences that govern its subcellular localization. year, we plan to use the robotic microscope to prospectively examine To facilitate the study, the full-length protein was subdivided into whether this disease-associated conformation of huntingtin is a better smaller overlapping pieces. Each huntingtin fragment was tagged predictor of neurodegeneration than inclusion body formation. with GFP at the amino terminus and with β-galactosidase at the car- Selected References boxyl terminus. We used the GFP tag to image the location of the Saudou F, Finkbeiner S, Devys D, Greenberg ME (1998) Huntingtin attached huntingtin fragment in living neurons, and the β-galactosi- acts in the nucleus to induce apoptosis but death does not correlate dase tag was used to develop a novel and sensitive luminescence with the formation of intranuclear inclusions. Cell 95:55–66. assay for its presence in different subcellular fractions. Finkbeiner S (2000) Calcium regulation of the brain-derived neu- We found that a domain in the amino terminus of huntingtin, adjacent rotrophic factor gene. Cell. Mol. Life Sci. 57:394–401. to the polyglutamine stretch, plays a role in governing the localization of huntingtin and regulating the conformation of the polyglutamine Arrasate Iraqui M, Brooks E, Chang P, Mitra S, Finkbeiner S (2002) stretch. The presence of this domain of huntingtin promoted its move- Prospective analysis of huntingtin conformation and degeneration in ment from a subcellular fraction enriched in cytosolic proteins to one neurons. Soc. Neurosci. 29:293.12 (abstract). enriched with components of the nucleus and the endosomal system. Bradley J, Finkbeiner S (2002) An evaluation of specificity in activ- However, this region of huntingtin does not appear to mediate the ity-dependent gene expression in neurons. Prog. Neurobiol. translocation of huntingtin into the nucleus. Our imaging studies 67:469–477. showed that these fragments of huntingtin remained primarily in the Chang P, Arrasate M, Brooks L, Xia J, Truant R, Finkbeiner S (2002) cytoplasm of neurons. This domain of huntingtin contains four pro- A domain within huntingtin adjacent to the polyglutamine stretch tein-interaction domains known as HEAT repeats; however, the bind- that regulates its aggregation and subcellular localization. Soc. ing partners for these HEAT repeats have not been identified. When Neurosci. 29:293.11 (abstract). we further subdivided the domain of huntingtin, its localizing ability was lost, leading us to speculate that this domain may function as a Humbert S, Bryson EA, Cordelières FP, Connors NC, Datta SR, unit within huntingtin. Finkbeiner S, Greenberg ME, Saudou F (2002) The IGF-1/Akt path- way is neuroprotective in Huntington’s disease and involves hunt- Since this new domain of huntingtin is adjacent to the polyglutamine ingtin phosphorylation by Akt. Dev. Cell 2:831– 837. stretch, we suspected that it might affect the conformation of the polyglutamine stretch or the ability of huntingtin to aggregate into Kapadia S, Bradley J, Finkbeiner S (2002) Cooperative regulation of intranuclear inclusions. Using an antibody we developed that recog- neuronal gene expression by neurotrophic factors and calcium chan- nizes a conformation preferentially formed by disease-associated nels. Soc. Neurosci. 29:752.16 (abstract). Reports from the Laboratories 19