GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE

2002 ANNUAL REPORT

GLADSTONE INSTITUTE OF

NEUROLOGICAL DISEASE

THE J. DAVID GLADSTONE INSTITUTES

University of California, San Francisco

San Francisco General Hospital Medical Center

GLADSTONE INSTITUTEOF NEUROLOGICAL DISEASE

2002 ANNUAL REPORT

Copyright 2003by The J. David Gladstone InstitutesAll rights reserved

Editors: Gary Howard and Stephen OrdwayDesigners: John C. W. Carroll and John HullPhotographers: Stephen Gonzales and Christopher GoodfellowProject Coordinator: Sylvia A. Richmond

THE J. DAVID GLADSTONE INSTITUTES

P.O. Box 419100, San Francisco, CA 94141-9100Telephone (415) 826-7500 • Facsimile (415) 826-6541http://www.gladstone.ucsf.edu/

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

Neurodegenerative disorders rob peopleof their ability to remember, speak, write,ambulate, and control their lives. Theseconditions are on the rise because peopleare living longer, and aging stronglyincreases the risk of being afflicted bythese conditions. The enormous cost ofcaring for individuals with these condi-tions threatens our health care system. A

medical breakthrough is clearly needed, and the surest way to sucha breakthrough is to determine exactly how these diseases result inthe dysfunction and degeneration of nerve cells. In addition, neuro-logical diseases raise a range of fascinating questions that are of fun-damental scientific interest. While the investigation of neurologicaldiseases has promoted basic neuroscientific discoveries for over acentury, there has never been a more promising and exciting con-vergence of basic and disease-related neuroscience than now.

Investigators at the Gladstone Institute of Neurological Disease(GIND) have continued to examine the possibility that many, if notall, neurodegenerative disorders are caused by the accumulation ofproteins that have assumed pathogenic conformations. Although dif-ferent proteins accumulate in different neurodegenerative disorders,the ways in which their neurotoxic assemblies damage nerve cellsmay overlap. This possibility raises hope that it will be feasible todevelop treatments that can prevent, stall, or even reverse more thanone of these conditions. Other studies have focused on the molecu-lar mechanisms of neural plasticity, which is critical for the devel-opment of the nervous system as well as for its adaptations to envi-ronmental stimuli and for the remodeling of its circuitry after injury.

Scientific Discoveries

The laboratory of Dr. Steven Finkbeiner studies how a genetic muta-tion leads to Huntington’s disease and how the nervous systemadapts to brief experiences by making long-lasting changes in itsstructure and function. Huntington’s disease is a fatal inherited neu-rodegenerative disorder that is associated with increasingly disrup-tive involuntary movements and a progressive loss of motor control.It is caused by abnormal polyglutamine expansions in the proteinhuntingtin, which affect protein folding. Last year, Dr. Finkbeinerdeveloped a robotic microscope for the large-scale analysis of neu-ronal cell cultures expressing different forms of huntingtin or relat-ed proteins. This year, he used this powerful new tool to reveal thatmutant huntingtin causes a defect in the formation and maintenanceof the branches (neurites) through which brain cells communicatewith each other. These new findings further validate the cell culturemodel Dr. Finkbeiner uses in his studies, since abnormalities in neu-rite extension and regeneration are also early pathological featuresof Huntington’s disease. Using an antibody they developed, Dr.Finkbeiner and his associates also found that cleavage within a spe-cific domain of mutant huntingtin promotes pathogenesis by gener-

ating a protein fragment that readily adopts a disease-associated con-formation. Longitudinal monitoring of neurons in culture with therobotic microscope allowed the investigators to demonstrate that aprotein kinase, Akt, promotes neuronal survival much more potent-ly than had been appreciated from initial reports that measured sur-vival at single time points. The potency of Akt makes it an especial-ly attractive therapeutic target to block neurodegeneration.

The laboratory of Dr. Fen-Biao Gao focuses on the genes and molec-ular pathways that regulate the development and maintenance ofneuronal dendrites. Dendrites are tree-like extensions of neurons thatreceive signals and participate in information processing and stor-age. These structures account for more than 90% of the surface ofsome neurons. In many neurological disorders, includingAlzheimer’s disease (AD) and fragile X syndrome, the number ofdendritic branches is altered. However, little is known about themechanisms that control dendritic branching in vivo. Last year, Dr.Gao’s laboratory selectively mutated genes in individual neuronsand studied the consequences of this manipulation on dendriticbranching in living fruit flies. This year, the investigators used thisapproach to study the pathogenesis of fragile X syndrome, the mostcommon form of inherited mental retardation in humans. The syn-drome is caused by mutations in the fragile X mental retardation 1(fmr1) gene. Their studies revealed that a close homologue of thefmr1 gene is highly expressed in the fruit fly nervous system, wherethe protein is localized to both dendrites and axons of specific neu-rons. Studies are under way to determine whether and how muta-tions in the fly gene alter the function and integrity of these neuronalbranches. Dr. Gao and his associates also set up automated behav-ioral tests to quantify the functional consequences of disrupting geneexpression in the fruit fly nervous system. Using such tests, they dis-covered that mutations in the presenilin gene alter the linear loco-motion of fly larvae. Presenilin is a major drug target in AD researchbecause it is necessary for the production of the neurotoxic amyloid-β peptides (Aβ), which are presumed to cause AD. Dr. Gao’s furtheranalysis of presenilin in the fruit fly may identify ways to inhibit itsAD-promoting activities while preserving its beneficial functions.

The laboratory of Dr. Yadong Huang investigates the lipid carrierapolipoprotein (apo) E and its relationship to AD. The three majorhuman isoforms of apoE differentially affect the risk of developingAD (E4 > E3 > E2). Roughly 10 years after the discovery of thislink, apoE4 remains the main known inherited risk factor for themost frequent form of AD. Yet, it is still uncertain how exactly itincreases the risk and accelerates the onset of this illness. Last year,Dr. Huang and his collaborators demonstrated that apoE4 has agreater proclivity to be broken down into fragments than apoE3,both in people with AD and in genetically modified (transgenic)mice expressing human apoE in the brain. The accumulation ofapoE4 fragments was associated with abnormal phosphorylation oftau and cytoskeletal derangements. Abnormally phosphorylated tauis the major constituent of neurofibrillary tangles, a pathological

Director’s Report4

LENNART MUCKE, M.D.

DIRECTOR’S REPORT

hallmark of AD. This year, Dr. Huang demonstrated that theincreased fragmentation of apoE is dependent, at least in part, on anintramolecular domain interaction that occurs in apoE4 but not inapoE3. Furthermore, when expressed by themselves in the brains oftransgenic mice, the apoE fragments elicited neurodegenerativealterations in various brain regions, including regions involved inlearning and memory. Inhibitors of the protease presumed to cleaveapoE within neurons might block the adverse effects of apoE4 andthereby benefit the many apoE4 carriers suffering from or at risk fordeveloping AD. Therefore, the isolation and further characterizationof this enzyme is an important goal of ongoing studies.

The laboratory of Dr. Robert W. Mahley maintains its long-termfocus on the mechanisms underlying the pathogenic activities ofapoE4 in AD and other conditions. ApoE3 promotes neurite out-growth, protects the nervous system against diverse injuries, andfacilitates regeneration of the nervous system after trauma. In con-trast, apoE4 is not protective and, in many instances, even worsensthe outcome of neural injuries. This year, Dr. Mahley and his asso-ciates extended their investigation of the most common apoE iso-forms to the production of Aβ peptides. They examined the effectsof apoE3 and apoE4 on the processing of the amyloid precursorprotein (APP) and on Aβ production in cultures of neuronal cells.Both apoE isoforms stimulated Aβ production, but apoE4 did so toa significantly greater extent than apoE3. Interestingly, this differ-ential effect of the apoE isoforms was mediated by stimulating cell-surface APP recycling rather than by altering cellular cholesterolcontent or activities of enzymes involved in Aβ production. Whenthe investigators disrupted the intramolecular domain interactionthat occurs in apoE4, but not in apoE3, the differential effect ofapoE3 and apoE4 on Aβ production was attenuated, suggesting thatthis effect involves conformational differences among the apoEvariants. Since Aβ plays a central role in the pathogenesis of AD,the stronger stimulatory effect of apoE4 on Aβ production couldcontribute to the increased AD risk associated with inheritance ofthis apoE isoform. In addition, apoE4 may increase AD risk throughAβ-independent mechanisms, as demonstrated in collaborativestudies with Dr. Huang (see above).

My own laboratory has continued to investigate the molecular path-ways that link genetic determinants or risk factors of AD to neu-rodegeneration and cognitive decline. We have demonstrated intransgenic mouse models that human Aβ causes reductions in calci-um-dependent proteins in granule cells of the dentate gyrus, a neu-ronal population critically involved in learning and memory. Ourdata suggest that the reductions in calcium-dependent proteins arecaused by small soluble neurotoxic Aβ assemblies rather than by thelarge deposits of Aβ that make up amyloid plaques. Notably, plaqueburden remains the most widely used pathological endpoint measurein the preclinical assessment of AD treatments even though it oftendoes not correlate well with cognitive deficits. Since the molecularalterations we identified correlated extremely well with learning

deficits in our mouse models, they constitute reliable molecular indi-cators of clinically relevant deficits and, hence, could improve theassessment of novel AD treatments. We also found marked reduc-tions in calcium-dependent proteins in granule cells of humans withAD, although this neuronal population is relatively resistant to AD-related cell death. This result demonstrates that neuronal populationsresisting cell death in AD can still be drastically altered at the molec-ular level. They also suggest that at least some of the neurologicaldeficits seen in AD may not reflect loss of brain cells. This conclu-sion has important implications because lost neurons are still hard orimpossible to replace, whereas molecular alterations in survivingbrain cells may be more readily reversible by pharmacological inter-ventions. We look forward to exploring this possibility further in thenext year.

The laboratory of Dr. Robert E. Pitas has followed up on their dis-covery of apoE-binding protein (EBP), a novel brain protein thatbelongs to a previously uncharacterized family of proteins withunknown function. The proteins are encoded by four homologousgenes in humans and mice and by one related gene in the fruit fly.The association of EBP with apoE3, initially identified by the yeasttwo-hybrid approach, has now been confirmed in other biochemicalassays. Dr. Pitas and his associates have also produced evidence thatEBP interacts with the cytoskeleton and have developed polyclonalantibodies that detect both human and mouse EBP. Staining ofmouse brain sections with these antibodies revealed neuron-specificexpression of EBP in many regions, including the hippocampus anddentate gyrus. Ablating the expression of apoE or expressing humanAPP and Aβ in neurons altered the expression pattern of EBP inmouse brains, suggesting that the expression of EBP is affected byfactors critically involved in the pathogenesis of AD. Interestingly,in cell cultures, EBP protected neuronal cells against oxidativestress. This neuroprotective function of EBP may relate to its inter-action with apoE, which also appears to fulfill neuroprotective func-tions. Studies are under way to ablate EBP expression in fruit fliesand mice to determine the physiological functions of EBP and theroles it might play in neurological disease.

The laboratory of Dr. Karl H. Weisgraber investigates the relation-ship between the three-dimensional structure of different apoE mol-ecules and their normal and pathological activities. While much isknown about the structure of lipid-free apoE, very little is knownabout apoE’s structure when it is bound to lipids, although apoEprobably exists in the body primarily in a lipid-bound (lipidated)state. This year, Dr. Weisgraber and his coworkers have made greatstrides in this area by producing crystals of lipidated apoE whosestructure can now be investigated by crystallography and other bio-physical approaches. In other studies, the investigators discoveredthat apoE4 is more likely than apoE3 to assume a particular foldingstate (molten globule) that could promote its binding to and translo-cation across membranes. By assuming this folding state, apoE4might escape from vesicular compartments within the cell and gain

Director’s Report 5

GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE

access to the cytoskeleton and to enzymes and other molecules in thecytoplasm. Collaborative studies are under way to determinewhether this process contributes to the cytoskeletal destabilizationand susceptibility to proteolytic cleavage that Drs. Huang andMahley have found to be associated with apoE4.

Education, Special Initiatives, and Recognition

The Gladstone Institutes and UCSF provide state-of-the-art researchfacilities and a highly interactive academic environment that is idealfor training in neuroscience and biomedical research. GIND investi-gators participate actively in the training of students and residentsfrom various UCSF departments and interdepartmental programs,including the Departments of Neurology and Physiology, theNeuroscience Program, the Biomedical Sciences Program, thePharmaceutical Sciences and Pharmacogenomics Program, and theMedical Scientist Training Program, as well as from graduate andundergraduate programs at UC Berkeley and other institutions.

Several members of our institute have collaborated to make ourtraining environment even more inspiring and rewarding for stu-dents of all biomedical disciplines. For example, Dr. Finkbeiner hasdeveloped a curriculum for the neurology house staff called theNeuroscience of Disease Seminar series. He has also served as theneuroscience liaison for UCSF’s Brain Interest Program, whichaims to foster interest in the brain and provides an opportunity forstudents to learn about different faculty, their careers, and theirinterests. Also noteworthy in this context is the continued successof our weekly GIND seminar series, which is organized by Dr. Gao.These well-attended seminars provide a stimulating forum for edu-cation in disease-related neuroscience and for scientific exchangeamong members of the institutes and colleagues from the greaterUCSF community.

GIND investigators have extended their efforts to promote educationand scientific exchange in disease-related neuroscience far beyondthe boundaries of our institute. They have organized and participat-ed in a number of national and international conferences that haveadvanced our field of research in various ways. Despite their busyschedules and expanding research efforts, GIND members have con-tinued to devote time to community outreach. As described in theEducation and Community Outreach section of this report, theseefforts included participation in activities aimed at educating thepublic about AD and neuroscientific research in general. Our syner-gism with the UCSF Memory and Aging Center, the local chapter ofthe Alzheimer’s Association, and the Hereditary Disease Foundationhas allowed us to maintain and expand fruitful links between ourresearch and the patients afflicted by the diseases we study.

I am delighted that our accomplishments have not gone unnoticed,as reflected by the honors and awards institute members receivedthis year. Dr. Gao received a McKnight Neuroscience of BrainDisorders Award from the McKnight Endowment Fund forNeurosciences, one of six awards to scientists whose research isdirected toward finding new ways to diagnose, treat, and cure disor-ders of the brain and central nervous system. He also received a

Klingenstein Fellowship Award in Neuroscience from theKlingenstein Foundation, which supports new approaches to neuro-scientific research. My own research efforts on AD were recognizedby a Metropolitan Life Foundation Award for Medical Research anda MERIT award from the National Institute on Aging. Any awardreflects the participation of many, and I consider all members of mylaboratory and my collaborators important contributors. I thank themfor their tremendous loyalty and support. We all share these awardstogether.

Dr. Tony Wyss-Coray, a member of the GIND since its inauguration,has been recruited to Stanford University, where he now holds anappointment as assistant professor in the Department of Neurologyand Neurological Sciences. The staff research investigator positionhe held at the GIND aims to give exceptional young scientists theopportunity to focus solely on developing a successful and creativeresearch program before accepting a standard faculty position. Dr.Wyss-Coray made the most of this opportunity and left the institutewell published and supported by a substantial amount of independ-ent NIH funding. We wish him and his terrific group the very best inall their future endeavors and look forward to welcoming the nextoutstanding candidate into the position he vacated. We are equallyexcited about the recruitment of a new faculty member to the GIND.The search for suitable candidates was launched in the fall and willbe conducted by an eight-member joint UCSF/Gladstone committee.This faculty recruitment represents the first step toward expandingthe GIND as we prepare for the relocation of our research programto UCSF’s new Mission Bay campus.

Gladstone investigators have continued to participate actively infinalizing the design of the new research facility that will accommo-date all three Gladstone Institutes at Mission Bay. The GIND willoccupy the third floor of the new Gladstone building, which isscheduled for completion in 2004. The consolidation of our labora-tories, cores, and animal care facilities into one building at the newcampus will further enhance our ability to contribute to research andtraining in neurology and neuroscience.

In closing, I would like to thank the participants of this year’sScientific Advisory Board meeting, Drs. Dale E. Bredesen (BuckInstitute for Age Research), Dennis Selkoe (Harvard MedicalSchool), and Eric Shooter (Stanford University) for their outstand-ing input. It contributed greatly to our success. The progress wemade in 2002 reflects the work of all the researchers at the GINDand of the administrative staff at the Gladstone Institutes. As out-lined in this report, we have shed new light on molecular processescausing devastating neurological diseases as well as on mechanismsthat might be utilized to prevent and cure them.

Lennart Mucke, M.D.Director

Director’s Report6

2002 ANNUAL REPORT

Description 7

The J. David Gladstone Institutes is the product of the wisdom andhard work of many individuals. The first was J. David Gladstone him-self, a Los Angeles real-estate entrepreneur. Others are the trustees.The original trustees, all of whom had known or worked closely withMr. Gladstone, were Richard S. Brawerman, his attorney and execu-tor of his estate; Richard D. Jones, his real-estate attorney; and DavidOrgell, his cousin and confidant. When Mr. Orgell died in 1987, hewas succeeded on the board by Albert A. Dorman, a southernCalifornia executive with experience in managing large organizations.

At the time of Mr. Gladstone’s death in 1971, the southern Californiareal-estate market was just beginning to flourish. His estate, left almostentirely for medical education and research, was relatively modest bylater standards. However, the trustees recognized the estate’s potentialfor growth and, through their inspired management, increased its worthseveralfold within the first decade. From the beginning, the decisionsof the trustees had profound and positive effects on the research organ-ization that evolved. They continue to manage and enlarge the assets ofthe J. David Gladstone Institutes and to oversee their use.

Gladstone Institute of Cardiovascular Disease

Close ties already existed between the UCSF School of Medicine andSFGH, the hospital of the City and County of San Francisco, when thetrustees leased vacant space from the City in 1977 in which to createlaboratories and offices. The partnership has flourished. Gladstonescientists collaborate with their colleagues at UCSF and SFGH andprovide service to those organizations as professors and staff physi-cians. The mutually beneficial association between the Gladstone,UCSF, and SFGH has created a productive and supportive environ-ment in which scientists conduct basic research while availing them-selves of clinical and academic opportunities.

To choose a director for the developing research facility, the trusteessought guidance from the scientific community. The choice wasRobert W. Mahley, M.D., Ph.D. At the time of Mr. Gladstone’s death,he was just completing his internship. However, by 1979, when hewas appointed director, Dr. Mahley had established himself as a lead-ing researcher in the field of lipoprotein metabolism and atherosclero-sis. He came to the Gladstone from the National Institutes of Health,where he headed the Laboratory of Experimental Atherosclerosis.Less than a year after his appointment, Dr. Mahley had assembled astaff of 25, and the new organization, then called the GladstoneFoundation Laboratories for Cardiovascular Disease, officiallyopened on September 1, 1979. Dr. Mahley is professor of pathologyand medicine at UCSF and is a member of the National Academy ofSciences and the Institute of Medicine.

By the end of 2002, the research staff of the GICD had grown to morethan 100 scientists, postdoctoral fellows, students, and research asso-ciates, occupying about 48,000 square feet of laboratory and officespace in buildings 9 and 40 on the SFGH campus. In 22 years of oper-ation, the institute has attained an international reputation for excel-

Primary research efforts at the J. David Gladstone Institutesfocus on three of the most important clinical problems of mod-ern times: cardiovascular disease, AIDS, and neurodegenera-

tive disorders. Cardiovascular disease, the nation’s leading killer,claims the lives of over one million Americans each year. Despitemore effective treatments, AIDS remains a leading cause of death inthe United States. Worldwide, more than 42 million people are livingwith HIV/AIDS, and more than 21 million have died as a direct resultof HIV infection. Alzheimer’s disease, the most recent focus of inves-tigation by Gladstone scientists, robs people of their ability to think,remember, and control their lives. It currently affects over 4 millionpeople in the United States alone and is predicted to affect 14 millionAmericans by 2050. The impact of these diseases on world healthinfuses Gladstone scientists with a sense of purpose and urgency.

Gladstone is composed of three institutes, each of which issues its ownannual report. The Gladstone Institute of Cardiovascular Disease(GICD), which opened in 1979, focuses on atherosclerosis and itscomplications. In 1992, the Gladstone Institute of Virology andImmunology (GIVI) was established to study HIV, the causative agentof AIDS. The 1993 discovery that apolipoprotein (apo) E—long stud-ied at GICD for its role in heart disease—plays a role in Alzheimer’sdisease as well led to the establishment of the Gladstone Institute ofNeurological Disease (GIND) in 1998. The three institutes are locatedat the San Francisco General Hospital (SFGH) campus of theUniversity of California, San Francisco (UCSF), and will soon moveinto a new facility at UCSF’s Mission Bay campus. While independ-ent, Gladstone is formally affiliated with UCSF, and Gladstone inves-tigators hold university appointments and participate in many univer-sity activities, including the teaching and training of graduate students.

Although autonomous in their areas of specialization, the institutesshare a common approach. Each institute is organized into researchunits consisting of scientists, postdoctoral researchers, research asso-ciates, and students. This structure is designed to accommodate smallgroups of scientists who work together closely but who also benefitfrom collegial interactions with other research groups. Collaborationsamong staff members with various areas of expertise create a stimu-lating environment that fortifies the scientific lifeblood of the organ-ization.

Each institute receives expert input on the progress of its science froman advisory board of distinguished scientists. The scientific advisoryboards provide a twofold service in reviewing the quality of theresearch and in advising the president, directors, and trustees.

The work of the scientific staff at all three institutes also extendsbeyond the laboratory to the wider community. The mission of theinstitutes includes the education of graduate and medical students,postdoctoral fellows, and visiting scientists; specialized training forpracticing physicians; and educational outreach to the local andextended community.

Description ofTHE J. DAVID GLADSTONEINSTITUTES

Executive DirectorRichard Hille

Chief Financial OfficerHal Orr, C.M.A.

TrusteesRichard S. BrawermanAlbert A. DormanRichard D. Jones

PresidentRobert W. Mahley, M.D., Ph.D.

Description8

2002 ANNUAL REPORT

lence. Its productivity is documented in the more than 900 scientificpapers published by GICD scientists.

Research at the GICD is conducted in five areas and is supported bythree core laboratories.

Lipoprotein Biochemistry and Metabolism. A major focus ofresearch in this area is to correlate the structure and function of theapolipoproteins involved in cholesterol transport, with particularemphasis on apoE. One of the structural tools that scientists in thisunit use is x-ray crystallography to determine the three-dimensionalstructures of proteins. The investigators in this unit are Dr. Mahleyand Karl H. Weisgraber, Ph.D.

Cell Biology. Studies in this unit examine how the body’s variouscells regulate the storage and use of cholesterol as it relates to thedevelopment of atherosclerosis. The focus is on the roles of apoB,apoE, and class A scavenger receptors in cellular cholesterol metab-olism and atherogenesis. The investigators in this unit are Robert E.Pitas, Ph.D., and Yadong Huang, M.D., Ph.D.

Molecular Biology. Scientists in this unit apply the latest DNA tech-niques to understand the regulation of genes important in controllingcholesterol, triglycerides, and apolipoprotein production. Studiesfocus on apoE and apoB, which mediate the interaction of lipopro-teins with cell-surface receptors. Enzymes controlling cholesterylester and triglyceride production represent a new area of research.This has led to studies of adipose tissue metabolism and obesity. Inaddition, transgenes and homologous recombination are used to cre-ate animal models of human diseases. The investigators in this unit

Gladstone Institute of Cardiovascular Disease

Scientific Advisory Board

Göran K. Hansson, M.D., Ph.D.Professor of Cardiovascular Research Center for Molecular MedicineKarolinska Institute, Karolinska Hospital

Joachim J. A. Herz, M.D.Professor of Molecular GeneticsUniversity of Texas Southwestern Medical Center

Aldons J. Lusis, Ph.D.Professor of Medicineand of Microbiology and Molecular GeneticsUniversity of California, Los Angeles

Karen Reue, Ph.D.Research BiologistWest Los Angeles Veterans Administration Medical CenterAssociate Professor of MedicineUniversity of California, Los Angeles

Donald M. Small, M.D.Chairman, Department of BiophysicsProfessor of Biophysics, Medicine and BiochemistryBoston University School of Medicine

Daniel Steinberg, M.D., Ph.D.Professor Emeritus, Department of MedicineUniversity of California at San Diego

Alan R. Tall, M.D.Professor of MedicineColumbia University College of Physicians and Surgeons

are John M. Taylor, Ph.D., Stephen G. Young, M.D., and Robert V.Farese, Jr., M.D.

Vascular Biology. This research aims to elucidate how mono-cytes/macrophages are attracted to sites of atherosclerotic lesionformation and to delineate the role of platelets in forming the occlu-sive thrombus that leads to myocardial infarction. Another researchgoal is to elucidate cell-signaling pathways that can be used to conferproliferative advantages to genetically modified cells. The investiga-tors in this unit are Israel F. Charo, M.D., Ph.D., and Bruce R.Conklin, M.D.

Clinical Molecular Genetics. Patient studies and national and inter-national population screening projects conducted by Gladstoneresearchers aim to identify unique genetic abnormalities that causehypercholesterolemia and premature myocardial infarction.Researchers in this unit operate the Lipid Disorders Training Center,which trains medical personnel to manage dyslipidemic patients,and the Lipid Clinic, which provides consultation on disease man-agement to SFGH patients and to private, referring physicians. Thisunit also conducts the Turkish Heart Study, which investigates car-diovascular risk factors in a developing nation with a high incidenceof heart disease. The investigators in this unit are Dr. Mahley andThomas P. Bersot, M.D., Ph.D.

Gladstone Genomics Core. The Genomics Core assists scientistswith the unprecedented research opportunities presented by thedecoding of the mouse and human genomes. Directed byChristopher S. Barker, Ph.D., this laboratory provides state-of-the-art technologies in the area of functional genomics for Gladstonescientists and other investigators at SFGH. The core focuses onDNA microarray technology, including the preparation of customoligonucleotide microarrays and customized microarray hybridiza-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 isheavily used by investigators of all three institutes. The core’sactivities are coordinated by Dr. Taylor.

Gladstone Microscopy Core. The Microscopy Core, under thedirection of Dr. Young, provides expertise, instrumentation, service,and training for the generation and capture of research data in theform of microscopic images and for the quantitation, analysis, andinterpretation of those images to all three institutes.

Gladstone Institute of Virology and Immunology

The GIVI resulted from the convergence of several factors. On theforefront of the battle against AIDS since the beginning of the pan-demic, SFGH is widely recognized as one of the world’s leadingclinical research centers for the study of HIV disease. The State ofCalifornia provided funding to build an AIDS research center atSFGH under the auspices of the UCSF School of Medicine.Additional funds were needed to finish and equip the center and toundertake the research. The success of the established relationshipof UCSF and the City with the Gladstone formed the foundation fora unique agreement by which the Gladstone would lease the center,establish the research program, and manage the ongoing studies.

Gladstone and UCSF were able to attract an outstanding physician-scientist to direct the new institute. Warner C. Greene, M.D., Ph.D.,an internationally recognized immunologist and virologist, official-ly took the helm in September 1991. Before coming to Gladstone,Dr. Greene was professor of medicine and investigator in theHoward Hughes Medical Institute at Duke University MedicalCenter. Currently, Dr. Greene is also a professor of medicine and of

Description 9


Gladstone-UCSF Laboratory of Clinical Virology. TheGladstone-UCSF Laboratory of Clinical Virology, directed by Dr.Grant, provides key virological testing in support of HIV-relatedclinical research projects at UCSF. Established in collaboration withthe UCSF AIDS Research Institute, this laboratory evaluatespatients who are failing combination antiviral therapy, studies HIVreplication in the central nervous system, and investigates mecha-nisms of primary HIV infection and sexual transmission. This labo-ratory is also developing state-of-the-art assays for genotypic andphenotypic drug resistance and assessment of viral loads using ultra-sensitive techniques to further enhance clinical AIDS research atSFGH and UCSF.

Antiviral Drug Research Division. The Antiviral Drug ResearchDivision evaluates potential new antiviral drugs. Using an animalmodel system, the SCID-hu mouse, the group is developing newmethods for drug evaluation and extending its work to the field ofviral pathogenesis. The laboratory is directed by Cheryl A. Stoddart,Ph.D., a staff research scientist in GIVI.

microbiology and immunology at UCSF, co-director of the UCSFCenter for AIDS Research, and a member of the executive commit-tees of the UCSF AIDS Research Institute and UCSF BiomedicalSciences Graduate Program.

Formally dedicated on April 19, 1993, the GIVI occupies 27,000square feet of space on the top two floors of SFGH’s building 3.Studies at the GIVI are conducted under the direction of an out-standing group of physician-scientists in five state-of-the-art labora-tories and three supporting laboratories.

Laboratory of Molecular Immunology. The Laboratory ofMolecular Immunology studies the mechanisms by which proteinswithin the immune cells harboring HIV may act to trigger the growthof the virus and how the virus’s own proteins subsequently amplify itsreplication and pathogenic effects in primary T cells and macrophages.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,including the NF-κB/Rel family of transcription factors.

Laboratory of Molecular Evolution. The Laboratory of MolecularEvolution focuses on evolution and its implications for medicine andepidemiology. Genetic variations in host susceptibility and in micro-bial replication capacity, virulence, and drug susceptibility typicallydetermine who develops disease and who remains healthy. The lab-oratory examines several consequences of molecular evolution,including HIV-1 drug resistance, selection pressures bearing onHIV-1 populations during transmission and in tissues, and nonpath-ogenic simian immunodeficiency virus infection in natural hostspecies. This laboratory is directed by Robert M. Grant, M.D.,M.P.H., an assistant investigator in GIVI and assistant professor ofmedicine at UCSF.

Laboratory of Viral Pathogenesis. The Laboratory of ViralPathogenesis focuses on the pathogenic mechanisms of HIV in vivo,with the specific intent of finding better ways to prevent or suppressHIV-induced disease. The work falls into two areas: effects of HIVon the central hematopoietic system and transmission of HIV acrossmucosal and placental barriers. Research in this laboratory is direct-ed by Joseph M. McCune, M.D., Ph.D., a senior investigator in GIVIand professor of medicine at UCSF.

Laboratory of Molecular Virology. The Laboratory of MolecularVirology 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-induced T-cell death, focusing on the role of apoptosis. This labora-tory is directed by Eric M. Verdin, M.D., a senior investigator inGIVI and professor of medicine at UCSF.

Laboratory of Cellular Immunology. The Laboratory of CellularImmunology investigates innate and adaptive cellular immuneresponses against HIV and simian immunodeficiency virus atmucosal and systemic sites. The work focuses on understanding hostimmune/pathogen interactions that might be manipulated by vacci-nation or therapeutic drugs. The laboratory is headed by Douglas F.Nixon, M.D., Ph.D., an associate investigator in GIVI and associateprofessor of medicine at UCSF.

Flow Cytometry Core Laboratory. The Flow Cytometry CoreLaboratory is dedicated to providing cutting-edge techniques influorescence-based cell sorting and analysis to Gladstone andUCSF scientists. This laboratory operates both a Becton-DickinsonFACS Vantage and FACS DiVa for sorting and a FACScan foranalysis. The laboratory is directed by Martin Bigos, M.S., a staffresearch scientist in GIVI.

Gladstone Institute of Virology and Immunology


Elizabeth H. Blackburn, Ph.D.Professor of Biochemistry and BiophysicsUniversity of California, San Francisco

Robert C. Gallo, M.D.Director, Institute of Human VirologyProfessor of Medicine, Microbiology and ImmunologyUniversity of Maryland at Baltimore

Beatrice H. Hahn, M.D.Professor of Medicine and MicrobiologyUniversity of Alabama at Birmingham

Edward W. Holmes, M.D.Vice Chancellor for Health SciencesDean, School of MedicineUniversity of California, San Diego

Stanley J. Korsmeyer, M.D.Sidney Farber Professor of Pathologyand Professor of MedicineHarvard Medical SchoolDirector, Program in Molecular OncologyDepartment of Cancer Immunology and AIDSDana-Farber Cancer Institute

Joseph R. Nevins, Ph.D.James B. Duke Professor and ChairmanDepartment of GeneticsDuke University Medical Center

Thomas A. Waldmann, M.D.Chief, Metabolism BranchNational Cancer InstituteNational Institutes of Health

Robin A. Weiss, Ph.D.Professor of Viral OncologyWohl Virion CentreWindeyer Institute of Medical SciencesUniversity College London

Description10

2002 ANNUAL REPORT

Gladstone Institute of Neurological Disease

The GIND resulted from the natural expansion of highly successfulresearch programs. Its predecessor, the Gladstone MolecularNeurobiology Program, was created in 1996 as a joint venture of theGladstone Institutes and the UCSF Department of Neurology. LennartMucke, M.D., recruited to head the new program, brought with him agroup of researchers with expertise in diverse areas of disease-relatedneuroscience. With its establishment, neuroscientists in the new pro-gram could leverage Gladstone’s wealth of experience with apoE byapplying it to the field of neurodegenerative disease research. Theseefforts were complemented with research on amyloid proteins, whichplay a seminal role in Alzheimer’s disease.

Significant findings were rapidly made in a broad range of researchareas, including molecular biology, cell biology, physical structure,signal transduction, experimental pathology, and behavioral neurobi-ology. In 1998, the trustees expanded the program to create the GIND.Its goal is to advance the understanding of the nervous system to thepoint where rational strategies can be developed for the treatment andprevention of Alzheimer’s disease, cerebrovascular disease, and othermajor neurological conditions. Productive synergies exist with scien-tists studying cardiovascular disease and AIDS in the other institutes.

As was the case with the two other institutes, Gladstone and UCSFwere able to attract an outstanding physician-scientist to direct thenew institute. Dr. Mucke was educated at the Max-Planck-Institute forBiophysical Chemistry in Germany, the Massachusetts GeneralHospital, and Harvard Medical School. He came to Gladstone fromThe Scripps Research Institute to first expand Gladstone’s researchefforts in disease-oriented neuroscience in the context of theMolecular Neurobiology Program and then to direct the new institute.Dr. Mucke teaches neurology and neuroscience at UCSF, where he isthe first holder of the Joseph B. Martin Distinguished Professorship inNeuroscience.

The GIND was formally dedicated on September 11, 1998. Its labora-tories are housed in buildings 1, 9, and 40 of the SFGH campus.Studies at the GIND are conducted in eight state-of-the-art laborato-ries and a behavioral core laboratory. The research focuses on sixmajor areas relating to neurodegenerative disorders, cognitive func-tion, and brain inflammation as outlined below.

Physiological and Pathophysiological Roles of AmyloidogenicProteins in the Brain. While amyloidogenic molecules, such as theamyloid β protein precursor and α-synuclein, may normally facilitatelearning and memory, they can be broken down into peptides oraltered in their conformation to form neurotoxic aggregates in cellsand tissues. Understanding how these toxic proteins form and actcould facilitate the design of better treatments for Alzheimer’s diseaseand other neurodegenerative disorders. Defining the normal functionof the amyloidogenic precursor molecules is of fundamental neurosci-entific interest. Investigators involved in research on this topic are Dr.Mucke, Robert W. Mahley, M.D., Ph.D., Yadong Huang, M.D., Ph.D.,and Karl H. Weisgraber, Ph.D.

Role of ApoE in Neurodegeneration and Cognitive Impairment.The apoE4 allele is the main known genetic risk factor for the mostcommon form of Alzheimer’s disease and for poor neurological out-come after head injury. Defining the effects of the three main humanapoE isoforms (E2, E3, and E4) on the structure and function of thebrain should provide crucial insights into the contribution of theapoE4 variant to neurological disease. Characterizing how changes inthe x-ray crystallographic three-dimensional structure of apoE affectits activity may result in the development of novel apoE-targeted drug

Gladstone Institute of Neurological Disease


Dale E. Bredesen, M.D.President and CEOBuck Institute for Age ResearchProfessor of NeurologyUniversity of California, San Francisco

Gerald D. Fischbach, M.D.Dean of Faculty of Medicine and for Health SciencesHarold and Margaret Hatch ProfessorColumbia University

Dennis J. Selkoe, M.D.Co-Director, Center for Neurologic DiseasesBrigham and Women’s HospitalProfessor of Neurology and NeuroscienceHarvard University

Eric M. Shooter, Ph.D.Professor of NeurobiologyStanford University

Sidney Strickland, Ph.D.Dean of Educational AffairsProfessor of Neurobiology and GeneticsRockefeller University

Marc Tessier-Lavigne, Ph.D.Susan B. Ford Professor of Humanities and SciencesProfessor of Biological SciencesStanford University

treatments for Alzheimer’s disease and other neurological conditions.Investigators involved in research on this topic are Drs. Mahley,Weisgraber, Huang, Mucke, and Robert E. Pitas, Ph.D.

Huntingtin and Other Polyglutamine-Repeat Proteins.Huntington’s disease, the most common inherited neurodegenerativedisorder, is caused by an abnormally long stretch of the amino acidglutamine within the protein called huntingtin. Abnormal polygluta-mine stretches within other proteins are responsible for several othermidlife neurodegenerative disorders. Determining how abnormalpolyglutamine stretches cause neurons to die may make it possibleto develop specific therapies for these disorders. It may also revealgeneral mechanisms of neurodegeneration that are relevant to otherneurological diseases. This topic is a major focus of Steven M.Finkbeiner, Ph.D.

Neural Plasticity. Plasticity is a property of the nervous system thatenables it to undergo long-lasting, sometimes permanent adaptiveresponses to brief stimuli. Plasticity is believed to be important forestablishing precise patterns of synaptic connections during early neu-ronal development and for learning and memory in adults.Disturbances in plasticity and synaptic function could contribute sig-nificantly to memory disorders characteristic of many neurodegenera-tive diseases, such as Alzheimer’s disease and Huntington’s disease.An understanding of the molecular mechanisms that regulate the for-mation, activity, degeneration, and regeneration of synapses and neu-ronal dendrites could form the basis for therapeutic strategies to pre-vent memory loss and cognitive decline in diverse diseases.Investigators involved in research on this topic include Drs.Finkbeiner, Mucke, and Fen-Biao Gao, Ph.D.

Description 11


Construction of Gladstone’s new facility at Mission Bay is onschedule. In late 2004, all three institutes will move to a newsix-story building, containing about 190,000 square feet of

space, adjacent to UCSF’s new basic science campus. The move is amilestone for Gladstone. “This decision was an investment in thefuture,” said Gladstone president Robert W. Mahley. “The move willenhance our ability to fulfill our mission of contributing to the healthand well-being of all people.”

The programming and schematic design phases for the facility havebeen completed, and the design development phase is proceeding onschedule. The San Francisco Redevelopment Agency has approvedthe schematic design. Utilities, lighting, trees, and sidewalks at thebuilding site have been installed. Preparation of the site has begun,and the steel for the building has been purchased.

In conjunction with NBBJ Architects of San Francisco, Todd Sklar,Gladstone’s project development and management consultant, hasdirected a team of more than 20 firms through a rigorous planningand review process. The review process has included significantinput from the institute directors, investigators, and others.

The first floor will hold the administrative offices and building oper-ations, a 150-person lecture hall, and four seminar rooms. The sec-ond floor will be left as shell space for future expansion or may bedeveloped and leased out temporarily. The third, fourth, and fifthfloors will house the offices and laboratories of the three institutes.

The plans provide for a pleasant work environment. The building’slong and relatively narrow footprint will maximize the amount ofnatural light in the offices and laboratories. Equipment areas will belocated in the center of each floor. Stairwells at the ends of the build-ing will have large windows to take advantage of the beautiful views

of downtown San Francisco and the bay. One main entrance will facethe entrance to UCSF’s campus, and the other will open onto a land-scaped plaza.

The new building will greatly enhance the research environment forGladstone scientists. Having all three institutes under one roof willfoster a sense of unity. Larger and more open laboratories will facil-itate communication and collaborations among the researchers. Theadditional space will allow Gladstone to increase its staff of scientistsand administrators from about 300 currently to more than 500 with-in the next 7 years.

The move will also strengthen the interactions between Gladstoneand UCSF. The Gladstone building will be directly across the streetfrom UCSF’s new basic science campus. “We foresee a constantexchange of ideas that will benefit researchers at both institutions,”said Dr. Mahley. “There will be a synergy that will promote theprogress of research and the advancement of our basic science.”

Behavioral Core Laboratory. Because many of our mouse modelsare designed to simulate aspects of human diseases resulting in mem-ory deficits, behavioral disturbances, or movement disorders, thedetailed behavioral characterization of these models plays an importantrole in the assessment of their clinical relevance. Behavioral alterationsin transgenic models can shed light on the central nervous systemeffects of diverse molecules and are used to assess novel therapeuticstrategies at the preclinical level. This core is engaged in collaborativestudies with investigators at all three Gladstone Institutes, as well aswith scientists in various UCSF departments and other institutions.

Neurobiological Function of Glial Cells and Their Role inNeurological Disease. Glial cells are specialized brain cells that sup-port the health and function of neurons. In response to brain injuries,these cells produce a large number of molecules that participate ininflammatory and immune responses. While acute glial responses mayhelp prevent neuronal damage and facilitate the removal of toxic amy-loid proteins, abnormal activation of these cells could contribute toneurological disease. Genetic and pharmacological strategies are usedto characterize the beneficial and detrimental roles of glial cells incerebral amyloidosis, neurodegeneration, and HIV-associated demen-tia. Investigators involved in this research include Drs. Mucke and Pitas.

New Gladstone Laboratories at the UCSF Mission Bay Campus

Model showing Gladstone’s location at Mission Bay.

Mission Bay12

2002 ANNUAL REPORT

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 Bayredevelopment area. Asterisk indicates approximate location of Gladstone.

Michael Sechman Associates

*

Members of the Institutet 13

Laboratory Associate

John A. Gray

Administrative Staffof the J. David Gladstone Institutes

President

Robert W. Mahley, M.D., Ph.D.

Vice Presidentfor Administrative Affairs

Daniel M. Oshiro, M.S.

Administrative Assistants

Catharine H. EvansKarina G. FantilloMariena D. GardnerLeslie T. ManuntagMarlette A. MarasiganKelley S. NelsonJennifer L. PolizzottoEmily K. O’Keeffe

Executive Assistants

Brian AuerbachDenise Murray McPhersonSylvia A. Richmond

Communications

Daniel M. Oshiro, M.S., OfficerSusan H. DanSylvia A. RichmondTeresa R. Roberts

EditorialGary C. Howard, Ph.D.Stephen B. Ordway

Graphics and PhotographyJohn C.W. Carroll, ManagerStephen GonzalesChristopher A. GoodfellowJohn R. Hull, M.F.A.

Public AffairsLaura Lane, M.S., Manager

Finance and Accounting

Marc E. Minardi, M.A., OfficerRichard S. Melenchuk, M.S., ManagerEmilia M. Franco Marga R. Guillén, M.P.A.Kai Yun W. SunKenneth J. Weiner

Director

Lennart Mucke, M.D.Joseph B. Martin Distinguished Professorof Neuroscience

Investigators

Steven Finkbeiner, M.D., Ph.D.Assistant Professor of Neurologyand Physiology

Fen-Biao Gao, Ph.D.Assistant Professor of Neurologyand Physiology

Robert W. Mahley, M.D., Ph.D.Professor of Pathology and Medicine

Robert E. Pitas, Ph.D.Professor of Pathology

Karl H. Weisgraber, Ph.D.Professor of Pathology

Staff Research Investigator

Yadong Huang, M.D., Ph.D.Assistant Professor of Pathology

Staff Research Scientist

Christopher S. Barker, Ph.D.

Research Scientists

Robert L. Raffaï, Ph.D.Kimberly A. Scearce-Levie, Ph.D.

Visiting Scientists

Andrea J. BarczakLiming Dong, Ph.D.Chandi Griffin, M.S.Dionysos SlagaTony Wyss-Coray, Ph.D.

Postdoctoral Fellows

Lorenzo Arnaboldi, Ph.D.Montserrat Arrasate, Ph.D.John Bradley, Ph.D.Shengjun Chang, Ph.D.Irene Cheng, Ph.D.Jeannie Chin, Ph.D.Luke A. Esposito, Ph.D.Christian Essrich, Ph.D.Danny M. Hatters, Ph.D.Paul C.R. Hopkins, Ph.D.Wenjun Li, Ph.D.Jorge J. Palop, Ph.D.Clare A. Peters-Libeu, Ph.D.

Jukka Puoliväli, Ph.D.Juan Santiago-Garcia, Ph.D.Miki Tamura, Ph.D.Kanyan Xu, Ph.D.Qin Xu, Ph.D.Shi-Ming Ye, Ph.D.

Students

Brigitte BogertSarah CarterPatrick ChangRuomeng DongJennifer HsuEmily LeschPeter LiCatherine MassaroSiddhartha MitraSean PintchovskiVikram RaoKimmy SuNeal T. SweeneyAmy TangBrigitte Watkins

Senior Research Associates

Kay S. ArnoldMaureen E. BalestraWalter J. BrechtZhong-Sheng Ji, Ph.D.Yvonne M. NewhouseGui-Qiu Yu, M.S.

Research Associates

Elizabeth S. Brooks, M.S.Blanca CabezasKenneth CheungNhue L. DoKristina Hanspers, M.S.Yanxia Hao, M.S.Gregor HareFaith M. HarrisBrian JonesShyamal G. KapadiaLisa N. KekoniusAlan LeeSam LoebMaya MathewRene D. MirandaHilda C. OrdanzaDavid S. PetersonBelma SadikovicKristina P. ShockleyRichard M. StewartMaryam A. TabarChunyao XiaFengrong Yan

Members of the Gladstone Institute of Neurological Disease

Members of the Institute14

2002 ANNUAL REPORT

Grants and Contracts

Rex F. Jones, Ph.D., OfficerFrank T. Chargualaf, M.B.A.Lynne M. CoulsonMartin C. RiosYvonne L. Young

Human Resources

Migdalia Martinez, M.S., OfficerJohn R. LeViathan, M.A., ManagerWendy M. FosterChad E. PophamAlyssa S. Uchimura

Information Services

Reginald L. Drakeford, Sr., OfficerJon W. Kilcrease, ManagerMatthew L. LyonSarah K. MaysJoseph R. SolanoyWaldo Yee

Intellectual Property/Technology Transfer

Joan V. Bruland, J.D., OfficerErin MaddenAnne Scott, M.S.

Office of the President

Susan H. DanTeresa R. Roberts

Operations

Deborah S. Addad, Officer

Facilities Vincent J. McGovern, M.S., ManagerDavid R. BourassaRandy A. DamronRoger A. Shore

Purchasing P.J. Spangenburg, ManagerTyler G. CamposP. Sidney OduahAlberto L. ReynosoBenjamin V. Young

Receptionist

Hope S. Williams

Student Assistants

Shannon P. Chi Christina N. Luna

Reports from the Laboratories 15

REPORTS FROMTHE 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 Laboratories16

Molecular Mechanisms of Plasticity and Neurodegeneration

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

Assistant InvestigatorSteven Finkbeiner, M.D., Ph.D.

Postdoctoral FellowsMontserrat Arrasate, Ph.D.John Bradley, Ph.D.

FINKBEINER LABORATORY

StudentsSarah CarterPatrick ChangJennifer HsuPeter LiSiddhartha MitraSean PintchovskiVikram RaoBrigitte Watkins

Research AssociatesElizabeth BrooksKenneth CheungShyamal Kapadia

Administrative AssistantKelley Nelson

Our laboratory is interested in two biological questions. First,how does the nervous system adapt to brief experiences bymaking long-lasting changes in its structure and function?

The molecular mechanisms that underlie this process, collectivelyknown as plasticity, are important for the proper development of thenervous system and for forming memories. We are especially inter-ested in the role of new gene expression in coupling transient neu-ronal activity to long-term changes in synaptic function. The secondquestion is how a genetic mutation leads to an adult-onset progres-sive neurodegenerative disease. We focus on Huntington’s disease(HD), the most common inherited neurodegenerative disorder in theUnited States. Unlike Alzheimer’s disease and Parkinson’s disease,the cause of HD is known. HD is caused by a mutation that leads toan abnormal polyglutamine expansion in the huntingtin protein.Since HD shares some common pathological features withParkinson’s disease and Alzheimer’s disease, we hope that, byunderstanding HD, we will understand the molecular mechanisms ofneurodegeneration more broadly.

Applications of Robotic Microscopy

Is it a cause or an effect? That question is often difficult to addressexperimentally but it must be answered to establish biological orpathological mechanisms and to identify appropriate therapeutictargets. The difficulty lies in understanding the true relationshipbetween the outcome of a long-term dynamic process and a partic-ular subsidiary change. The conundrum is especially common instudies of pathogenesis in which multiple changes occur in paral-lel. Some changes are a direct consequence of an inciting event andmediate morbidity, others are beneficial coping responses, and stillothers are neither helpful nor harmful. The interrelatedness ofmany molecular and cellular processes can also make it challeng-ing to isolate one process and to manipulate it to evaluate its role.

Last year, we described our invention of a computer-controlledrobotic microscope that performs automated imaging and analysisof living or fixed cells. The capabilities of the microscope havefundamentally altered the way we perform many of our experi-ments. For example, we used to study mechanisms of neurodegen-

eration in HD by manually examining immunocytochemicallystained neurons transfected with wildtype or disease-associatedforms of huntingtin. This approach was painstaking and relativelyinsensitive. With the microscope, we can observe neurons as theybegin to express transfected huntingtin and then monitor them overtime, observing their survival, degeneration, and eventual death.The new approach is 100-fold faster than our old assay, more sen-sitive, and less susceptible to user bias.

We also designed the microscope and the controlling computerprograms to be able to return to the same field of neurons or thesame neuron after the tissue-culture plate has been removed fromthe microscope stage. In fact, although the high-throughput capa-bilities of the microscope are very useful, the ability to follow cellslongitudinally may be the key capability that helps us unravelcause and effect mechanisms. By observing a neuron and the adap-tive or maladaptive changes that it undergoes over time, we canreconstruct dynamic relationships that we lose by examining a sin-gle snapshot. For example, longitudinal analysis of huntingtin-transfected neurons revealed a polyglutamine-expansion-depend-ent defect in dendrite and axon formation (Figure 1A). Initially,neurons transfected with either wildtype or mutant huntingtinextended neurites similarly; however, neurons transfected withmutant huntingtin eventually failed to extend neurites and thenretracted them before the neuron died. Dendritic dystrophy andaxon regeneration are early pathological features of HD and so ourability to recapitulate another feature of HD with our simple neu-ronal model adds to the validity of the model. We expect that lon-gitudinal analysis will be useful to trace pathogenic pathwaysbackwards through time to elucidate critical inciting events.

In the past year, we discovered that the ability to monitor the sameneuron or population of neurons over time permits us to use pow-erful statistical tools that are commonly used in clinical studies,such as Kaplan-Meier analysis and Cox proportional hazardsregression analysis. In collaboration with UCSF biostatistician Dr.Marc Segal, we have begun to adapt these tools to analyze data setsgenerated by the robotic microscope. These tools are powerful for

two reasons. First, Kaplan-Meier analysis is extraordinarily sensi-tive, in part because it can detect important differences that emergeor accrue over the course of an experiment. We used this approachto evaluate the ability of a protein kinase, Akt, to promote neuronalsurvival (Figure 1B). Longitudinal monitoring and Kaplan-Meieranalysis showed that Akt promotes neuronal survival much morepotently than had been appreciated from initial reports that meas-ured survival at single time points. The newly found potency ofAkt makes it an especially attractive therapeutic target.

Perhaps the most important reason that statistical tools such as Coxproportional hazards regression analysis are so powerful is thatthey enable us to identify and quantify variables that have predic-tive value. This method provides a way to use longitudinal data(the appearance of identified changes within a particular neuronand the knowledge of that neuron’s fate) to identify whether and towhat extent a change that appears on one day predicts a particularfate for that neuron on another day. We have applied this methodof analysis to neurons cotransfected with the gene for green fluo-rescent protein (GFP) and either an active form of Akt or a controlplasmid. Previously, we showed that the expression level of GFPcorrelated with the expression level of Akt in individual neuronseven though the overall expression of these two proteins variedfrom neuron to neuron. Therefore, we asked whether the amount of

GFP fluorescence within individual neurons, serving as a surrogatefor Akt expression within that neuron, could predict which neuronssurvived. We found that the levels of GFP in neurons cotransfect-ed with empty vector did not predict survival, whereas the levelsof 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 canpredict its fate.

The approach is especially well suited to studying neurodegenera-tive diseases. In HD, a mutation increases the risk to all neurons ofinitiating the biochemical processes that lead to death. However,stochastic processes appear to play a major role in determiningwhen a particular neuron is affected and eventually dies. Becausethese processes may take time to unfold, specific changes may bevisible in only a small fraction of neurons at one point in time.Thus, the significance and even the detection of some changes maynot be recognized with conventional methods. On the other hand,the ability to track the fate of individual neurons with the roboticmicroscope and apply these statistical tools makes it possible toidentify highly significant and predictive relationships even if thechanges are transient or affect a small fraction of neurons at a time.Another reason the approach could be useful to study pathogenesisis its potential for identifying therapeutic targets. The approachshould help us distinguish factors that predict survival or dysfunc-tion and thereby identify therapeutic targets before we fully under-stand the molecular pathways that link them.

Molecular Mechanisms of Synaptic Plasticity

It is widely held that new gene transcription must occur in neuronsfor long-term memories to form. Our laboratory is interested in anumber of questions concerning the role of new gene expression inplasticity. What synaptic signals trigger new gene transcription?What are the gene targets of these signals and how do the encodedproteins promote synaptic plasticity? How do genes transcribed inthe nucleus strengthen only a subset of synapses made by a neuronthat 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 influencesynaptic plasticity and potently regulate neuronal gene transcription.Ca2+ influx through either of two ion channels, the N-methyl-D-aspartate (NMDA) receptor or the L-type voltage-sensitive Ca2+

channel (L-VSCC), appears to be particularly effective in regulatingneuronal gene transcription. Neurotrophins are a family of peptidesthat are essential for the normal growth, development, and differen-tiation of neuronal subpopulations in the central and peripheral nerv-ous systems. One neurotrophin, brain-derived neurotrophic factor(BDNF), has also been shown to regulate synaptic plasticity andlearning and memory during adulthood.

Earlier, we had shown that Ca2+ influx through the L-VSCC direct-ly induces BDNF gene transcription. We hypothesized that BDNFand Ca2+ influx signals might cooperate more broadly to regulateneuronal gene transcription and synaptic plasticity. The hypothesisattracted us because it offered a potential biochemical mechanism totransduce rapidly varying electrical activity (through the productionof transient Ca2+ influx signals) into longer-lasting biochemical sig-nals (BDNF signaling). BDNF-induced signaling cascades generatevery long-lasting adaptive responses, such as those envisioned forlearning and memory.

To test this hypothesis, we introduced reporter genes into corticalneurons and examined the responses to Ca2+ influx and BDNF. To



Figure 1. Applications of robotic microscopy. (A) Mutant huntingtin (Htt) inducesneurite retraction. Striatal neurons were transfected with amino-terminal hunt-ingtin fragments fused to GFP and containing either a normal (Htt-25Q-GFP) ordisease-associated (Htt-103Q-GFP) stretch of polyglutamines. After automatedimaging approximately daily, corresponding microscope fields were examined toreconstruct the pattern of neurite extension of individual neurons over thecourse of the experiment. (B) Akt potently protects neurons. Cultured corticalneurons were transfected with GFP together with either a control vector or anactive 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-vations. Neurons transfected with Akt survived significantly better than thosetransfected with control vector. The extraordinary statistical significanceachieved here reflects the large number of observations and the power ofKaplan-Meier analysis. (C, D) The level of Akt expression predicts neuronallongevity. The expression of the GFP marker directly varies with the expressionof cotransfected Akt. Therefore, we used the level of GFP fluorescence as a sur-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 ofGFP did not predict longevity in neurons transfected with empty vector (D).Censored data were removed from both Akt and control graphs for clarity.

our surprise, we discovered that the efficacy with which BDNFelicits neuronal gene expression is highly dependent on the level ofL-VSCC activity. For example, modest plasma membrane depolar-ization significantly potentiated the ability of BDNF to induceexpression of the immediate-early gene c-fos (Figure 2A).Conversely, application of any one of three structurally distinct andspecific L-VSCC antagonists significantly reduced BDNF-inducedc-fos expression. In contrast, application of both NMDA andBDNF led to additive gene expression responses, and coapplicationof an NMDA receptor antagonist had no effect on responses toBDNF. These results suggest that Ca2+ influx signals synergisticallyregulate BDNF responses in a channel-specific fashion.

Ca2+ influx through L-VSCCs could potentiate BDNF signals at anysite from the plasma membrane (where BDNF initiates a signal) tothe nucleus (where the c-fos promoter is located). The c-fos pro-moter contains two DNA elements that can each mediate responsesto either Ca2+ influx or growth factor signals. One element is theCa2+ and cyclic AMP response element (CaRE) and the other is theserum response element (SRE). We tested whether luciferasereporter genes containing only CaREs or SREs could mediate syn-ergistic responses to BDNF and L-VSCC activation. We found thatonly the SRE or Elk-1, one of the transcription factors that binds theSRE, showed clear evidence of synergistic activation. These resultssuggest 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-1through the Ras pathway, Ca2+ influx might potentiate BDNFresponses by upregulating this pathway. We examined the effects ofL-VSCC activation and BDNF on extracellular signal–regulatedkinase (ERK), a downstream component of the Ras pathway andthe major Elk-1 kinase. We found that L-VSCC activation andBDNF had additive effects on ERK activation (Figure 2B). Theseresults suggest that the site of synergy is in the nucleus, downstreamof ERK but upstream of transcription initiation.

Recently, a family of factors called histone deacetylases (HDACs)has been identified. These factors regulate gene expression througheffects on specific transcription factors and on chromatin structure.Deacetylation of specific histones suppresses gene expression bymaking the associated DNA less available to the transcriptionmachinery. Importantly, Ca2+ influx can promote the translocationof two HDAC family members from the nucleus to the cytoplasm,thereby removing their suppressive effects. We found that pharma-cological agents known to inhibit a subset of HDACs (e.g., tricho-statin A or tripoxin) do not by themselves induce expression of a c-fos-luciferase reporter gene in neurons. However, when addedtogether with BDNF, HDAC inhibitors significantly potentiate neu-ronal gene expression, similar to L-VSCC activation (Figure 2C).

In summary, we have elucidated a new signaling pathway in neu-rons by which Ca2+ influx through L-VSCCs cooperates withBDNF to regulate gene expression. We hypothesize that synergyarises from the integration of two pathways: BDNF signals thatactivate Elk-1 bound to the SRE and L-VSCCs signals to HDACsthat derepress the chromatin structure of the SRE-containing pro-moter.

Molecular Mechanisms of Huntington’s Disease

Abnormal deposits of huntingtin known as intranuclear inclusionsare a hallmark of HD. Although our previous work showed thatinclusion formation was not required for huntingtin-induced degen-eration, nuclear localization was nonetheless important. Reduction


2002 ANNUAL REPORT

Figure 2. Synaptic signals can cooperate to synergistically regulate neuronal genetranscription. (A) Cortical neurons were left unstimulated or were stimulated witheither 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-scriptase polymerase chain reaction. Costimulation with KCl and BDNF had a syn-ergistic effect on c-fos induction (the shaded portion of the bar corresponds to theamount 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-rylation and activation of ERK or CREB, two factors that critically determine theactivity of the c-fos promoter. Thus, the site of synergy is downstream of ERK andis probably in the nucleus. The additional lane of BDNF (10 ng/ml, 45 minutes)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)Synergy may depend on Ca2+ influx–mediated inhibition of HDACs. Cortical neu-rons were transfected with a luciferase reporter gene under the control of the c-fospromoter ( fos-luc).The addition of the HDAC inhibitor trichostatin A (TSA, 400 nM)had almost no effect alone compared with untreated neurons. BDNF modestlyinduced c-fos-luciferase; however, application of TSA and BDNF synergisticallyinduced neuronal gene expression. Ca2+ influx induces the nuclear export of atleast two HDACs and could indicate that synergy arises from the spatial coinci-dence of two signals at the promoter: Ca2+-mediated HDAC inhibition and BDNF-induced activation of critical nuclear enhancer transcription factors (e.g., Elk-1).

polyglutamine expansions, we discovered that, in the absence of theadjacent domain, the polyglutamine expansion appeared to be morelikely to adopt a conformation recognized by our antibody and lesslikely to aggregate. This result is interesting because cleavage of hunt-ingtin at a site within the domain that we have defined has been pro-posed to play a pathogenic role. Our findings suggest that cleavagepromotes pathogenesis by generating a fragment of huntingtin that canmore readily adopt a disease-associated conformation. In the comingyear, we plan to use the robotic microscope to prospectively examinewhether this disease-associated conformation of huntingtin is a betterpredictor of neurodegeneration than inclusion body formation.

Selected References

Saudou F, Finkbeiner S, Devys D, Greenberg ME (1998) Huntingtinacts in the nucleus to induce apoptosis but death does not correlatewith the formation of intranuclear inclusions. Cell 95:55–66.

Finkbeiner S (2000) Calcium regulation of the brain-derived neu-rotrophic factor gene. Cell. Mol. Life Sci. 57:394–401.

Arrasate Iraqui M, Brooks E, Chang P, Mitra S, Finkbeiner S (2002)Prospective analysis of huntingtin conformation and degeneration inneurons. Soc. Neurosci. 29:293.12 (abstract).

Bradley J, Finkbeiner S (2002) An evaluation of specificity in activ-ity-dependent gene expression in neurons. Prog. Neurobiol.67:469–477.

Chang P, Arrasate M, Brooks L, Xia J, Truant R, Finkbeiner S (2002)A domain within huntingtin adjacent to the polyglutamine stretchthat regulates its aggregation and subcellular localization. Soc.Neurosci. 29:293.11 (abstract).

Humbert S, Bryson EA, Cordelières FP, Connors NC, Datta SR,Finkbeiner S, Greenberg ME, Saudou F (2002) The IGF-1/Akt path-way is neuroprotective in Huntington’s disease and involves hunt-ingtin phosphorylation by Akt. Dev. Cell 2:831–837.

Kapadia S, Bradley J, Finkbeiner S (2002) Cooperative regulation ofneuronal gene expression by neurotrophic factors and calcium chan-nels. Soc. Neurosci. 29:752.16 (abstract).



in the nuclear accumulation of huntingtin eliminated degenerationin our HD neuronal model, and since we reported this finding, sev-eral laboratories have reproduced the observation in other cell mod-els and in transgenic and knock-in mice.

The factors that govern the subcellular localization of huntingtin areunknown. In fact, although huntingtin was discovered almost adecade ago, its normal function remains a mystery. Therefore, weperformed a large-scale screen of the huntingtin protein (3144amino acids) for sequences that govern its subcellular localization.To facilitate the study, the full-length protein was subdivided intosmaller overlapping pieces. Each huntingtin fragment was taggedwith GFP at the amino terminus and with β-galactosidase at the car-boxyl terminus. We used the GFP tag to image the location of theattached huntingtin fragment in living neurons, and the β-galactosi-dase tag was used to develop a novel and sensitive luminescenceassay for its presence in different subcellular fractions.

We found that a domain in the amino terminus of huntingtin, adjacentto the polyglutamine stretch, plays a role in governing the localizationof huntingtin and regulating the conformation of the polyglutaminestretch. The presence of this domain of huntingtin promoted its move-ment from a subcellular fraction enriched in cytosolic proteins to oneenriched with components of the nucleus and the endosomal system.However, this region of huntingtin does not appear to mediate thetranslocation of huntingtin into the nucleus. Our imaging studiesshowed that these fragments of huntingtin remained primarily in thecytoplasm of neurons. This domain of huntingtin contains four pro-tein-interaction domains known as HEAT repeats; however, the bind-ing partners for these HEAT repeats have not been identified. Whenwe further subdivided the domain of huntingtin, its localizing abilitywas lost, leading us to speculate that this domain may function as aunit within huntingtin.

Since this new domain of huntingtin is adjacent to the polyglutaminestretch, we suspected that it might affect the conformation of thepolyglutamine stretch or the ability of huntingtin to aggregate intointranuclear inclusions. Using an antibody we developed that recog-nizes a conformation preferentially formed by disease-associated


Molecular Mechanisms Underlying Dendritic Morphogenesisand Their Involvement in Neurological Diseases

Fen-Biao Gao, Ph.D.

Assistant InvestigatorFen-Biao Gao, Ph.D.

Postdoctoral FellowsWenjun Li, Ph.D.Miki Tamura, Ph.D.Kanyan Xu, Ph.D.

GAO LABORATORY

StudentsBrigitte BogertEmily LeschNeal T. SweeneyKimmy SuAmy Tang

Research AssociatesNhue L. DoAlan LeeBelma Sadikovic

Administrative AssistantTyson Jue

Signaling between neurons requires specialized subcellular struc-tures, including axons and dendrites. Dendrites can be highlybranched and may account for more than 90% of the postsy-

naptic surface of some neurons. Only recently have dendrites beenappreciated as having much more active roles in neuronal function. Inaddition, the number of dendritic branches and dendritic spines isaltered in many neurological disorders, such as Alzheimer’s diseaseand fragile X syndrome. Despite the importance of dendrites in neu-ronal function and dysfunction, the molecular mechanisms underlyingdendritic morphogenesis in vivo remain unknown. We continue toinvestigate how dendritic outgrowth is regulated and how alterationsin neuronal morphology contribute to disease processes.

Using Drosophila as a Model Systemto Study Disease Mechanisms

As a genetic model system, the fruit fly Drosophila melanogaster hasgreatly contributed to our understanding of normal animal develop-ment. More recently, Drosophila has been used to dissect the molecu-lar mechanisms underlying a number of human neurological disor-ders. Studies of diseases in model organisms will offer insights intonormal developmental processes and will likely lead to new therapeu-tic interventions for these seemingly intractable diseases.

One of the major reasons we use the peripheral nervous system(PNS) of Drosophila as our primary model system is its simplicity.In each abdominal hemisegment of Drosophila embryos or larvae,there are only 44 PNS sensory neurons, which can be grouped intodorsal, lateral, and ventral clusters. In the dorsal cluster, there are sixmultiple dendritic (MD) neurons, four external sensory neurons, onebipolar dendritic neuron, and one internal sensory neuron. Theseneurons elaborate their dendrites in a two-dimensional plane justbeneath the epidermal cell layer and can be labeled by green fluo-rescent protein using the UAS-GAL4 system. Therefore, we candirectly visualize the dendrites and axons of dorsal MD neurons inliving Drosophila embryos and larvae and follow their growth,branching, and remodeling in real time. Our previous studies haveshown that the dendritic branching pattern of the MD neurons varies

little from embryo to embryo, suggesting that a genetic programcontrols dendritic morphogenesis. Indeed, a genetic screen has iden-tified a number of important proteins that control different aspects ofdendrite development, including Flamingo, a G protein–coupledreceptor–like protein with seven transmembrane segments and alarge amino-terminal domain containing nine cadherin repeats, andSequoia, a tramtrack-related novel zinc finger protein.

As the building block of the nervous system, individual neuronsneed to maintain their structural and functional integrity throughoutadult life. In a number of age-related neurodegenerative diseases,neuronal morphology is altered long before cell death occurs fromaccumulated insults. It is highly likely that the progressive decline inthe function of individual neurons plays a major role in the progres-sion of these neurological diseases. Thus, we believe that under-standing alterations in neuronal morphology and function at the sin-gle neuron level throughout life will contribute to the understandingof disease mechanisms.

To study how neuronal morphology is regulated by various factors,our laboratory uses the mosaic analysis with a repressible marker(MARCM) technique to visualize single wildtype or mutant PNS neu-rons in living Drosophila larvae. This single-neuron assay system pro-vides several advantages. First, each dorsal cluster contains only sixMD neurons, which elaborate their dendrites in a two-dimensionalplane. We can study dendritic growth and branching of the same iden-tifiable single MD neuron in vivo and compare wildtype and mutantneurons with ease and precision. Second, the dendritic field of a par-ticular MD neuron in the dorsal cluster is stereotyped, with limitedvariation between abdominal segments of the same larva or betweendifferent larvae at the same stage. Therefore, we can analyze quantita-tively the effects of loss-of-function mutations and overexpression ofgenes of interest on the dendritic morphology of each MD neuron invivo. Third, we can continuously image the dendrites and spine-likeprocesses of a single wildtype or mutant MD neuron in a living ani-mal over several days and study age-dependent alterations in vivo.Using this single-neuron assay system, we found that Flamingo has a

cell-autonomous function in controlling dendritic extension and axon-al elongation in vivo. Similar approaches are being used to study thefunctions of disease genes in neural development (see below).

Genetic Dissection of Disease Gene Function in Drosophila

We are interested in understanding the functions of several genes thathave been implicated in neurological disorders. During the past year,we have continued to focus on the fragile X syndrome. This is themost common form of inherited mental retardation in humans, with anestimated incidence of 1 in 4000 males and 1 in 8000 females. Thesyndrome is characterized by learning disabilities and mild to severemental retardation that is often associated with autistic behavior,hyperactivity, attention deficit disorder, facial dysmorphism, andenlarged testes in postpubertal males. The genetic defect of fragile Xsyndrome was discovered in 1991, when a single gene, known as thefragile X mental retardation 1 (fmr1) gene, was identified. Fmr1 islocated at a fragile site near the end of the long arm on the X chromo-some. A CGG trinucleotide repeat is present in the 5′ untranslatedregion of fmr1 mRNA. This CGG repeat is normally highly polymor-phic in length, ranging from a few to 60 repeats, and is often inter-rupted with AGG repeats. However, most patients with fragile X syn-drome have over 200 CGG repeats and a hypermethylated CpG islandwithin the promoter region that results in the transcriptional silencingof fmr1. In a few cases of sporadic fragile X syndrome without CGGrepeat expansion, deletion or missense point mutations were found inthe coding region of fmr1, further supporting the notion that the lossof fmr1 activity is the cause of fragile X syndrome.

Fmr1 encodes a putative RNA-binding protein that contains tworibonucleoprotein K homology (KH) domains and an arginine- andglycine-rich domain (RGG box). FMR1 is most abundantlyexpressed in the brain and in the testis. In both human and murinebrains, FMR1 is highly expressed in neuronal perikaryon and den-drites, with little expression in glial cells. FMR1 preferentially bindsto poly(G), poly(U), and a subset of brain mRNAs in vitro. More

recent studies have identified a number of mRNAs that containG-quartet structures and are present in FMR1 messenger-ribonu-cleoprotein (mRNP) complexes. However, the in vivo bindingspecificity and the in vivo RNA targets of FMR1, as well as itsphysiological function, remain largely unknown.

The Drosophila Genome Project has identified only one fmr1 genehomolog (dfmr1) in flies, which encodes a protein that shares a highdegree of amino acid identity with human FMR1 in several keydomains (Figure 1). For instance, the KH domains and the ribosomeinteraction domains of the two proteins are more than 70% identical,suggesting functional conservation among species. Our in situ andimmunostaining analyses indicate that dFMR1 is highly expressed inthe Drosophila nervous system and localized to both dendrites andaxons of MD neurons. Through a large-scale genetic screen, we iden-tified loss-of-function mutations in the dfmr1 gene. Using a westernblot with an anti-dFMR1 antibody, we have analyzed larvae that werehomozygous for the mutated chromosome (Figure 2). None of thethree dfmr1 mutant lines studied, dfmr11, dfmr12, and dfmr14,expressed the dFMR1 protein. We cloned and sequenced the genomicDNA at the dfmr1 locus and identified an 11-nucleotide deletion in thedfmr11 allele and a single nucleotide mutation in the dfmr14 allele thatresulted in a stop codon (Figure 2). We did not find mutations in thedfmr1 coding region of the dfmr12 allele, which suggests that themutations that result in the loss of dFMR1 expression may be in thepromoter or introns. Dfmr1 mutant flies are viable and develop toadulthood at the predicted Mendelian ratio.

Establishment of Behavioral Assays in Drosophila to Studythe Roles of Disease Genes in Neuronal Function

Besides using genetic approaches to investigate the function of diseasegenes, we have expanded our efforts into behavioral studies. Beforemetamorphosis, Drosophila larvae stop feeding and begin to wanderin search of a pupation site. Larvae crawl by anterior extension andposterior retraction. The wandering behavior of the third-instar larvae



Figure 1. Schematic representation of the domain structures and homology ofDrosophila and human FMR1 proteins. The Drosophila Genome Project hasrevealed only one fly homolog of the human fmr1 gene (hfmr1), referred to asdfmr1. Dfmr1 is located on the third chromosome and encodes a protein with681 amino acids (aa) that shares a high degree of conservation with hFMR1.Most notably, the two KH domains and the ribosome interaction domain are

about 70% identical between dFMR1 and hFMR1. The RGG box, anotherRNA-binding motif found in hFMR1, is also present in dFMR1. In addition, aregion that mediates protein–protein interactions between hFMR1 andhFXR1/hFXR2 shows about 50% identity with dFMR1. The high degree ofsequence conservation suggests that dFMR1 is the functional homolog ofhFMR1 in flies.

is relatively simple and stereotypic and can be separated into twophases: linear locomotion and relatively nonlocomotive turning eventsbetween two periods of linear locomotion. During turning events, lar-vae search for a new direction and afterward crawl in a different or thesame direction as previous linear locomotion. We have set up the com-puter-assisted dynamic image-analysis system to analyze quantitative-ly the locomotion behavior of Drosophila larvae at the wanderingstage. The parameters analyzed include the average speed of linearlocomotion, average directional change, and average duration of lin-ear locomotion. To simplify our analysis, larvae locomotion behavioris recorded in a defined environment without cues for phototaxis,chemotaxis, and presumably geotaxis. Because a large number of ani-mals can be recorded and analyzed fairly quickly with the computersoftware, subtle differences in specific aspects of locomotion behav-ior can be detected with statistical significance. Our preliminary stud-ies indicate that mutations in the presenilin gene, which has beenimplicated in Alzheimer’s disease, reduce the speed of larval linearlocomotion by 30%, but do not affect their directional change. Thesestudies will help analyze the functional consequence of mutated genesand dissect the neuronal circuits that control a particular behavior.

Selected References

1. Gao F-B, Brenman JE, Jan LY, Jan YN (1999) Genes regulatingdendritic outgrowth, branching and routing in Drosophila. Genes Dev.13:2549–2561.

2. Gao F-B, Kohwi M, Brenman JE, Jan LY, Jan YN (2000) Controlof dendritic field formation in Drosophila: The roles of Flamingo andcompetition between homologous neurons. Neuron 28:91–101.

3. Brenman JE, Gao F-B, Jan LY, Jan YN (2001) Sequoia, a tramtrack-related zinc finger protein functions as a pan-neural regulator for den-drite and axonal morphogenesis in Drosophila. Dev. Cell 1:667–677.

4. Gao F-B (2002) Understanding fragile X syndrome: Insights fromretarded flies. Neuron 43:859–862.

5. Sweeney NT, Li W, Gao F-B (2002) Genetic manipulation of sin-gle neurons in vivo reveals essential roles of Flamingo in neuronalmorphogenesis. Dev. Biol. 247:76–88.


2002 ANNUAL REPORT

Figure 2. Generation of dfmr1 mutant fly lines. (A) A P-element is inserted inthe first intron of the dfmr1 gene. The ATG start codon is in the third exon.Since the upstream activation sequence (UAS) is engineered into the P-ele-ment, the endogenous dfmr1 gene can be overexpressed when Gal4 mole-cules are present. Mutations could be introduced in the dfmr1 gene, which

would abolish dfmr1 activity and the overexpression phenotype. (B) Westernblot analysis demonstrates dFMR1 in wildtype (wt) flies but not in three dfmr1mutants. (C) Point mutations or small deletions generated by the chemicalmutagen ethyl methanesulfonate were found in dfmr1 in different mutantlines. nt, nucleotide.


Staff Research InvestigatorYadong Huang, M.D., Ph.D.

Postdoctoral FellowsShengjun Chang, Ph.D.Qin Xu, Ph.D.

HUANG LABORATORY

Senior Research AssociateWalter J. Brecht

Research AssociateFaith M. Harris

Summer StudentRuomeng Dong

Administrative AssistantJennifer Polizzotto

Apolipoprotein E Proteolysis and Alzheimer’s Disease

Yadong Huang, M.D., Ph.D.

Apolipoprotein (apo) E4 is a major risk factor for Alzheimer’sdisease. Biochemical, cell biological, and transgenic animalstudies have suggested several potential mechanisms to

explain apoE4’s contribution to the pathogenesis of Alzheimer’s dis-ease. These include the modulation of the deposition and clearanceof amyloid β peptide (Aβ) and the formation of plaques, impair-ment of the antioxidative defense system, dysregulation of the neu-ronal signaling pathways, disruption of cytoskeletal structure andfunction, the alteration of the phosphorylation of tau, and the for-mation of neurofibrillary tangles (NFTs). However, the mechanismsof these apoE4-mediated effects are still largely unknown. Likewise,it is not known which of these pathophysiological effects of apoE4 isthe primary effect and which are subsequent or downstream effects.

Studies from our laboratory have demonstrated a biological event thatcould play a major role in apoE4-related neuropathology. Specifically,we found that apoE is subject to cleavage in both human and mousebrains by a neuron-specific protease that generates bioactive carboxyl-terminal-truncated fragments of apoE. ApoE4 is more susceptible tothis cleavage than apoE3. The carboxyl-terminal-truncated fragmentsof apoE are neurotoxic, leading to cell death and the formation ofintracellular NFT-like inclusions in some of the dying cells. SinceapoE is synthesized by neurons under diverse pathological conditions,this cleavage could be an initial event in apoE4-related neuropatholo-gy. Thus, our hypothesis is that apoE4 produced in neurons inresponse to aging, oxidative stress, brain injuries, or Aβ deposition isuniquely susceptible to proteolytic cleavage and that the resultingbioactive carboxyl-terminal-truncated fragments, probably togetherwith other Alzheimer’s disease–related factors (e.g., Aβ), inducecytoskeletal alterations and other neuropathology (Figure 1).

In the past year, we tested this hypothesis by addressing the followingthree questions. What regulates apoE expression in neurons? Why isapoE4 more susceptible than apoE3 to proteolysis? Does expressionof carboxyl-terminal-truncated apoE4 in neurons cause neuronal andbehavioral deficits in transgenic mice?

Neuronal Production of ApoE and Its Regulationby Astroglia

To investigate how apoE expression is regulated in neurons, we trans-fected neuronal cell lines with a large fragment of human apoEgenomic DNA, which includes a 5-kilobase (kb) 5′ flanking region, a3-kb 3′ flanking region, and a 3-kb tissue-specific control element.Although the baseline expression of apoE in these transfected cellswas very low, conditioned medium from an astrocytic cell line (C6) ormouse primary astrocytes increased apoE mRNA expression by 3–4-fold (Figure 2A) and apoE protein expression by 4–10-fold (Figure2B). These results suggest that astrocytes secrete a factor or factorsthat regulate apoE expression in neuronal cells. This regulationappears to be controlled by the extracellular signal–regulated kinase(ERK) pathway (Figure 2C). The ERK pathway inhibitor U0126, at alevel that causes no cytotoxicity, almost abolished apoE expression inneuronal cells, whereas other mitogen-activated protein kinase(MAPK) pathway inhibitors—c-jun N-terminal kinase (JNK)-inhibitor-1 and SB203580 (for the p38 pathway)—had no significanteffect on apoE expression (Figure 2C). Furthermore, the human neu-ronal precursor NT2/D1 cells expressed apoE constitutively (Figure2D). As these cells differentiated into neurons induced by retinoic

Figure 1. Working model of apoE proteolysis andAlzheimer’s disease. ER, endoplasmic reticulum.

acid, their apoE expression increased initially and then diminished(Figure 2D). However, treatment of the fully differentiated neuronswith astrocyte-conditioned medium rapidly upregulated apoE expres-sion (Figure 2D). These observations and previous studies showingthat excitotoxic stress induces neuronal expression of apoE led us tohypothesize that, in response to brain injury, gliosis, or Aβ neurotoxi-city, neurons are induced to express apoE for purposes of repair orremodeling. However, in apoE4 carriers, these events trigger prote-olytic processing of apoE, which is detrimental to repair and remodel-ing processes.

Domain Interaction Is Responsible for ApoE4’sSusceptibility to Proteolysis

To determine whether domain interaction, which is mediated by for-mation of a salt bridge between Arg-61 and Glu-255, is responsible forthe susceptibility of apoE4 to proteolysis, we incubated recombinantapoE4-Thr-61 or apoE4-Ala-255, both of which lack domain interac-tion, with lysates from apoE knockout mouse brains or Neuro-2a cellsat 37°C for 3 hours and analyzed the proteolysis of apoE by anti-apoEwestern blotting. ApoE4-Thr-61 and apoE4-Ala-255 were both muchmore resistant to proteolysis than wildtype apoE4 (Figure 3), suggest-ing that apoE4 domain interaction is responsible for its susceptibilityto proteolysis. This conclusion is supported by our preliminary studyin transgenic mice expressing apoE4-Thr-61 or apoE4-Ala-255 inneurons, in which apoE4 proteolysis is abolished.

Neuronal Deficit in Transgenic Mice ExpressingApoE4(∆272–299) in Neurons

To determine whether expression of carboxyl-terminal-truncated apoEin transgenic mice induces neuropathological changes, we createdtransgenic mouse lines expressing apoE4(∆272–299) at various levelsin central nervous system (CNS) neurons, including levels similar tothose observed in human cortex. To avoid the cytotoxic effect of thetruncated apoE4 (observed in vitro in transfected neuronal cells) onembryonic development, we used a neuron-specific Thy-1 promoterthat induces transgene expression at day 15 after birth. The highexpressers died from severe neurodegeneration at 2–4 months of age,while the low expressers were viable and fertile. Anti-apoE immuno-staining of brain sections showed carboxyl-terminal-truncated apoE4in neurons in the neocortex (Figure 4A), hippocampus (Figure 4B andC), and cerebellum (data not shown). Inclusion bodies containingtruncated apoE4 were observed in neurons of mice at 2–4 months ofage (Figure 4A–C). Hematoxylin-eosin staining revealed degenera-tion of neurons expressing the truncated apoE4 in CA1 (Figure 4D)and CA3 (Figure 4E) neurons of the hippocampus. Importantly,expression of a shorter truncated form of apoE4, apoE4(∆241–299) atsimilar levels did not induce neuropathology (Figure 4F), suggestingthat the lipid-binding domain within the truncated apoE4 fragments isresponsible for the neurotoxic effect.

Western blotting with anti-phosphorylated tau (p-tau) demonstratedaccumulation of monomeric p-tau and sodium dodecyl sulfate(SDS)–resistant p-tau aggregates in the brains of transgenic miceexpressing high levels of the truncated apoE4 at 2–4 months of age,which is 6–11-fold higher than in nontransgenic littermates at thesame age. Notably, the amounts of SDS-resistant p-tau aggregates inthe brains of 2–4-month-old transgenic mice expressing high levels ofthe truncated apoE4 were similar to those in 18-month-old neuron-specific enolase (NSE)–apoE4 transgenic mice, suggesting that thetruncated apoE4 enhances tau phosphorylation in transgenic mice.


2002 ANNUAL REPORT

Figure 2. Expression of apoE in neuronal cells and its regulation by astroglia.Mouse neuroblastoma Neuro-2a cells were stably transfected with a humanapoE4 genomic DNA construct. The regulation of apoE4 expression by the con-ditioned medium from an astrocytic cell line (C6 CM) or mouse primary astro-cytes (1° astrocyte CM) was determined at the mRNA (A) and protein (B) levelsby real-time polymerase chain reaction and anti-apoE western blotting, respec-tively. (C) Effects of various MAPK pathway inhibitors on the regulation of apoE4expression in Neuro-2a cells by C6 CM. (D) Human neuronal precursor NT2/D1cells were induced to differentiate by incubation with retinoic acid (RA) for 0, 1,or 4 weeks (w). Secretion of apoE into the medium was determined by anti-apoE western blotting. After induction with RA for 4 weeks, some cells weretreated with C6 CM for 24 hours and then apoE secretion was determined (D,three far right lanes).

Figure 3. Domain interaction is responsible for apoE4’s susceptibility to pro-teolysis. Recombinant human apoE4, apoE3, apoE4-Thr-61, or apoE4-Ala-255 (1 µg protein) was incubated with apoE knockout mouse brain lysates (20µl) at 37°C for 3 hours. Fragmentation of apoE was determined by anti-apoEwestern blotting.

Gallyas silver staining revealed NFT-like structures in neurons in theneocortex of apoE4(∆272–299) transgenic mice (Figure 4G). Thesedata suggest that apoE4 fragments increase tau phosphorylation invivo in transgenic mice. We are expanding the transgenic lines inwhich low levels of the carboxyl-terminal-truncated apoE areexpressed to explore in more detail the effects of the truncated apoE4on neuronal and behavioral deficits.

In summary, our findings suggest that apoE synthesized in neuronsundergoes proteolytic processing, probably in the secretory pathway,to generate carboxyl-terminal-truncated fragments that cause neu-rodegeneration, with apoE4 being more susceptible than apoE3 to thecleavage. This process may not occur to a significant extent underphysiological conditions, since little apoE is normally expressed inneurons. However, in response to aging, oxidative stress, brain injury,or Aβ deposition, neurons may turn on or increase their apoE expres-sion for purposes of repair or remodeling, thereby passively activatingthis proteolytic process, especially for apoE4 carriers. We are testingthis hypothesis now. We are also attempting to identify the putativeprotease that cleaves apoE at its carboxyl terminus, as it may serve asa therapeutic target for the prevention and treatment of neurodegener-ative diseases associated with apoE4.



Figure 4. Neuronal deficits in transgenic mice expressing apoE4(∆272–299) butnot in mice expressing apoE4(∆241–299) at 2–4 months of age. Brain sectionsfrom transgenic mice expressing apoE4(∆272–299) (A–E) or apoE4(∆241–299)(F) were stained with anti-apoE (A–C) or hematoxylin-eosin (D–F). Note the for-

mation of truncated apoE4-containing inclusion bodies in cortical (A), CA1 (B), orCA3 (C) neurons and neurodegeneration in the CA1 (D, upper panel) and CA3 (E,upper panel) regions. (G) Gallyas silver staining of a brain section from anapoE4(∆272–299) mouse revealed NFT-like structures in cortical neurons.

Selected References

Mahley RW, Huang Y (1999) Apolipoprotein E: From atherosclerosisto Alzheimer’s disease and beyond. Curr. Opin. Lipidol. 10:207–217.

Huang Y, Liu XQ, Wyss-Coray T, Brecht WJ, Sanan DA, Mahley RW(2001) Apolipoprotein E fragments present in Alzheimer’s diseasebrains induce neurofibrillary tangle-like intracellular inclusions inneurons. Proc. Natl. Acad. Sci. USA 98:8838–8843.

Brecht WJ, Harris FM, Tesseur I, Wyss-Coray T, Yu G-Q, Mucke L,Mahley RW, Huang Y (2002) ApoE proteolysis and hyperphosphory-lation of tau in transgenic mice expressing apoE4 in neurons. Soc.Neurosci. 29:592.13 (abstract).

Buttini M, Yu G-Q, Shockley K, Huang Y, Jones B, Masliah E,Mallory M, Yeo T, Longo FM, Mucke L (2002) Modulation ofAlzheimer-like synaptic and cholinergic deficits in transgenic mice byhuman apolipoprotein E depends on isoform, aging, and overexpres-sion of amyloid β peptides but not on plaque formation. J. Neurosci.15:10539–10548.

Harris FM, Brecht WJ, Tesseur I, Mahley RW, Wyss-Coray T, HuangY (2002) Astroglial regulation of apoE expression in neuronal cells.Soc. Neurosci. 29:193.16 (abstract).

Ji Z-S, Miranda RD, Newhouse YM, Weisgraber KH, Huang Y,Mahley RW (2002) Apolipoprotein E4 potentiates amyloid β peptide-induced lysosomal leakage and apoptosis in neuronal cells. J. Biol.Chem. 277:21821–21828.


Apolipoprotein E: Impact on Neurodegenerationand Alzheimer’s Disease Pathobiology

Robert W. Mahley, M.D., Ph.D.

Senior InvestigatorRobert W. Mahley, M.D., Ph.D.

Postdoctoral FellowShiming Ye, Ph.D.

MAHLEY LABORATORY

Senior Research AssociatesMaureen E. BalestraWalter J. BrechtZhong-Sheng Ji, Ph.D.

Research AssociatesRene D. MirandaDavid S. Peterson

Executive AssistantSylvia A. Richmond

Administrative AssistantCatharine H. Evans

Along-term major focus of this laboratory relates to under-standing the function of apolipoprotein (apo) E in neurobiol-ogy. By the mid-1980s, clues indicating that apoE played an

important role in neurological diseases had begun to surface. ApoEwas produced in abundance in the brain and served as the principallipid transport vehicle in cerebrospinal fluid. It was induced at highconcentrations in peripheral nerve injury, appeared to play a key rolein repair by redistributing lipids to regenerating axons and to Schwanncells during remyelinization, and modulated neurite outgrowth in cul-tured rabbit dorsal root ganglion cells or Neuro-2a cells. The stage wasset for the discovery by Roses and associates (Duke University) thatapoE4 is a major susceptibility gene associated with approximately40–65% of cases of sporadic and familial Alzheimer’s disease andincreases the occurrence and lowers the age of onset of the disease.Furthermore, the apoE4 allele is associated with poor clinical outcomein patients with acute head trauma, whereas apoE2 may be protectiveagainst neurodegenerative diseases.

A key to understanding the role of apoE in neurological diseasesresides in determining how apoE modulates neuronal repair, remodel-ing, or protection (Figure 1). Injurious agents can cause neuronal dam-age, requiring neuronal repair of synaptodendritic connections. Wewould suggest that apoE3 and apoE2 may be effective in mediatingthe repair process and in protecting neurons from excessive damage,whereas apoE4 may be relatively ineffective. In addition, as reviewedbelow, apoE4 may have detrimental effects on central nervous systemneurons. The mechanisms responsible for the isoform-specific effectsof apoE are the focus of the laboratory.

ApoE Potentiation of Aβ-Induced Lysosomal Leakage andApoptosis in Neuronal Cells

We assessed the isoform-specific effects of apoE on the response ofNeuro-2a cells to the amyloid β peptide (Aβ1–42). As determined bythe intracellular staining pattern and the release of β-hexosaminidaseinto the cytosol, apoE4-transfected cells treated with aggregatedAβ1–42 showed a greater tendency toward lysosomal leakage thanneo- or apoE3-transfected cells. Aβ1–42 caused significantly greater

cell death and more than twofold greater DNA fragmentation inapoE4-secreting than in apoE3-secreting or control cells. H2O2 orstaurosporine enhanced cell death and apoptosis in apoE4-transfectedcells but not in apoE3-transfected cells. A caspase-9 inhibitor abol-ished the potentiation of Aβ1–42-induced apoptosis by apoE4. Similarresults were obtained with conditioned medium from cells secretingapoE3 or apoE4. Cells preincubated for 4 hours with a source ofapoE3 or apoE4, followed by removal of apoE from the medium andfrom the cell surface, still exhibited the isoform-specific response toAβ1–42, indicating that the potentiation of apoptosis required intra-cellular apoE, presumably in the endosomes or lysosomes. Studies ofphospholipid [dimyristoylphosphatidylcholine (DMPC)] bilayer vesi-cles encapsulating 5-(and-6)-carboxyfluorescein dye showed thatapoE4 remodeled and disrupted the phospholipid vesicles to a greaterextent than apoE3 or apoE2 (Figure 2). In response to Aβ1–42, vesi-cles containing apoE4 were disrupted to a greater extent than thosecontaining apoE3. These findings are consistent with the idea that

Figure 1. Hypothetical role of apoE in the pathogenesis of neurodegeneration andAlzheimer’s disease.

apoE4 forms a reactive molecular intermediate that avidly binds phos-pholipid and may insert into the lysosomal membrane, destabilizing itand causing lysosomal leakage and apoptosis in response to Aβ1–42.These studies are conducted in association with Zhong-Sheng Ji,Ph.D., and Yadong Huang, M.D., Ph.D.

ApoE Enhances Aβ Peptide Productionin Cultured Neurons

Many studies have suggested that apoE has isoform-specific effects onthe deposition or clearance of Aβ peptides. Few studies, however,focus on whether apoE isoforms also influence Aβ production. Weexamined the effects of lipid-poor apoE isoforms on the processing ofamyloid precursor protein (APP) and on Aβ production in rat neuro-blastoma B103 cells stably transfected with human wildtype APP695(B103-APP). ApoE3 and apoE4 both stimulated Aβ production inB103-APP cells, but apoE4 did so to a significantly greater extent(Figure 3A). The enhanced Aβ production by apoE4 was abolished atlow temperatures (22°C), which block endosome recycling. The dif-ferential effects of apoE3 and apoE4 on Aβ production were mediat-ed not by altering cellular cholesterol content or α- and β-secretase

activities, but by stimulating cell-surface APP recycling, as deter-mined by monitoring the internalization of radiolabeled 1G7 anti-body against the amino terminus of APP (Figure 3B). Furthermore,preincubation of the B103-APP cells with a low concentration (25nM) of the receptor-associated protein, which blocks the low densitylipoprotein receptor–related protein pathway, abolished the differen-tial effects of apoE3 and apoE4 on Aβ production. This result sug-gests that this pathway may be involved in the more pronouncedstimulatory effect of apoE4 on Aβ production. Finally, replacementof Arg-61 with threonine in apoE4, which abolishes the intramolecu-lar domain interaction of apoE4, also attenuated the differentialeffects of apoE3 and apoE4 on Aβ production, suggesting that apoE4domain interaction is involved in this process. Thus, apoE4 not onlyaffects Aβ deposition or clearance but also modulates APP process-ing and Aβ production, which may provide an alternative explanationas to why apoE4 is associated with increased risk for Alzheimer’s dis-ease. These studies are conducted in collaboration with Shiming Ye,Ph.D., and Dr. Huang.

Bioactive Carboxyl-Terminal-Truncated Fragments of ApoE4Alter the Cytoskeleton and Cause Neurodegeneration

Several lines of evidence indicate that apoE4 has a major effect on thecytoskeleton of neurons and disrupts cytoskeletal structure and func-tion. Strittmatter and associates demonstrated differential effects ofapoE isoforms on tau. Others, including our laboratory, have shownthat apoE4 enhances tau phosphorylation. We have shown that, in thepresence of a lipid source, apoE3 stimulates neurite outgrowth in cul-tured neurons, whereas apoE4 inhibits neurite outgrowth and causesmicrotubular instability. ApoE4 also has detrimental effects in apoE-null transgenic mice overexpressing apoE4, including behavioralabnormalities and significant decreases in immunostained synapto-physin and microtubule-associated protein 2 in the hippocampus andthe cortex. These findings suggested that apoE4 affects the cytoskele-tal system and neural plasticity.

Our most recent studies have examined the cellular and molecularmechanisms by which apoE3 and apoE4 differentially modulate thecytoskeleton of neurons. Carboxyl-terminal-truncated fragments ofapoE occur in cultured neurons and in the brains of patients withAlzheimer’s disease and induce the formation of neurofibrillary tan-gle–like inclusions in neurons and neurodegeneration. These inclu-



Figure 2. Release of fluorescent dye from DMPC phospholipid vesicles by apoE3and apoE4. After a 10-second baseline measurement, apoE was added to theDMPC and the fluorescence released over 50 seconds was measured. ApoE4markedly destabilizes the phospholipid bilayer vesicles.

Figure 3. ApoE3 and apoE4 exert isoform-specific effects on Aβ productionthrough their differential effects on intracellular APP recycling. (A) Blockageof APP recycling by culturing cells at low temperature abolished the apoE4-enhanced Aβ production. Recombinant human apoE3 or apoE4 (7.5 µg/ml)was incubated with B103-APP cells at either 22°C or 37°C for 24 hours. Theconditioned media were assayed for Aβ by enzyme-linked immunosorbentassay. Values are the mean ± SD of two experiments, each repeated 4–6

times for each condition. * p < 0.05. (B) ApoE4 increased the internalizationof cell-surface APP to a greater extent than apoE3. Internalization of cell-sur-face APP after apoE treatment was determined by measuring the uptake ofradioiodinated 1G7 antibody. The results are expressed as a ratio of theradioactivity associated with the internalized versus cell-surface pools of APP.Values are the mean ± SD of two experiments, each repeated three times foreach condition. * p < 0.05.

sions were composed of phosphorylated tau (p-tau), phosphorylatedneurofilaments of high molecular weight, and truncated apoE.Truncated apoE4, especially apoE4(∆272–299), induced neurofibril-lary tangle–like inclusions in up to 75% of transfected neuronal cells(Figure 4), but not in transfected nonneuronal cells. ApoE4 was moresusceptible to truncation than apoE3 and resulted in much greaterintracellular inclusion formation. These results suggest that apoE4preferentially undergoes intracellular processing, creating a bioactivefragment that interacts with cytoskeletal components and induces neu-rofibrillary tangle–like structures and cell death. The preferential pro-teolytic cleavage of apoE4 may represent an important mechanism forapoE4 in neurodegenerative disorders. These studies are being con-ducted in collaboration with Dr. Huang (see his report for moredetails).

Selected References

Buttini M, Orth M, Bellosta S, Akeefe H, Pitas RE, Wyss-Coray T,Mucke L, Mahley RW (1999) Expression of human apolipoprotein E3or E4 in the brains of Apoe–/– mice: Isoform-specific effects on neu-rodegeneration. J. Neurosci. 19:4867–4880.

Mahley RW, Huang Y (1999) Apolipoprotein E: From atherosclerosisto Alzheimer’s disease and beyond. Curr. Opin. Lipidol. 10:207–217.

Buttini M, Akeefe H, Lin C, Mahley RW, Pitas RE, Wyss-Coray T,Mucke L (2000) Dominant negative effects of apolipoprotein E4revealed in transgenic models of neurodegenerative disease.Neuroscience 97:207–210.

Mahley RW, Rall SC Jr (2000) Apolipoprotein E: Far more than a lipidtransport protein. Annu. Rev. Genomics Hum. Genet. 1:507–537.

Raber J, Wong D, Yu G-Q, Buttini M, Mahley RW, Pitas RE, MuckeL (2000) Apolipoprotein E and cognitive performance. Nature404:352–354.

Huang Y, Liu XQ, Wyss-Coray T, Brecht WJ, Sanan DA, Mahley RW(2001) Apolipoprotein E fragments present in Alzheimer’s diseasebrains induce neurofibrillary tangle-like intracellular inclusions inneurons. Proc. Natl. Acad. Sci. USA 98:8838–8843.

Ji Z-S, Miranda RD, Newhouse YM, Weisgraber KH, Huang Y,Mahley RW (2002) Apolipoprotein E4 potentiates amyloid β peptide-induced lysosomal leakage and apoptosis in neuronal cells. J. Biol.Chem. 277:21821–21828.


2002 ANNUAL REPORT

Figure 4. Bioactive carboxyl-terminal-truncated apoE(∆272–299) accumulates intracellularly in neurons and forms a cytosolicinclusion complexed with p-tau and other cytoskeletal proteins. Carboxyl-terminal-truncated apoE fragments occur in neuronalinclusions in brains of Alzheimer’s disease patients. GFP, green fluorescent protein.


Director andSenior InvestigatorLennart Mucke, M.D.

Research ScientistKimberly A. Scearce-Levie, Ph.D.

Postdoctoral FellowsIrene Cheng, Ph.DJeannie Chin, Ph.D.Luke A. Esposito, Ph.D.

MUCKE LABORATORY

Christian Essrich, Ph.D.Jorge J. Palop, Ph.D.Jukka Puoliväli, Ph.D.

Visiting ScientistTony Wyss-Coray, Ph.D.

StudentCatherine Massaro

Senior Research AssociateGui-Qiu Yu, M.S.

Research AssociatesGregor HareBrian JonesHilda C. OrdanzaKristina P. ShockleyFengrong Yan

Laboratory AssociateJohn A. Gray

Executive AssistantDenise Murray McPherson

Administrative AssistantLeslie Manuntag

Neurobiology of Dementia

Lennart Mucke, M.D.

In this laboratory, we study processes that result in memory lossand other major neurological deficits, with an emphasis onAlzheimer’s disease (AD) and related neurodegenerative disor-

ders. Our long-term goal is to advance the understanding of thehealthy and the diseased central nervous systems to a point whererational strategies can be developed to prevent and cure these con-ditions. AD and Parkinson’s disease are the most frequent neu-rodegenerative disorders. They erode people’s ability to think andcontrol their movements, two of the most critical and fascinatingfunctions of the central nervous system. Both conditions are on therise and can be neither prevented nor cured. These facts underlinethe significance and urgency of our research efforts.

Molecules similar to those involved in neurodegenerative diseasesare highly expressed in the nervous system of diverse species andappear to function in learning, synaptic plasticity, and regeneration.We are particularly curious about the functions and pathogenic rolesof amyloid precursor proteins (APP), apolipoprotein (apo) E, and α-synuclein, which play key roles in AD and Lewy body diseases suchas Parkinson’s disease.

Mutations in human APP (hAPP) that increase the production ofamyloid-β peptides ending at residue 42 (Aβ42) cause autosomaldominant forms of early-onset familial AD (FAD). Aβ42 and otherAβ peptides also accumulate in the brains of sporadic cases of AD,suggesting a central role of these APP metabolites in the pathogene-sis of AD in general. In AD-related transgenic models, we previous-ly discovered that Aβ peptides can damage synapses and disruptneural memory circuits independent of their deposition into the amy-loid plaques that form in AD brains. The plaque-independent toxic-ity of Aβ was inhibited by apoE3, but not apoE4, which may relateto the differential effects of apoE isoforms on AD risk (E4 > E3) andage of onset (E4 < E3).

During the past year, we focused on the identification of molecularindicators and mediators of hAPP/Aβ-induced neuronal dysfunctionand degeneration.

Aβ-Dependent Deficits in Learning and Memory Are CloselyRelated to Neuronal Depletion of a Calcium-Binding Protein

The bright prospects of increasing life expectancy in many popula-tions around the world are tempered by an alarming increase in aging-related neurodegenerative disorders. AD, the most frequent of theseconditions, causes inexorable loss of memory and other cognitivefunctions. Although the etiology of most AD cases remains elusive,the analysis of AD-related transgenic mouse models is beginning tounravel the pathogenic importance of specific AD-associated mole-cules. These models are also used increasingly to assess novel ADtreatments. Amyloid plaques are the primary pathological outcomemeasure in these studies, although their contribution to AD-relatedcognitive deficits is controversial. In fact, it remains to be determinedwhich of the many pathological and biochemical alterations in AD andin transgenic models of the disease contribute most critically to thedecline in neuronal functions.

We therefore investigated the relationship between morphological,biochemical, and behavioral alterations in transgenic mice in whichneuronal expression of hAPP is directed by the platelet-derivedgrowth factor β chain promoter. Mice expressing FAD-mutanthAPP (hAPPFAD) have high levels of human Aβ in the hippocampalformation. This brain region includes the dentate gyrus, which iscritically involved in spatial learning and memory. We analyzed theexpression of calcium-dependent proteins in these brain regionsbecause pathological and cell culture studies suggest that alterationsin neuronal calcium homeostasis play an important role in thepathogenesis of AD.

Calbindin, a 28-kDa calcium-binding protein, is particularly abundantin neurons of the dentate gyrus and highly responsive to alterations incalcium influx. Most hAPPFAD mice had significantly lower neuronalcalbindin levels in the dentate gyrus than nontransgenic controls(Figure 1A). Interestingly, granule cells are relatively resistant to AD-associated cell death. Yet, we found marked reductions in neuronal cal-bindin levels in the dentate gyrus of AD cases, with the most striking

depletions seen in the most severely demented subjects (Figure 1B).Double-labeling of brain sections from hAPPFAD mice for calbindinand the neuronal marker Neu-N (Figure 2) indicated that the calbindinreduction in the dentate gyrus primarily reflects a decrease in neuronalcalbindin levels rather than a loss of calbindin-producing neurons.These results demonstrate that neuronal populations resisting cell deathin AD can still be drastically altered at the molecular level. It is likelythat such molecular alterations have functional consequences.

hAPPFAD mice showed significant interindividual variations in cal-bindin reductions that were detectable at the protein and mRNA lev-els (Figure 3A). However, calbindin levels did not correlate withhAPPFAD levels, suggesting that the calbindin reductions are notcaused by the expression of hAPPFAD per se. To assess whether reduc-

tions in calbindin were caused by Aβ, we analyzed their relationshipwith Aβ deposits (plaques), levels of soluble Aβ1–42 and Aβ1–x(approximates total Aβ), and Aβ1–42/Aβ1–x ratios. Calbindin reduc-tions in hAPPFAD mice did not correlate with the extent of Aβ deposi-tion but correlated with the Aβ1–42/Aβ1–x ratio, which reflects theabundance of Aβ ending at residue 42 relative to other, mostly short-er, Aβ peptides.

These results are consistent with mounting evidence that AD-relat-ed neuronal deficits are caused by nonfibrous Aβ assemblies ratherthan by plaques. They are also consistent with studies suggestingthat, above an absolute threshold concentration, the formation ofneurotoxic Aβ assemblies depends more on relative than absolutelevels of Aβ1–42. Although Aβ production is dependent on hAPP


2002 ANNUAL REPORT

Figure 1. Calbindin depletion in the granular layer of the dentate gyrus inhAPPFAD mice and humans with AD. Hippocampal sections from hAPPFADmice and a nontransgenic (NTG) mouse (A) and from humans with or with-out AD (B) were immunolabeled for calbindin. Each panel reflects findings

from a different subject. Comparable calbindin reductions were identified inhAPPFAD mice and AD cases. Numbers in parentheses indicate Blessedscore, which increases with the severity of the dementia.

Figure 2. Calbindin depletion in the dentate gyrus of hAPPFAD mice is notdue to loss of granule cells. Double-immunolabeling of sagittal vibratomesections for calbindin and the neuronal marker Neu-N confirmed the reduc-

tion of calbindin in granule cells of hAPPFAD mice but did not reveal obviouschanges in the density of their nuclei compared to nontransgenic controls.NTG, nontransgenic.

A

B

levels, the formation of neurotoxic Aβ assemblies may be stronglyaffected by proteins that bind or degrade Aβ. This may explain whyreductions in calbindin correlated with the relative abundance ofAβ1–42 but not with hAPPFAD levels. The exact mechanisms bywhich Aβ assemblies reduce calbindin remain to be determined.They could involve destabilization of the neuronal calcium home-ostasis by chronic inflammation, formation of pores in cell mem-branes, and alterations in the function of calcium channels and othermembrane proteins.

To further assess the pathophysiological significance of Aβ-inducedcalbindin reductions, we analyzed hAPPFAD mice and nontrans-genic controls in a Morris water maze test, which provides putativemeasures of learning and memory. In contrast to nontransgenicmice, hAPPFAD mice showed a tight correlation between calbindinlevels and deficits in learning and memory in the spatial componentof this test (Figure 3B).

Our findings that hAPPFAD/Aβ is sufficient to reduce neuronal cal-bindin levels in vivo and that this effect is tightly associated withbehavioral deficits has practical implications, particularly in light ofincreasing efforts to assess novel therapies for AD in transgenicmouse models. The behavioral testing of mice is time consuming,and test results obtained in different laboratories can vary widely.Reliable surrogate markers of behavioral deficits could help cir-cumvent these obstacles and facilitate the preclinical assessment ofAD treatments. Our results suggest that calbindin levels in the den-tate gyrus are a better diagnostic marker of Aβ-induced neuronaldysfunction than plaque load (Figure 3C), which remains the mostwidely used pathological endpoint measure in the preclinicalassessment of novel AD treatments. Experiments are in progress tofurther validate this notion.

Although it is likely that diverse molecular alterations contribute toAD-associated neuronal dysfunction, the tight association betweenmolecular and functional alterations we identified makes one won-der whether reductions in calbindin not only indicate but also medi-ate hAPPFAD/Aβ-dependent behavioral deficits. Calcium-bindingproteins play key roles in calcium signaling and homeostasis, whichare critical to neuronal function. Calbindin can buffer intracellularcalcium, activate enzymes, modulate ion channels, increase calciumentry into neurons, facilitate spatial transfer or release of calciumwithin the neuronal cytoplasm, and prolong intracellular calciumsignals. Since calbindin can also protect neurons against Aβ-induced toxicity, the reduction of calbindin by Aβ could be part ofa vicious cycle promoting progressive neuronal dysfunction inhAPPFAD mice and in AD.

These findings raise the intriguing possibility that reductions in cal-bindin in hAPPFAD mice and humans with AD not only reflect cog-nitive deficits but also contribute to them. We will continue toinvestigate the roles of these and related molecular alterations aspotential indicators and mediators of neurodegenerative disease.

Blocking Aβ Production Increases the Resistanceof hAPPFAD-Producing Neurons to Secondary Insults

In some cell-culture models, expression of APPFAD or mutant pre-senilins is sufficient to kill neurons within a relatively short time.However, even though the mutant proteins are expressed from birthin humans with FAD mutations, it typically takes at least two tothree decades for the disease to manifest itself. Aging in humansand other species is associated with increasing oxidative stress,DNA damage, and decline in mitochondrial function, all of whichmight trigger neurodegeneration in neurons primed for excessiveactivation of proapoptotic pathways. This scenario would seem

more consistent with cell-culture models in which expression ofFAD-mutant proteins does not induce apoptosis by itself but ratherincreases the susceptibility of cells to apoptosis induced by otherinsults. We previously developed such a model by stably expressingdifferent hAPP constructs in neuronal cell lines (B103 cells) at near-physiological levels. Cells expressing hAPPFAD were significantlymore susceptible to cell death induced by secondary insults thancells expressing hAPPWT. As outlined below, this difference is like-ly due to increased production of Aβ42 in hAPPFAD cells rather thanto a FAD mutation–related loss of neuroprotective APP functions.



Figure 3. Calbindin reductions in the dentate gyrus accurately reflect Aβ-induced deficits in learning and memory. (A) Levels of calbindin (CB)immunoreactivity (IR) in the dentate gyrus of hAPPFAD mice correlated tightlywith calbindin protein and mRNA levels in the dentate gyrus of the oppositehemibrain, as determined by western blot analysis and quantitative fluorogenicreverse transcriptase polymerase chain reaction, respectively. (B) Reductionsin calbindin correlated tightly with behavioral deficits. hAPPFAD mice (filleddots) and nontransgenic littermate controls (open dots) were trained in aMorris water maze. The average time it took the mice to locate the platform inall hidden platform trials (left) and the percentage of time they searched in thetarget quadrant after removal of the platform (right) were used as putativemeasures of spatial learning and memory retention, respectively. (C) Aβ likelyelicits both plaque-dependent and plaque-independent neuronal alterations.Cumulatively, our data suggest that plaque-independent neurotoxicity plays acritical role in the pathogenesis of Aβ-induced cognitive deficits (see text andreferences). Reductions of calbindin in the dentate gyrus accurately reflect thisform of Aβ toxicity and may even contribute to it.

A mutation that prevents hAPP from entering the distal secretorypathway, where most Aβ peptides are generated, markedlyincreased the resistance of APPFAD cells to apoptosis (Figure 4).Preventing Aβ42 production with a mutation (M596I) that blockscleavage of hAPP by β-secretase also drastically increased theresistance of hAPPFAD cells, underlining the potential therapeuticvalue of drugs that specifically block this enzyme activity.Treatment of hAPPFAD cells with an inhibitor of γ-secretase, theother key enzyme required for Aβ production, had a similar effect.

There is substantial concern that inhibiting cleavage of Notch andother γ-secretase activities might limit the therapeutic usefulness ofγ-secretase inhibitors, but it has been postulated that partial inhibi-tion of γ-secretase might allow reduction of Aβ production whileleaving intact other γ-secretase activities. Indeed, repeated doses ofactive γ-secretase inhibitor within an appropriate dose range partial-ly decreased Aβ production in our experiments and increased theresistance of hAPPFAD cells to levels found in hAPPWT cells. Thistreatment also slightly increased the resistance of hAPPWT cells,which produce some Aβ, suggesting that susceptibility to apoptosisis proportional to the amount of Aβ42 produced and that partial inhi-

bition of γ-secretase may indeed increase the survival of neurons,even in cells expressing hAPPWT at near-physiological levels.

Interestingly, hAPPWT inhibited apoptosis induced by DNA-dam-aging insults, even when it was retained in the endoplasmic reticu-lum and intermediate compartment (Figure 4). This finding sug-gests that its neuroprotective effect does not involve secreted formsof APP or the transduction of signals by APP that is anchored in thesurface membrane. Studies are under way to determine whether theantiapoptotic effect of APP in the endoplasmic reticulum and inter-mediate compartment is mediated by interactions of APP or itsintracellular domain with adapter proteins, inhibitors of the Notchintracellular domain, p53, or other transcription factors that mightmodulate cell death and survival signals in the nucleus.

Selected References

Hsia A, Masliah E, McConlogue L, Yu G, Tatsuno G, Hu K,Kholodenko D, Malenka R, Nicoll R, Mucke L (1999) Plaque-inde-pendent disruption of neural circuits in Alzheimer’s disease mousemodels. Proc. Natl. Acad. Sci. USA 96:3228–3233.

Mucke L, Masliah E, Yu G-Q, Mallory M, Rockenstein EM, TatsunoG, Hu K, Kholodenko D, Johnson-Wood K, McConlogue L (2000)High-level neuronal expression of Aβ1-42 in wild-type human amy-loid protein precursor transgenic mice: Synaptotoxicity withoutplaque formation. J. Neurosci. 20:4050–4058.

Masliah E, Rockenstein E, Veinbergs I, Sagara Y, Mallory M,Hashimoto M, Mucke L (2001) β-Amyloid peptides enhance α-synu-clein accumulation and neuronal deficits in a transgenic mouse modellinking Alzheimer’s disease and Parkinson’s disease. Proc. Natl. Acad.Sci. USA 98:12245–12250.

Wyss-Coray T, Lin C, Yan F, Yu G-Q, Rohde M, McConlogue L,Masliah E, Mucke L (2001) TGF-β1 promotes microglial amyloid-βclearance and reduces plaque burden in transgenic mice. Nat. Med.7:612–618.

Buttini M, Shockley K, Yu G-Q, Masliah E, Mallory M, Yeo T, LongoFM, Mucke L (2002) Modulation of Alzheimer-like synaptic andcholinergic deficits in transgenic mice by human apolipoprotein Edepends on isoform, aging and overexpression of Aβ but not onplaque formation. J. Neurosci. 22:10539–10548.

Raber J, Bongers G, LeFevour A, Buttini M, Mucke L (2002)Androgens protect against apoE4-induced cognitive deficits. J.Neurosci. 22:5204–5209.

Wyss-Coray T, Mucke L (2002) Inflammation in neurodegenerativedisease—a double-edged sword. Neuron 35:419–432.

Palop JJ, Jones B, Kekonius L, Chin J, Yu G-Q, Raber J, Masliah E,Mucke L (2003) Neuronal depletion of calcium-dependent proteins inthe dentate gyrus is tightly linked to Alzheimer’s disease-related cog-nitive deficits. Proc. Natl. Acad. Sci. USA. In press.


2002 ANNUAL REPORT

Figure 4. Inhibition of Aβ production increases the resistance of hAPPFAD cellsto apoptosis. Stably transfected B103 cells were plated in selection medium,differentiated in N2 medium, exposed to camptothecin (to induce apoptosis),and analyzed by cell death detection ELISA. The extent of apoptosis isexpressed relative to control cultures that express the same construct butwere exposed to vehicle instead of camptothecin. * p < 0.001 relative to allother cell lines (Tukey-Kramer posthoc test). WT, wildtype hAPP; FAD, hAPPcarrying the V717I mutation; WT/ER and FAD/ER, hAPPWT and hAPPFAD con-structs carrying a mutation that results in the retention of hAPP in the endo-plasmic reticulum and intermediate compartment; FAD/β–, hAPPFAD constructcarrying a mutation that prevents cleavage by β secretase.


Senior InvestigatorRobert E. Pitas, Ph.D.

Postdoctoral FellowsLorenzo Arnaboldi, Ph.D.Paul C. R. Hopkins, Ph.D.Juan Santiago-García, Ph.D.

PITAS LABORATORY

Research AssociateRichard M. Stewart

Administrative AssistantEmily K. O’Keeffe

Figure 1. EBP is predicted to contain two coiled-coil domains and, near the carboxylterminus, two transmembrane (TM) helices. EBP has no significant sequence iden-

tity with characterized protein families. Due to the frequent involvement of coiled-coildomains in multimerization, EBP is shown here as a dimer. aa, amino acids.

Molecular Mechanisms Contributing to Neurological Disorders

Robert E. Pitas, Ph.D.

Our laboratory is interested in identifying previously unchar-acterized proteins and mechanisms that contribute to thedevelopment of neurological disorders. These studies may

lead to new strategies for treatment. Mutations in several proteinshave been linked to the development of Alzheimer’s disease (AD);however, the mechanism by which many of these mutant proteinscontribute to the disease has not been elucidated. One of the mostimportant risk factors for the development of AD is inheritance of atleast one apolipoprotein (apo) E4 allele. ApoE is a lipid-binding pro-tein that is produced in the brain and other organs. ApoE has beenexamined for its interaction with proteins known to be important forthe pathogenesis of AD, including the amyloid beta (Aβ) peptide andtau components of the defining lesions of AD: neuritic plaques andneurofibrillary tangles. However, the extent to which these interac-tions increase the risk of developing AD remains uncertain. We aretesting the hypothesis that apoE4 contributes to AD and other neu-rological diseases through its interactions with intracellular proteinsthat are important for the maintenance of neuronal plasticity orthrough its effects on the metabolism of lipids in the central nervoussystem (CNS). We have, therefore, begun to identify and character-ize brain-derived apoE-binding proteins and to fully characterizelipid abnormalities that occur in the brain during the initiation andprogression of neurological disorders.

ApoE-Binding Proteins in the Brain

We have continued to investigate a CNS protein (EBP) that we iden-tified by its binding to apoE in a yeast two-hybrid screen of a human

brain cDNA library. BLAST searches showed that EBP is a memberof a previously uncharacterized family of proteins with unknownfunction. The proteins are encoded by four homologous genes inhumans with equivalent genes in mice. There is one related gene inDrosophila, which is predicted to produce a protein with 45% aminoacid identity with human EBP. The mRNAs that encode the humanand mouse forms of this protein are present almost exclusively in thebrain. EBP is predicted to contain a high content of α-helix and toform coiled-coil domains. It contains two predicted transmembranedomains near the carboxyl terminus (Figure 1). The association ofEBP with apoE3, initially identified by the yeast two-hybridapproach, has been confirmed. Fluorescence quenching experimentsusing recombinant EBP and apoE3 showed that the fluorescence ofapoE is increased by coincubation with EBP, indicating an interac-tion between the two proteins. Analysis of coincubated apoE3 andEBP by size-exclusion chromatography also demonstrated an inter-action and suggests that the apoE3–EBP complex has a 1:1 stoi-chiometry. Using assays that separate various components of thecytoskeleton by differential centrifugation, we have determined thatEBP interacts with the cytoskeleton. Furthermore, in the presence ofEBP but not in control cells, apoE3 also associates with thecytoskeleton and is found in the cell pellet of cells transfected toexpress EBP.

We previously reported the results of in situ hybridization studies ofmouse brain sections that revealed panneuronal expression of EBP.These findings were confirmed in primary cultures derived from

fetal mouse brain. Reverse transcription–polymerase chain reactionanalysis demonstrated that EBP mRNA is expressed in neurons butnot in astrocytes.

To further examine the expression pattern of EBP, we have developedpolyclonal antibodies to a mixture of three peptides corresponding tosequences near the amino terminus of the protein and to the recom-binant protein. These antibodies detect both human and mouse EBP.In mouse brain sections, EBP was expressed only in neurons, withthe highest level of expression in Purkinje cells of the cerebellum,neurons of the hippocampus, cerebral cortex, interpeduncular nucle-us, habenular complex, cochlear nucleus, and olfactory bulb. In thehippocampus, EBP was highly expressed in cell bodies of thepyramidal neurons in the CA1, CA2, and CA3 layers and in the den-tate gyrus. EBP was also observed in cell bodies within the molecu-lar and granular layers of the hippocampus.

We have begun to analyze EBP expression in brain sections fromcontrol and Apoe–/– mice (Figure 2A and B) and from Apoe–/– miceexpressing human apoE3 or apoE4 (Figure 2C and D). In the hip-pocampus of wildtype mice, predominant immunoreactivity for EBPwas seen in the cell bodies of neurons in the hippocampus.Interestingly, in Apoe–/– mice, the EBP staining pattern was muchmore diffuse, suggesting that apoE expression affects the distribu-tion of EBP. These results were confirmed and extended by examin-ing the EBP expression in the hippocampus of Apoe–/– mice express-ing apoE3 or apoE4 in astrocytes. Diffuse staining was observed, asin the Apoe–/– mice; even more interestingly, however, EBP aggre-gates were seen in the hippocampus in apoE4 mice, but not inapoE3, Apoe–/–, or wildtype mice (Figure 2).

In a different line of investigation, we analyzed the expression ofEBP in transgenic mice expressing amyloid precursor protein (APP)carrying the Swedish and Indiana mutations, which leads to highlevels of Aβ peptide formation, cognitive impairment, and neurode-generation. We also observed EBP aggregates in the hippocampus of

these mice but not in littermate controls. Currently, we are investi-gating the nature of these EBP deposits and their relationship to neu-rodegeneration.

Interestingly, we have also found that stable expression of EBP pro-tects Neuro-2a cells from H2O2-induced cell death. The resultsobtained with stably transfected cells were confirmed and extendedusing Neuro-2a cells transiently transfected to express EBP. Thetransiently transfected cells are a mixed population of nonexpressingcells and cells with a range of EBP expression (Figure 3). These cellswere either not treated or were incubated with H2O2, fixed,immunostained with anti-EBP and a fluorescein-labeled secondantibody, and analyzed with a fluorescence-activated cell sorter. Weobserved a decrease in the relative number of nonexpressing cellsand a preferential survival of EBP-expressing cells after H2O2 treat-ment. We are currently determining the mechanism by which EBPprotects against cell death induced by oxidative stress.

Since EBP was previously uncharacterized, is highly conservedthroughout evolution, and is expressed exclusively by neurons, wehave initiated studies to assess its function in mice using gene knock-out technology and in Drosophila using double-stranded RNA-medi-ated gene silencing, also called RNA interference (RNAi). Wesequenced ~19 kilobases (kb) of the murine EBP gene and deter-mined the gene structure. The gene consists of five exons; exon 1contains a 5′ untranslated region, and exons 2–5 contain the codingregion. From online databases and our sequence, we determined thatthe mouse gene spans more than 40 kb. We designed and constructeda conditional knockout vector to delete exon 2, which contains thetranslation start site. LoxP sites were introduced to allow cell- or tis-sue-specific elimination of expression by breeding with Cre-deletermice. This vector was introduced into mouse embryonic stem cells.Targeted cells were selected, verified, and injected into C57BL/6blastocysts at the Gladstone Blastocyst Core. Chimeric mice havebeen generated. These mice will be bred to obtain mice with germlinetransmission and then crossed with Cre-deleter mice.


2002 ANNUAL REPORT

Figure 2.Immunostaining of EBP inmouse brain sections.Vibratome sections wereimmunostained withanti-EBP. Sections fromhippocampus of wildtype(WT) (A) or Apoe–/– (B)mice or from Apoe–/– miceexpressing human apoE3(C) or apoE4 (D) inastrocytes are shown.Black arrowheads indicateEBP aggregates in apoE4transgenic mice. Theseaggregates are shown athigher magnificationin the inset.

In experiments performed in collaboration with Dr. Fen-Biao Gao,we suppressed the expression of mRNA for the Drosophila homo-logue of EBP using the RNAi approach. The consequence of organ-ism-wide ablation of expression is a delay in the onset of pupationand mortality during the early stages of metamorphosis, a time thatcorrelates with increased expression of EBP in the CNS. We are cur-rently investigating the effects of cell-specific ablation of expression.

Lipid Metabolism and Neurological Disease

Several lines of evidence suggest that altered lipid metabolism isassociated with the development of neurological disease. A relative-ly consistent finding in humans with AD is a decrease in the phos-phatidylethanolamine (PE) content of the brain. PE exists in twoforms, one having two fatty acids linked to the glycerol backbone ofthe PE by ester bonds (diacyl PE) and the other having an alkylgroup at position one of the phospholipid in an α, β-unsaturatedether linkage (plasmalogen or alkenylacyl PE). The decrease in thePE content in AD brains is due primarily to a decrease in the plas-malogen component of PE. However, it is unclear whether or not thedecrease in plasmalogen is related to the etiology of the disease or issimply a result of the changing cellular composition of the brain.

As a new initiative in our laboratory, we have begun to examine thisquestion in transgenic mouse models with certain features of AD.We have established the techniques required for a detailed charac-

terization of CNS lipids. This involves extracting lipids from brain,separating all lipid classes by thin-layer chromatography, and quan-titating each phospholipid class (phosphatidylcholine, PE, sphin-gomyelin, phosphatidylserine, and phosphatidylinositol). The PE isthen separated into the diacyl and plasmalogen forms and quantitat-ed, and the composition is determined by gas-liquid chromatogra-phy. In collaboration with Dr. Lennart Mucke, we have performedinitial studies in control mice and transgenic mice from the J20Aline, which express human APP with both the Swedish and Indianamutations. These mice develop Aβ deposits and cognitive impair-ments. We have analyzed the hippocampus, cortex, and cerebellumseparately. Preliminary results showed a decrease in PE plasmalogenin the hippocampus of 18-month-old transgenic mice (32% versus41% in controls). We are currently analyzing the lipid content ofJ20A mouse brains to determine the temporal pattern of the changesin PE composition. These studies will show whether the changesprecede or follow the appearance of Aβ deposits and the relationshipof the changes in composition to the onset of cognitive decline. Wewill then extend these analyses to examine the effect of apoE3 andapoE4 expression in the brain on lipid composition with and withoutthe coexpression of mutant APP.

Selected References

Raber J, Wong D, Buttini M, Orth M, Bellosta S, Pitas RE, MahleyRW, Mucke L (1998) Isoform-specific effects of human apolipopro-tein E on brain function revealed in ApoE knockout mice: Increasedsusceptibility of females. Proc. Natl. Acad. Sci. USA 95:10914–10919.

Buttini M, Orth M, Bellosta S, Akeefe H, Pitas RE, Wyss-Coray T,Mucke L, Mahley RW (1999) Expression of human apolipoproteinE3 or E4 in the brains of Apoe–/– mice: Isoform-specific effects onneurodegeneration. J. Neurosci. 19:4867–4880.

Buttini M, Akeefe H, Lin C, Mahley RW, Pitas RE, Wyss-Coray T,Mucke L (2000) Dominant negative effects of apolipoprotein E4revealed in transgenic models of neurodegenerative disease.Neuroscience 97:207–210.

Raber J, Wong D, Yu G-Q, Buttini M, Mahley RW, Pitas RE, MuckeL (2000) Apolipoprotein E and cognitive performance. Nature404:352–354.

Santiago-García J, Mas-Oliva J, Innerarity TL, Pitas RE (2001)Secreted forms of the amyloid-β precursor protein are ligands for theclass A scavenger receptor. J. Biol. Chem. 276:30655–30661.

Hopkins PCR, Santiago-García J, Hahn SL, Stewart RM, Do NL,Gao FB, Pitas RE (2002) A neuronal intracellular apoE-binding pro-tein. Soc. Neurosci. 29:883.10 (abstract).



Figure 3. Neuro-2a cells were transiently transfected to express EBP and thenincubated in the presence or absence of 150 µM H2O2 for 24 hours. Cells werelabeled with anti-EBP and analyzed by fluorescence-activated cell sorting.


Structure and Function of Apolipoprotein E

Karl H. Weisgraber, Ph.D.

Senior InvestigatorKarl H. Weisgraber, Ph.D.

Visiting ScientistLiming Dong, Ph.D.

Research ScientistRobert L. Raffaï, Ph.D.

WEISGRABER LABORATORY

Postdoctoral FellowsDanny M. Hatters, Ph.D.Clare A. Peters-Libeu, Ph.D.

Senior Research AssociatesKay S. ArnoldYvonne M. Newhouse

Research AssociatesSam LoebMaya Mathew, M.S.Maryam A. TabarChunyao Xia, M.D.

Executive AssistantBrian Auerbach

Our research focuses on the structural and functional relation-ships of apolipoprotein (apo) E in lipoprotein metabolism,heart disease, and neurodegenerative diseases, including

Alzheimer’s disease. ApoE is a 299–amino acid, single-chain proteinwith two structural domains that also define functional domains(Figure 1). The three common human isoforms, apoE2, apoE3, andapoE4, differ at two positions in the molecule and have very differentmetabolic properties and effects on disease. ApoE3 (Cys-112, Arg-158) binds normally to low density lipoprotein (LDL) receptors and isassociated with normal lipid metabolism, whereas apoE2 (Cys-112,Cys-158) binds defectively to LDL receptors and, under certain cir-cumstances, is associated with the genetic disorder type III hyper-lipoproteinemia. ApoE4 (Arg-112, Arg-158) binds normally to LDLreceptors but is associated with elevated cholesterol levels andincreased risk for cardiovascular disease. In addition, apoE4 is a majorrisk factor for Alzheimer’s disease and predictor for poor outcomefrom head injury. Our objective is to determine how the structural andbiophysical properties of apoE influence its metabolic properties andcontribute to its isoform-specific effects in disease and injury.

X-Ray Crystallography

The structures of the amino-terminal domains of apoE2, apoE3, andapoE4 in the lipid-free state have been determined; all three structuresadopt a four-helix-bundle motif (Figure 1). However, subtle differ-ences in side-chain conformations and in salt-bridge arrangements ofthe isoforms affect their functions and characteristics. In addition,since apoE likely performs most, if not all, of its functions in a lipid-associated state, a major focus is to determine the influence of lipidbinding on apoE structure and function.

The successful crystallization of apoE complexed with the phospho-lipid dimyristoylphosphatidylcholine (DMPC) was a breakthrough instudying the interaction of apoE with lipid. This exciting result raisesfor the first time the possibility of obtaining detailed structural infor-mation on protein–lipid complexes. This is important for apoE func-tion because high-affinity binding to LDL receptors requires lipidassociation.

Figure 1. The two-domain structure of apoE. As determined by x-ray crystal-lography, the amino-terminal domain assumes a four-helix-bundle folding motif.The structure of the carboxyl-terminal domain is not known and is depicted asa series of α-helices, consistent with circular dichroism measurements. Thereceptor binding region of apoE is located in the amino-terminal domain onhelix 4. The carboxyl-terminal domain contains the elements for binding tospherical lipoprotein particles (boxed area). Amino acid differences at position112 distinguish apoE3 (Cys) and apoE4 (Arg). ApoE3 displays a lipoproteinpreference for HDL, whereas apoE4 displays a preference for VLDL. The con-cept of domain interaction was introduced to account for the influence of thepolymorphic site at position 112 in the amino-terminal domain on the lipid-binding properties of the carboxyl-terminal domain.

ApoE4•DMPC crystals displayed a fiber-like diffraction patternwith a unit cell spacing of 54 Å along the fiber axis and cell spac-ings of ~150 Å and ~300 Å, respectively, for the two axes approxi-mately perpendicular to the fiber axis. These findings are consistentwith the model that the apoE•DMPC discs (~150 Å in diameter and55 Å thick) stack to form long, fiber-like rod structures. The stack-ing of the discs appears to be well defined along the fiber axis, asindicated by the resolution extending to about 7–9 Å along this axis.The connection between the resulting rod-like fibers is less definedand extends to only ~15 Å, indicating a weaker stacking interactionalong the long cell axes where the sides of the discs touch each other.Recently, improved crystals were obtained by substituting dipalmi-toylphosphatidylcholine for DMPC. These crystals diffract toapproximately 8 Å along all three axes.

ApoE Isoform Differences

We have identified three structural and biophysical differencesamong the three apoE isoforms: (1) apoE4 domain interaction; (2)protein stability and folding; and (3) cysteine content (Figure 2). Ourworking hypothesis is that one or more of these differences areresponsible for the isoform-specific effects of apoE4 on plasmalipoprotein metabolism, atherosclerosis, and neurodegeneration. Totest this hypothesis, we will engineer each of these isoform differ-ences individually into the mouse Apoe gene by gene targeting. Inthis way, we can determine the contribution of each difference to theisoform-specific effects that are known to occur in plasma and thebrain (Figure 2). Sorting out the relative contributions of these dif-ferences has important implications in developing effective, apoE4-based therapeutic strategies.



Figure 2.Effect of apoE4 structure onfunction. To assess the impactof apoE4 structure on function,our long-term objective is tointroduce each of the knownisoform differences individuallyinto the mouse apoE gene bygene targeting. This approachwill provide mouse models toassess the relative contribu-tions of these differences toknown isoform-specific effectsin plasma (lipoprotein metabo-lism and atherosclerosis) andbrain (amyloid-β metabolismand fibrillogenesis, cognitivebehavior, lipid transport, andneuronal repair).

Figure 3. ApoE4 domain interaction. In apoE3, with cysteine at position 112,the Arg-61 side chain is positioned in a cleft between two helices and cannotinteract with the carboxyl-terminal domain. In apoE4, with arginine at position

112, however, the Arg-61 side chain projects into the aqueous environment,where it can interact with Glu-255, thereby mediating domain interaction,resulting in a different overall tertiary structure for apoE4.

ApoE4 Domain Interaction. ApoE4 binds preferentially to verylow density lipoproteins (VLDL), whereas apoE3 binds preferen-tially to high density lipoproteins (HDL) (Figure 1). We determinedthat the two domains in apoE4 interact and that this interaction isrestricted to apoE4. The interaction is mediated by Arg-61 (amino-terminal domain) and Glu-255 (carboxyl-terminal domain) and isresponsible for the VLDL binding preference of apoE4 (Figure 3).

We are collaborating with Drs. Fred Cohen and Irwin Kuntz (UCSF)and using their DOCK program to identify small molecules that willbind to apoE4 in the vicinity of Arg-61 but not to apoE3 and there-by interfere with domain interaction. We expect that such moleculeswill represent a therapeutic approach by converting apoE4 into an“apoE3-like” molecule (Figure 3). Sixty-five candidate compoundswere identified from the DOCK computer search and tested in anemulsion binding assay. In the initial screen, 13 of the 65 compoundswere found to reduce the apoE4 emulsion binding levels to theapoE3 range, indicating that they were interfering with apoE4domain interaction. In a second round of screening, 8 of the 13 pos-itive compounds repeated their interference with apoE4 domaininteraction. Two of these compounds, GIND-25 and -105, reducedemulsion binding, with neither compound affecting apoE3 binding(Figure 4).

In several species, including the mouse, apoE contains arginine andglutamic acid at positions equivalent to positions 112 and 255,respectively, in human apoE. However, these species lack the criti-cal human Arg-61 required for domain interaction. Their apoE con-tains threonine and, like human apoE3, displays a preference forHDL. Based on these observations, we used a “knock-in” gene tar-geting approach to introduce an arginine codon into the mouse geneto “humanize” mouse apoE at position 61 and introduce domaininteraction.

In heterozygous targeted mice, as in human apoE4 heterozygotes,the plasma level of the Arg-61 form is 20–40% lower than that ofwildtype apoE. This characteristic pattern reflects the more rapidclearance of apoE4 from plasma. The “Arg-61” mouse apoE alsodisplays the expected preference for VLDL. These results demon-strate that domain interaction was successfully introduced in vivo.Since the effects of Arg-61 apoE on plasma lipoprotein metabolism

in the Arg-61 mice were similar to those of apoE4 in humans, wewere eager to determine if domain interaction affects the centralnervous system of these mice. In a pilot study, aged Arg-61 mice(14–18 months old) and wildtype, age-matched controls were inject-ed with kainic acid to elicit cytotoxic neuronal injury. Mice (10mice/group) were injected intraperitoneally with either kainic acid(18 mg/kg) or saline as a control. After 6 days, the mice were sacri-ficed, and brain sections were stained with a monoclonal anti-mouseantibody to synaptophysin, a marker for dendrites, followed by afluorescein isothiocyanate-conjugated secondary antibody. Imageswere captured by laser-scanning confocal microscopy and analyzedand quantified for synaptic content. Our preliminary results show asignificant loss of synaptophysin immunoreactivity in the kainicacid–treated Arg-61 mice compared with the controls. These excit-ing results mirror those obtained by other investigators at theGladstone Institute of Neurological Disease using this injury modelin human apoE4 and apoE3 transgenic mice. More importantly, thestudy demonstrates that the effects of apoE4 domain interaction canbe observed in the central nervous system and that domain interac-tion plays a key role in the loss of synaptic connections after kainicacid injury.

Protein Stability and Folding. In addition to structural differencesamong protein isoforms, biophysical properties are important deter-minants of their functional properties. An emerging concept in pro-tein folding is that the stable folding intermediate, or molten glob-ule, represents a third thermodynamic state that a protein mayassume. A molten globule has a semi-rigid structure that is almostas compact as the native structure. It retains most of the secondarystructure and much of the tertiary structure of the native state.Owing to the partial loss of tertiary structure, it usually contains anexposed hydrophobic surface. Until recently, it had been assumedthat the molten globule was a relatively rare occurrence. However,there is a large body of experimental evidence that the molten glob-ule state is a common feature of most proteins and that molten glob-ules can exist in cells and play key roles in a wide variety of phys-iological processes, including translocation across membranes,increased affinity for membranes, binding to liposomes and phos-pholipids, protein trafficking, extracellular secretion, and regulationof the cell cycle.

To compare the physical characteristics of the apoE isoforms, we con-ducted guanidine and thermal denaturation studies of apoE2, apoE3,and apoE4 and their 22- and 10-kDa fragments. Guanidine denatura-tion demonstrated that the two domains unfold independently in thethree isoforms. However, the amino-terminal fragments of the apoEisoforms differed in stability. ApoE4 denatured at the lowest guanidineconcentration and temperature, while apoE2 denatured at the highestconcentration and temperature. Furthermore, the denaturation datasuggested that apoE4, unlike apoE2 and apoE3, did not fit a two-statedenaturation equilibrium. The lack of cooperative unfolding suggeststhat apoE4 forms a stable, partially folded intermediate.

Since folding intermediates are often more stable at an acidic pH, weexamined urea denaturation of the three 22-kDa fragments at pH 4.0.Fitting the data with a two- or three-stage model demonstrated thatapoE2 exhibits cooperative two-stage unfolding, whereas bothapoE3 and apoE4 display three-stage unfolding, indicating the pres-ence of stable folding intermediates under these conditions. Analysisof denaturation curves revealed that the stable intermediate of apoE4represents approximately 90% of the mixture at 3.75 M urea, where-as the intermediate of apoE3 represents approximately 30% at thisurea concentration and was increased to approximately 80% at 4.75M urea. The apoE2 fragment did not display a folding intermediate.


2002 ANNUAL REPORT

Figure 4. Effect of candidate compounds on binding of apoE to emulsion parti-cles. The binding of 125I-labeled apoE3 and apoE4 to triolein and phospholipidemulsion particles was determined in the absence and presence of candidatecompounds and the percentage of protein that bound to the emulsions wasdetermined. The results are the average of two determinations.

In collaboration with Drs. Anthony Fink and Keith Oberg(University of California, Santa Cruz), we used Fourier transmit-tance reflective infrared analysis to examine the characteristics ofthe apoE4 folding intermediate in the absence and presence of ureaat pH 4.0. In the absence of urea, the secondary structure of theapoE4 22-kDa fragment was estimated to consist of 75% α-helixand 3% β-sheet, consistent with previous estimates. In 3.75 M urea,apoE4 consisted of 46% α-helix and 17% β-sheet. Thus, the apoE4intermediate contained 61% of the original helical content and hadan increased β-sheet structure. Pepsin proteolysis of apoE4 at pH 4.0in 0 M and 3.75 M urea showed cleavages between helices 2 and 3,within helix 3, and between helices 3 and 4 in the presence of urea.These results indicate that there is a conformational change in apoE4at pH 4.0 in the presence of urea. We speculate that the four-helixbundle is partially unfolded, similar to the unfolded structure whenthis fragment binds to lipid.

Characterization of the apoE4 stable folding intermediate indicatesthat it is a molten globule. Since molten globules have been impli-cated in a variety of physiological processes, including membranebinding and translocation, we examined the ability of apoE4 andapoE3 to bind to and disrupt DMPC vesicles at pH 4.0, with andwithout urea. Under both conditions, apoE4 was more effective thanapoE3, suggesting that the apoE4 molten globule may be involved inmembrane translocation.

Cysteine Content. The three major isoforms differ in cysteine con-tent: apoE4, apoE3, and apoE2 contain 0, 1, and 2 cysteines, respec-

tively. As a result, apoE2 and apoE3 can form both disulfide-linkedhomodimers and heterodimers with apoA-II. In apoE3/3 subjects,approximately 50% of the apoE3 exists in one of these disulfide-linked forms. In addition, the apoE3 homodimer is present in cere-brospinal fluid. These disulfide-linked dimers affect lipid-bindingproperties and interaction with LDL receptors, but their effect inneurobiology has not been systematically assessed. Since mouseapoE also lacks cysteine, a future objective will be to use gene tar-geting to introduce a cysteine codon to produce the functional equiv-alent of apoE3 and determine its effect on neurodegeneration.

Selected References

Dong L-M, Weisgraber KH (1996) Human apolipoprotein E4domain interaction. Arginine 61 and glutamic acid 255 interact todirect the preference for very low density lipoproteins. J. Biol.Chem. 271:19053–19057.

Weisgraber KH, Mahley RW (1996) Human apolipoprotein E: TheAlzheimer’s disease connection. FASEB J. 10:1485–1494.

Raffaï RL, Dong L-M, Farese RV Jr, Weisgraber KH (2001)Introduction of human apolipoprotein E4 “domain interaction” intomouse apolipoprotein E. Proc. Natl. Acad. Sci. USA 98:11587– 11591.

Morrow JA, Hatters DM, Lu B, Höchtl P, Oberg KA, Rupp B,Weisgraber KH (2002) Apolipoprotein E4 forms a molten globulestate: A potential basis for its association with disease. J. Biol. Chem.277:50380–50385.




Behavioral Core Laboratory

Senior InvestigatorLennart Mucke, M.D.

Research ScientistKimberly A. Scearce-Levie, Ph.D.

BEHAVIORAL CORE LABORATORY

Postdoctoral FellowJukka Puoliväli, Ph.D.

Research AssociateLisa Kekonius

Administrative AssistantLeslie Manuntag

The Behavioral Core Laboratory was established to analyzenervous system functions in experimental mouse models ofhuman neurological diseases. Through collaborative inter-

actions and consultations, it has served scientists at all threeGladstone Institutes and other investigators at the SFGH campus,as well as colleagues at the Gallo Center, the Mount Zion andParnassus campuses of UCSF, UC Berkeley, and other institutions.

Our focus has been the behavioral evaluation of mouse models ofhuman dementing illnesses. Understanding what impairs learningand memory in these models is providing important insights intoboth central nervous system functions and clinically relevant dis-ease processes. For example, together with Dr. Bruce Miller andhis colleagues at the UCSF Memory and Aging Center, we areinvestigating specific links between cognitive impairments inpatients with dementia and behavioral deficits in mouse models ofthese diseases. A major long-term goal of this interaction is thedevelopment of suitable tests and novel treatment strategies toimprove cognition in patients suffering from Alzheimer’s disease(AD) and related conditions.

Behavioral Tests

We routinely use a comprehensive battery of tests (Table 1) to fullycharacterize the neurological condition of mouse models and tovalidate complex learning paradigms. In assessing complex behav-iors, it is crucial to determine if there are specific deficits in morebasic functions. For example, it is important to distinguish learn-ing impairments from performance deficits. Vestibular deficits areoften associated with increased horizontal locomotor activity,including circling, reduced rearing (raising of both forefeet off theground and extension of the body), abnormal posture, and poorswimming ability. In assessing olfactory memory, reduced abilityto detect a particular odorant may be a confounding factor. In thewater maze test, which assesses spatial learning and memory,vision and motivation are required to locate a hidden platform byusing visual cues outside the maze. To assess visual and motiva-tional problems, we test the ability of mice to locate a visible plat-form. The swim speed of the mice is also measured as an indicatorof motivation, motor function, and coordination, all of which can

influence the time required to locate the platform (latency).Metabolic alterations are confounding factors in certain tests, suchas the holeboard test, in which mice learn to locate a food or waterreward by poking their heads into a baited hole.

Complex behaviors are regulated by many genes, and different strainsof mice vary in their ability to master different tests. Therefore, theprotocols for the behavioral testing of disease models established ondifferent genetic backgrounds often must be adapted to make the taskneither so easy that all mice can perform it equally well nor so diffi-cult that none of the mice can perform it successfully. The followingsection and the selected references highlight some of the studies towhich this core facility has made significant contributions.

Effects of Apolipoprotein E and Amyloid Peptideson Learning and Memory

The three major human apolipoprotein (apo) E isoforms (E2, E3, andE4) differ in their effect on AD. Compared with apoE2 and apoE3,apoE4 increases the risk of AD and lowers the age of onset. ApoE4appears to interact with female gender, further increasing the risk ofAD and diminishing the effectiveness of treatments in women. Toassess how interactions between gender and apoE isoforms affect cog-nition, we studied female and male mice lacking mouse apoE(Apoe–/–) and expressing human apoE3 or apoE4 in the brain at com-parable levels. As they aged, female, but not male, apoE4 (Apoe–/–)mice developed progressive impairments in spatial learning and mem-ory in the water maze test, compared with age- and sex-matched miceexpressing apoE3, endogenous mouse apoE, or no apoE at all.

Subsequent studies revealed that androgens and androgenreceptor–dependent pathways protect against the detrimental effectsof apoE4 on cognition. Even brief periods of androgen treatmentreduced memory deficits in female apoE4 mice. In addition, apoE4male mice, which performed normally in a water maze test at baseline,developed prominent deficits in spatial learning and memory afterblockade of androgen receptors, whereas apoE3 male mice did not.Cognitive performance likely depends on a critical balance betweenplasma androgen levels and cytosolic androgen receptor levels in thebrain, and the higher endogenous plasma testosterone levels in maleapoE4 mice may provide a relative protection.



Table 1. Behavioral tests


2002 ANNUAL REPORT

While apoE4 is the best established genetic susceptibility factor forAD, neurotoxic assemblies of the amyloid β peptide (Aβ) haveemerged as the likeliest primary cause of the illness. When we com-bined the expression of apoE4 with the expression of human Aβ,both male and female mice developed deficits in learning and mem-ory, consistent with the fact that both men and women develop AD.Interestingly, apoE3 prevented or delayed Aβ-induced cognitivedeficits, whereas apoE4 did not. These results may relate closely tothe accelerated onset of AD in human apoE4 carriers and provideuseful preclinical outcome measures for therapeutic strategies aimedat Aβ, apoE4, or their pathogenic interactions. As described in thereports by Drs. Lennart Mucke, Robert Mahley, and Yadong Huang,we have made much progress in identifying the mechanisms bywhich these molecules erode cognitive functions.

Selected References

Raber J, Wong D, Buttini M, Orth M, Bellosta S, Pitas RE, MahleyRW, Mucke L (1998) Isoform-specific effects of human apolipoproteinE on brain function revealed in Apoe knockout mice–Increased sus-ceptibility of females. Proc. Natl. Acad. Sci. USA 95:10914– 10919.

Raber J, Akana SF, Bhatnagar S, Dallman MF, Wong D, Mucke L(2000) Hypothalamic–pituitary–adrenal dysfunction in Apoe–/– mice:Possible role in behavioral and metabolic alterations. J. Neurosci.20:2064–2071.

Raber J, Wong D, Yu G-Q, Buttini M, Mahley RW, Pitas RE, MuckeL (2000) Alzheimer’s disease: Apolipoprotein E and cognitive per-formance. Nature 404:352–354.

Smith SJ, Cases S, Jensen DR, Chen HC, Sande E, Tow B, SananDA, Raber J, Eckel RH, Farese RV Jr (2000) Obesity resistance andmultiple mechanisms of triglyceride synthesis in mice lackingDGAT. Nat. Genet. 25:87–90.

Masliah E, Rockenstein E, Veinbergs I, Sagara Y, Mallory M,Hashimoto M, Mucke L (2001) β-Amyloid peptides enhance α-synuclein accumulation and neuronal deficits in a transgenic mousemodel linking Alzheimer’s disease and Parkinson’s disease. Proc.Natl. Acad. Sci. USA 98:12245–12250.

Raber J, Bongers G, LeFevour A, Buttini M, Mucke L (2002)Androgens protect against apoE4-induced cognitive deficits. J.Neurosci. 22:5204–5209.


Genomics is the study of gene populations. The goal of theGenomics Core is to assist Gladstone scientists in their stud-ies of the unprecedented volume of information resulting

from the Human Genome Project and related projects describing thegenomes of other model organisms. Services are provided toGladstone scientists and, as resources allow, to the greater UCSF com-munity as well as investigators at other institutions. This past year hasbeen one of continued growth and change within the Genomics Coreas it expands to meet the needs of the Gladstone research community.

More specifically, the core makes available genomics technology suchas DNA microarrays. These techniques allow scientists to examine thetissues of a transgenic mouse or human with a disease by assessing theexpression levels of all the genes that could be expressed in each tis-sue. DNA microarrays contain oligonucleotides corresponding to thesequences of known or partially characterized genes on distinctregions of glass slides. By hybridizing tissue samples to these microar-rays, the relative abundance of tens of thousands of genes can bedetermined in a single experiment. The ability to monitor many bio-chemical responses, known and unknown, with a single assay is agreat advantage of the technology.

A major event this year was the inauguration of our printed oligonu-cleotide microarray service. Major roadblocks to printed microarrayshave been the steep learning curve and high failure rate associatedwith obtaining quality data. During the past two years, the core hasdevoted a large part of its resources to developing printed oligonu-cleotide microarrays and the technology to process them quickly, reli-ably, and consistently. Early this year, we completed a successful testof our systems with the assistance of the Verdin laboratory at theGladstone Institute of Virology and Immunology (GIVI). Sampleswere prepared according to our directions and then run on 60 arraysover 6 weeks without a single failure. Most laboratories running thesearrays expect to have failure rates as high as 50%. This successful finaltest allowed us to introduce our printed microarray service in June.

Another major event was the introduction of larger printed arrays. Newmouse and human oligonucleotide libraries based on new design algo-rithms were obtained from Operon and have replaced the first-genera-tion libraries. These new libraries contain 16,443 and 21,329 genes,respectively. This means that, for the first time, the size of the printedarrays produced in our laboratory is similar to those produced byindustry leader Affymetrix. In addition, for the first time, we designednew oligonucleotides for another laboratory. Using our PIK70 soft-ware, we designed 460 oligonucleotides against novel genes that hadbeen mapped onto the mouse genome and provided them to theAkhurst laboratory at the UCSF Cancer Center for inclusion in arraysprepared by that facility. In the coming months, we expect to replacePIK70 with software based on newer algorithms. We will continue to

design additional custom oligonucleotides for interested investigatorsto include in arrays produced in this laboratory and others.

Many investigators want to perform experiments when only verysmall amounts of RNA are available. The current generation of pro-tocols for both printed and Affymetrix arrays require 10–20 µg oftotal RNA as starting material. This amount can be prohibitive formany projects. This year, in collaboration with the UCSF SandlerCenter Functional Genomics Core Facility, we developed methods toamplify RNA from small samples. We modified the T7 RNA poly-merase method of Eberwine so that researchers can start with as lit-tle as 50 ng of total RNA. This technique has been applied success-fully to both Affymetrix and custom-printed arrays. However, it islaborious and expensive. We have been providing materials and serv-ices, on a fee-for-service basis, to NuGEN Technologies, a localbiotechnology company that is developing a novel amplificationmethod that is faster, more robust, and less expensive. We are com-pleting arrangements to be a beta test site for this company. If thistechnology lives up to its early promise, we hope to make it availablefor use by our clients in 2003.

Investigators Using Genomics Core Resources

Israel F. Charo, M.D., Ph.D. (Gladstone Institute of CardiovascularDisease). Researchers in the Charo laboratory have initiated a study toexamine the regulation of chemokines and chemokine receptors. Theyare comparing gene expression in the lymph nodes and lungs of CCR2knockout mice with and without Mycobacterium tuberculosis infec-tion. Additional comparisons are being made with correspondingsamples from wildtype mice.

Bruce R. Conklin, M.D. (Gladstone Institute of CardiovascularDisease). The Conklin laboratory has isolated heart samples from sev-eral different transgenic and nontransgenic mouse lines. These lineswere generated to investigate transcriptional regulation in the contextof mild to severe cardiac muscle remodeling and myopathy. Theresults of microarray analyses will allow the investigators to formulatenew hypotheses regarding cardiomyopathy and heart remodeling.

Steven Finkbeiner, M.D., Ph.D. (Gladstone Institute of Neurologi-cal Disease). The Finkbeiner laboratory seeks to understand how Ca2+

regulates the transcription of neuronal genes. They focus on genesmodulated by either the N-methyl-D-aspartate receptor or the L-typevoltage-sensitive calcium channel. Results of these studies could leadto the identification of gene targets that are important for synapticplasticity and could reveal the molecular mechanisms by which thecommon second messenger Ca2+ induces distinct but stimulus-specif-ic adaptive responses.

Warner C. Greene, M.D., Ph.D. (GIVI). The human T-cell lym-photropic virus-1 Tax oncoprotein activates viral gene expression and

Gladstone Genomics Core

Christopher S. Barker, Ph.D.

GLADSTONE GENOMICS CORE

Staff Research ScientistChristopher S. Barker, Ph.D.

Research AssociatesBlanca CabezasKristina Hanspers, M.S.Yanxia Hao, M.S.

Visiting ScientistsAndrea J. BarczakChandi Griffin, M.S.Dionysos Slaga

AdministrativeAssistantEmily K. O’Keeffe


2002 ANNUAL REPORT

alters the expression of a wide array of host genes by inducing hosttranscription factors, such as cyclic AMP response element bindingprotein (CREB), NF-κB, and AP-1. Researchers in the Greene labhave generated Jurkat T-cell lines inducibly expressing wildtype andtwo mutant Tax proteins (M22 and M47, which are defective in NF-κB and CREB activation, respectively). They are using these cell linesto identify and differentiate genes that are regulated by Tax-inducedNF-κB and/or CREB.

Joseph M. McCune, M.D., Ph.D. (GIVI). The McCune laboratoryhas initiated studies to analyze genes upregulated by interleukin 7 inselected subpopulations of human lymphocytes and to analyze genesthat might be differentially expressed in recent thymic emigrants.These experiments require amplification of transcripts from relativelysmall numbers of phenotypically homogeneous populations of cells(isolated by fluorescence-activated cell sorting in the Flow CytometryCore Laboratory) using the new methods described above.

Lennart Mucke, M.D. (Gladstone Institute of NeurologicalDisease). The amyloid precursor protein (APP) and one of its metabo-lites, the amyloid-β peptide (Aβ), play a central role in Alzheimer’sdisease, but it remains unknown how Aβ (or other APP fragments)elicits the progressive decline in the function and survival of braincells associated with this illness. Dr. Christian Essrich in the Muckelab has analyzed transgenic mouse models of Alzheimer’s diseasewith DNA microarrays to determine the effects of APP and Aβ ongene expression in the hippocampus, a brain region critically involvedin learning and memory. This investigation revealed expressionchanges in several gene clusters that might be involved in the neu-rodegenerative and behavioral alterations in these mice. A number ofthese gene expression changes have already been confirmed by quan-titative fluorogenic reverse transcriptase polymerase chain reaction,western blot analysis, or immunohistochemistry. Notably, changes inthe levels of some calcium-dependent gene products in subregions ofthe hippocampus correlated tightly with deficits in learning and mem-ory, suggesting a mechanistically informative relationship to the cog-nitive decline observed in Alzheimer’s disease.

Douglas F. Nixon, M.D., Ph.D. (GIVI). The Nixon laboratory is com-paring the differential gene expression in primary human CD4+ Tcells exposed to HIV-1. This is intended as a first step in characterizingthe changes in apoptotic gene expression that occur in macrophagesand CD4+ T cells in response to HIV-1 exposure.

Eric M. Verdin, M.D. (GIVI). The Verdin laboratory has initiated twoprojects. In the first project, researchers led by postdoctoral fellowHerbert Kasler are looking for novel targets of the class IIa histonedeacetylases (HDACs) in T cells. Class IIa HDACs are calcium sig-nal–dependent corepressors of transcription that play important rolesin muscle differentiation, neuronal survival, and negative selection ofT cells. Recently, they demonstrated a critical role for HDAC7 in theregulation of Nur77, a gene involved in the negative selection ofdeveloping T cells in the thymus. Since then, they have used a num-ber of convergent approaches, with DNA microarrays as a readout, toidentify other genes in T cells that might be regulated by class IIaHDACs. Thus far, they have identified a functional cassette of at leasteight class IIa HDAC-regulated genes besides Nur77 that have beenimplicated in negative selection. Other genes were identified thatappear to be relevant to muscle differentiation and neuronal survival.A substantial number of additional microarray experiments will beperformed in the coming year to confirm and extend these findings.

In the second project, graduate student Prerana Jayakumar is studyinggenes that are transcriptionally regulated by HIV-1 Tat, a proteininvolved in transcription initiation and elongation from the HIV long

terminal repeat. Tat regulates several aspects of cellular function,including cellular activation, apoptosis, and general transcription. Tatcan also transduce into cells and exert effects even from the cell sur-face. Given these varied effects on cellular function, an analysis of cel-lular gene expression in response to various forms of the Tat proteinwill provide insights into HIV-mediated transcriptional regulationwithin the cell and increase our understanding of HIV pathogenesis.These experiments will allow identification of genes that are tran-scriptionally regulated by Tat and should provide insights into themechanism of Tat-mediated effects of HIV-1.

Stephen G. Young, M.D. (Gladstone Institute of CardiovascularDisease). Hepatic steatosis has been clinically associated withincreased risk for cirrhosis, but the mechanisms that mediate thisinjury remain unclear. Dr. Young has developed a strain of mice(Reversa mice) in which the hepatic microsomal triglyceride transferprotein (MTP) is inactivated. While the mice and their liver functionappear normal, he noticed an accumulation of neutral lipids withinhepatocytes when MTP is inactivated. In MTP knockout mice, sus-ceptibility to hepatic injury is increased when challenged by a secondinsult. The Young laboratory has begun to use DNA microarrays onRNA from these livers to determine how the accumulation of intra-cellular lipids enhances this susceptibility to injury.

Kenneth Aldape, M.D. (Department of Pathology, M.D. AndersonCancer Center, Houston, TX). The core provided sample preparationservices to Dr. Aldape’s laboratory for use in microarray expressionstudies through the SFGH General Clinical Research Center. Thegoals of this study were to identify candidate genes that may be impor-tant for the pathogenesis of glioblastomas, to identify genes importantfor prognosis, and to group glioblastomas according to expression pat-terns to identify new targets that might be exploited therapeutically.

Harold Bernstein, M.D., Ph.D. (Department of Pediatrics, UCSF).Muscle hypertrophy occurs in the heart and skeletal muscle as anadaptive process in response to various physiological and pathologicalstresses. The abnormal hemodynamic states associated with congeni-tal heart disease frequently lead to hypertrophy. Although it may becompensatory in the short term, such pathological hypertrophy even-tually outstrips the heart’s metabolic resources, leading to cell deathand heart failure. Therefore, this project seeks to understand the roleof the cell-cycle machinery in the hypertrophic response.

Harold Chapman, M.D. (Cardiovascular Research Institute, UCSF).The project is focused on the interaction between integrin and theurokinase receptor. The goal is to identify the downstream genes reg-ulated by expression of the urokinase receptor and to shed light on theurokinase receptor signal pathway, especially in the mouse B7 andB12 lung cell lines.

Maryka Quik, Ph.D. (The Parkinson’s Institute, Sunnyvale, CA).This group has initiated a project to study the mechanism underlyingthe occurrence of L-dopa-induced dyskinesia by investigating alter-ations in signal transduction systems using microarray techniques.

Shaun R. Coughlin, M.D., Ph.D. (Cardiovascular ResearchInstitute, UCSF). This study will examine signaling pathways reg-ulated by the protease thrombin and its cognate receptors, termedprotease-activated receptors. RNA will be isolated from primarycultures of human umbilical vein endothelial cells, which expressprotease-activated receptors 1–3. There are currently four membersof this family of G protein–coupled receptors, and the signalingpathways regulated by each receptor exhibit similarities to as wellas differences from the others. Examination of the genes that areup- or downregulated by each ligand/receptor pair would help iden-tify unique pathways and identify new genes in these pathways.



Joel D. Ernst, M.D. (Cardiovascular Research Institute, UCSF).Infection with the intracellular bacterium Mycobacterium tuberculosisleads to a paradoxical situation in which infection persists even thoughsignificant levels of the protective cytokine, interferon γ (IFNγ), areproduced. It has recently been found that M. tuberculosis persists part-ly because it can disrupt the IFNγ signaling pathway. The expressionof several genes that regulate macrophage function is controlled byIFNγ. Some of the better-characterized genes are interferon-inducibleprotein, monokine induced by gamma, and IFNγ-inducible GTPase.As the function of these genes is the activation of macrophages andchemoattraction of immune cells, it is important to determine how M.tuberculosis affects the IFNγ-dependent induction of these genes. Apreliminary study was carried out to identify a pool of genes that isregulated chiefly by IFNγ. In this study, wildtype C57BL/6 mice andIFNγ–/– mice were infected intravenously with M. tuberculosisH37Rv, and RNA was isolated from lungs of infected mice 14 dayslater (at which time IFNγ is produced by primed T cells). The RNAwas analyzed with Affymetrix U74A.v2 microarrays. About 150genes, at least 25 of which were functionally relevant, were morehighly expressed in the wildtype controls than in the IFNγ–/– mice.

Nurith Kurn, Ph.D. (NuGEN Technologies, San Carlos, CA).NuGEN Technologies has developed a proprietary technology toamplify RNA and DNA samples. The core has provided microarrays,RNA samples, and consultation services to enable the application ofthis technology to DNA microarrays.

Patrick McQuillen, M.D. (Department of Pediatrics, UCSF). Thegoal of this project is to understand the mechanisms that make theinfant brain unusually vulnerable to injury. The primary hypothesisof this study was that subplate neurons are vulnerable to earlyhypoxic ischemic brain injury and that the death of those cellsaccounts for the unique patterns of injury from ischemic hypoxia inthe developing brain. A rat brain model was used to identify genes

differentially expressed by subplate neurons before and afterischemic hypoxia.

Dean Sheppard, M.D. (Department of Medicine, UCSF). The goalof this project is to identify subsets of genes that are involved in anti-gen-induced airway hyperresponsiveness and bleomycin-inducedpulmonary fibrosis in mice. The investigators started by analyzingbaseline gene expression in different strains of mice and have fin-ished 45 arrays for baseline samples and expect to start evaluatingtreated lung samples soon. By evaluating patterns of gene expressionin inbred strains of mice with different degrees of sensitivity to eacheffect, they hope to increase their ability to identify mechanisticallyimportant genes.

Ajith Welihinda, Ph.D. (SangStat Corporation, Fremont, CA).SangStat has initiated a proof-of-principle experiment to determine ifthey will expand their research efforts to include microarray technol-ogy. The core primarily assists with preliminary experiments on a fee-for-service basis to facilitate their drug discovery efforts.

Selected Publications

Lee JH, Kaminski N, Dolganov G, Grunig G, Koth L, Solomon C,Erle D, Sheppard D (2001) Interleukin-13 induces dramatically dif-ferent transcriptional programs in three human airway cell types. Am.J. Respir. Cell Mol. Biol. 25:474–485.

Dahlquist KD, Salomonis N, Vranizan K, Lawlor SC, Conklin BR(2002) GenMAPP, a new tool for viewing and analyzing microarraydata on biological pathways. Nat. Genet. 31:19–20.

DeFreitas MF, Hamrick SEG, Ferriero DM, McQuillen PS (2002)Subplate neuron cell death and mRNA expression profiling followingoxygen glucose deprivation. Developmental Cerebral Blood Flow andMetabolism Symposium, Hershey, PA (abstract).

Outreach46

EDUCATION AND COMMUNITY OUTREACH

The Gladstone Institute of Neurological Disease (GIND) pro-vides a highly interactive academic environment and state-of-the-art research facilities that are ideal for training in

neuroscience and biomedical research. Our postdoctoral fellowsprogram offers opportunities for multidisciplinary training indiverse areas of basic and disease-related neuroscience. It com-bines rigorous scientific education and hands-on research experi-ence with a strong emphasis on mentoring and career development.Several GIND investigators collaborate closely with colleagues atUC Berkeley and Stanford, further broadening the exposure oftrainees to cutting-edge technologies and scientific concepts.

GIND investigators hold joint faculty appointments in differentUCSF departments and graduate programs, and have taught stu-dents of the Neuroscience Program, which is a component ofthe Program in Biological Sciences, the Biomedical SciencesProgram, the Pharmaceutical Sciences and PharmacogenomicsProgram, and the Medical Scientist Training Program.Undergraduate students from different programs at UCBerkeley have also benefited from training in GIND laborato-ries. Dr. Lennart Mucke, the director of the GIND, also partici-pates in the training of medical students and neurology resi-dents at UCSF’s Department of Neurology, the Memory andAging Center, and SFGH.

The Gladstone-UCSF community offers a large number of lec-tures, seminars, and journal clubs, featuring local experts as wellas outstanding scientists from around the world. The weekly GINDseminar series has continued to provide a stimulating and highlyinteractive forum for the presentation and discussion of innovativeresearch in basic and disease-related neuroscience.

Several GIND laboratories have contributed actively to theScience & Health Education Partnership, a collaboration betweenUCSF and the San Francisco Unified School District promoting apartnership between scientists and educators in support of high

quality science education for high school students. The programwas initiated in 1987 by UCSF professor Bruce Alberts, currentpresident of the National Academy of Sciences.

Translation of knowledge gained from basic research into educa-tion, prevention, and treatment programs is indeed an importantaspect of our mission. The GIND is actively engaged in efforts totranslate scientific discoveries into better treatments for major dis-eases of the nervous system.

For example, GIND has expanded its collaborative interactionswith UCSF’s Memory and Aging Center to ensure that advances inneurodegenerative disease research are promptly but cautiouslytranslated into better clinical care of patients suffering fromAlzheimer’s disease and related conditions.

We also take an active role in educating the public about neurode-generative disorders and neuroscientific research in general andparticipate regularly in fund-raising efforts of charitable organiza-tions. GIND investigators have continued to participate in publiclecture series aimed at educating lay people about researchadvances in our fields of study. Most of these lectures have focusedon the causes of neurodegenerative diseases and on emerging treat-ments that give rise to justifiable hope among patients and thoseproviding for their care.

Members of the GIND regularly participate in the Memory Walk,an annual walk-a-thon organized by the Alzheimer’s Association toraise awareness and funds for the Association’s efforts to helpAlzheimer patients and their families. Since 1989, Memory Walkparticipants have raised more than $100 million for Alzheimer pro-grams and services. Over $525,000 was raised with this year’sMemory Walk on Treasure Island in San Francisco Bay.

Through contributions such as those highlighted above we demon-strate our commitment to the community as well as to the pursuitof our scientific goals.

1. D’Hooge R, Nagels G, Westland CE, Mucke L, De Deyn PP(1996) Spatial learning deficit in mice expressing human 751-amino acid β-amyloid precursor protein. Neuroreport7:2807–2811.

2. Fleming LM, Weisgraber KH, Strittmatter WJ, Troncoso JC,Johnson GVW (1996) Differential binding of apolipoprotein Eisoforms to tau and other cytoskeletal proteins. Exp. Neurol.138:252–260.

3. Mahley RW, Nathan BP, Bellosta S, Pitas RE (1996)Apolipoprotein E: Structure, function, and possible roles inmodulating neurite extension and cytoskeletal activity. In:Apolipoprotein E and Alzheimer’s Disease (Roses AD,Weisgraber KH, Christen Y, eds) Springer-Verlag, Berlin, pp49–58.

4. Mahley RW, Nathan BP, Pitas RE (1996) Apolipoprotein E.Structure, function, and possible roles in Alzheimer’s disease.Ann. N.Y. Acad. Sci. 777:139–145.

5. Masliah E, Sisk A, Mallory M, Mucke L, Schenk D, Games D(1996) Comparison of neurodegenerative pathology in trans-genic mice overexpressing V717F β-amyloid precursor proteinand Alzheimer’s disease. J. Neurosci. 16:5795–5811.

6. Mohajeri MH, Bartsch U, van der Putten H, Sansig G, MuckeL, Schachner M (1996) Neurite outgrowth on non-permissivesubstrates in vitro is enhanced by ectopic expression of the neu-ral adhesion molecule L1 by mouse astrocytes. Eur. J.Neurosci. 8:1085–1097.

7. Pitas RE (1996) Microtubule formation and neurite extensionare blocked by apolipoprotein E4. Semin. Cell Dev. Biol.7:725–731.

8. Raber J, Bloom FE (1996) Arginine vasopressin release byacetylcholine or norepinephrine: Region-specific and cytokine-specific regulation. Neuroscience 71:747–759.

9. Roses AD, Einstein G, Gilbert J, Goedert M, Han S-H, HuangD, Hulette C, Masliah E, Pericak-Vance MA, Saunders AM,Schmechel DE, Strittmatter WJ, Weisgraber KH, Xi P-T (1996)Morphological, biochemical, and genetic support for anapolipoprotein E effect on microtubular metabolism. Ann. N.Y.Acad. Sci. 777:146–157.

10. Weisgraber KH, Mahley RW (1996) Human apolipoprotein E:The Alzheimer’s disease connection. FASEB J. 10:1485–1494.

11. Weisgraber KH, Dong LM (1996) Role of apolipoprotein E inAlzheimer’s disease: Clues from its structure. In: ApolipoproteinE and Alzheimer’s Disease (Roses AD, Weisgraber KH, ChristenY, eds) Springer-Verlag, Berlin, pp 11–19.

12. Wyss-Coray T, Masliah E, Toggas SM, Rockenstein EM,Brooker MJ, Lee HS, Mucke L (1996) Dysregulation of signaltransduction pathways as a potential mechanism of nervoussystem alterations in HIV-1 gp120 transgenic mice and humanswith HIV-1 encephalitis. J. Clin. Invest. 97:789–798.

13. Zhao J, Paganini L, Mucke L, Gordon M, Refolo L, Carman M,Sinha S, Oltersdorf T, Lieberburg I, McConlogue L (1996) β-

Secretase processing of the β-amyloid precursor protein intransgenic mice is efficient in neurons but inefficient in astro-cytes. J. Biol. Chem. 271:31407–31411.

14. Gutman CR, Strittmatter WJ, Weisgraber KH, Matthew WD(1997) Apolipoprotein E binds to and potentiates the biologicalactivity of ciliary neurotrophic factor. J. Neurosci.17:6114–6121.

15. Mahley RW (1997) Apolipoprotein E: Structure and function inlipid metabolism and neurobiology. In: The Molecular andGenetic Basis of Neurological Disease, 2nd edition (RosenbergRN, Prusiner SB, DiMauro S, Barchi RL, eds)Butterworth–Heinemann, Boston, pp 1037–1049.

16. Masliah E, Westland CE, Rockenstein EM, Abraham CR,Mallory M, Veinberg I, Sheldon E, Mucke L (1997) Amyloidprecursor proteins protect neurons of transgenic mice againstacute and chronic excitotoxic injuries in vivo. Neuroscience78:135–146.

17. Mattson MP, Barger SW, Furukawa K, Bruce AJ, Wyss-CorayT, Mark RJ, Mucke L (1997) Cellular signaling roles of TGFβ,TNFα and βAPP in brain injury responses and Alzheimer’s dis-ease. Brain Res. Rev. 23:47–61.

18. McGeer PL, Walker DG, Pitas RE, Mahley RW, McGeer EG(1997) Apolipoprotein E4 (apoE4) but not apoE3 or apoE2potentiates β-amyloid protein activation of complement invitro. Brain Res. 749:135–138.

19. Pitas RE (1997) Cerebrospinal fluid lipoproteins, lipoproteinreceptors, and neurite outgrowth. Nutr. Metab. Cardiovasc.Dis. 7:202–209.

20. Raber J, Chen S, Mucke L, Feng L (1997) Corticotropin-releas-ing factor and adrenocorticotrophic hormone as potential cen-tral mediators of OB effects. J. Biol. Chem. 272:15057–15060.

21. Raber J, Koob GF, Bloom FE (1997) Interferon-α and trans-forming growth factor-β1 regulate corticotropin-releasing fac-tor release from the amygdala: Comparison with the hypothal-amic response. Neurochem. Int. 30:455–463.

22. Wyss-Coray T, Borrow P, Brooker MJ, Mucke L (1997)Astroglial overproduction of TGF-β1 enhances inflammatorycentral nervous system disease in transgenic mice. J.Neuroimmunol. 77:45–50.

23. Wyss-Coray T, Masliah E, Mallory M, McConlogue L,Johnson-Wood K, Lin C, Mucke L (1997) Amyloidogenic roleof cytokine TGF-β1 in transgenic mice and in Alzheimer’s dis-ease. Nature 389:603–606.

24. Xu X, Raber J, Yang D, Su B, Mucke L (1997) Dynamic reg-ulation of c-Jun N-terminal kinase activity in mouse brain byenvironmental stimuli. Proc. Natl. Acad. Sci. USA94:12655–12660.

25. Bush TG, Savidge TC, Freeman TC, Cox HJ, Campbell EA,Mucke L, Johnson MH, Sofroniew MV (1998) Fulminantjejuno-ileitis following ablation of enteric glia in adult trans-genic mice. Cell 93:189–201.

Publications 47


PUBLICATIONS

26. Buttini M, Westland CE, Masliah E, Yafeh AM, Wyss-CorayT, Mucke L (1998) Novel role of human CD4 molecule iden-tified in neurodegeneration. Nat. Med. 4:441–446.

27. Coward P, Wada HG, Falk MS, Chan SDH, Meng F, Akil H,Conklin BR (1998) Controlling signaling with a specificallydesigned Gi-coupled receptor. Proc. Natl. Acad. Sci. USA95:352–357.

28. Ji Z-S, Pitas RE, Mahley RW (1998) Differential cellular accu-mulation/retention of apolipoprotein E mediated by cell sur-face heparan sulfate proteoglycans. Apolipoproteins E3 andE2 greater than E4. J. Biol. Chem. 273:13452–13460.

29. Krucker T, Toggas SM, Mucke L, Siggins GR (1998)Transgenic mice with cerebral expression of human immuno-deficiency virus type-1 coat protein gp120 show divergentchanges in short- and long-term potentiation in CA1 hip-pocampus. Neuroscience 83:691–700.

30. Mahley RW, Weisgraber KH, Farese RV Jr (1998) Disordersof lipid metabolism. In: Williams Textbook of Endocrinology,9th edition (Wilson JD, Foster DW, Kronenberg HM, LarsenPR, eds) WB Saunders, Philadelphia, pp 1099–1153.

31. Mahley RW (1998) Expanding roles for apolipoprotein E inhealth and disease. In: Atherosclerosis XI (Jacotot B, Mathé D,Fruchart J-C, eds) Elsevier, Amsterdam, pp 117–124.

32. Marshall DCL, Wyss-Coray T, Abraham CR (1998) Inductionof matrix metalloproteinase-2 in human immunodeficiencyvirus-1 glycoprotein 120 transgenic mouse brains. Neurosci.Lett. 254:97–100.

33. Masliah E, Raber J, Alford M, Mallory M, Mattson MP, YangD, Wong D, Mucke L (1998) Amyloid protein precursorstimulates excitatory amino acid transport: Implications forroles in neuroprotection and pathogenesis. J. Biol. Chem.273:12548–12554.

34. Mucke L, Buttini M (1998) Molecular basis of HIV-associatedneurologic disease. In: Molecular Neurology (Martin JB, ed)Scientific American, New York, pp 135–154.

35. Pitas RE, Ji Z-S, Weisgraber KH, Mahley RW (1998) Role ofapolipoprotein E in modulating neurite outgrowth: Potentialeffect of intracellular apolipoprotein E. Biochem. Soc. Trans.26:257–262.

36. Pitas RE, Ji Z-S, Supekova L, Mahley RW (1998) Divergentmetabolism of apolipoproteins E3 and E4 by cells. In: Progressin Alzheimer’s and Parkinson’s Diseases (Fisher A, Hanin I,Yoshida M, eds) Plenum, New York, pp 17–23.

37. Raber J, Wong D, Buttini M, Orth M, Bellosta S, Pitas RE,Mahley RW, Mucke L (1998) Isoform-specific effects ofhuman apolipoprotein E on brain function revealed in ApoEknockout mice: Increased susceptibility of females. Proc. Natl.Acad. Sci. USA 95:10914–10919.

38. Raber J (1998) Detrimental effects of chronichypothalamic–pituitary–adrenal axis activation. From obesityto memory deficits. Mol. Neurobiol. 18:1–22.

39. Raber J, Sorg O, Horn TFW, Yu N, Koob GF, Campbell IL,Bloom FE (1998) Inflammatory cytokines: Putative regulatorsof neuronal and neuro-endocrine function. Brain Res. Rev.26:320–326.

40. Toggas SM, Mucke L (1998) Transgenic models to assess thepathogenic potential of viral products in HIV-1-associated CNS

disease. In: The Neurology of AIDS (Gendelman HE, LiptonSA, Epstein L, Swindells S, eds) Chapman & Hall, New York,pp 156–167.

41. Bush TG, Puvanachandra N, Horner CH, Polito A, Ostenfeld T,Svendsen CN, Mucke L, Johnson MH, Sofroniew MV (1999)Leukocyte infiltration, neuronal degeneration, and neurite out-growth after ablation of scar-forming, reactive astrocytes inadult transgenic mice. Neuron 23:297–308.

42. Buttini M, Orth M, Bellosta S, Akeefe H, Pitas RE, Wyss-Coray T, Mucke L, Mahley RW (1999) Expression of humanapolipoprotein E3 or E4 in the brains of Apoe–/– mice: Isoform-specific effects on neurodegeneration. J. Neurosci .19:4867–4880.

43. D’Hooge R, Franck F, Mucke L, De Deyn PP (1999) Age-relat-ed behavioural deficits in transgenic mice expressing the HIV-1 coat protein gp120. Eur. J. Neurosci. 11:4398–4402.

44. Hsia AY, Masliah E, McConlogue L, Yu G-Q, Tatsuno G, HuK, Kholodenko D, Malenka RC, Nicoll RA, Mucke L (1999)Plaque-independent disruption of neural circuits inAlzheimer’s disease mouse models. Proc. Natl. Acad. Sci. USA96:3228–3233.

45. Huang F, Buttini M, Wyss-Coray T, McConlogue L, KodamaT, Pitas RE, Mucke L (1999) Elimination of the class A scav-enger receptor does not affect amyloid plaque formation orneurodegeneration in transgenic mice expressing human amy-loid protein precursors. Am. J. Pathol. 155:1741–1747.

46. Huang Y, Mahley RW (1999) Apolipoprotein E and human dis-ease. In: Plasma Lipids and Their Role in Disease (Barter PJ,Rye K-A, eds) Harwood Academic Publishers, Amsterdam, pp257–284.

47. Mahley RW, Huang Y (1999) Apolipoprotein E: From athero-sclerosis to Alzheimer’s disease and beyond. Curr. Opin.Lipidol. 10:207–217.

48. Mahley RW, Ji Z-S (1999) Remnant lipoprotein metabolism:Key pathways involving cell-surface heparan sulfate proteo-glycans and apolipoprotein E. J. Lipid Res. 40:1–16.

49. Mahley RW, Rall SC Jr (1999) Is ε4 the ancestral human apoEallele? Neurobiol. Aging 20:429–430.

50. Redfern CH, Coward P, Degtyarev MY, Lee EK, Kwa AT,Hennighausen L, Bujard H, Fishman GI, Conklin BR (1999)Conditional expression and signaling of a specificallydesigned Gi-coupled receptor in transgenic mice. Nat.Biotechnol. 17:165–169.

51. Xu X, Yang D, Wyss-Coray T, Yan J, Gan L, Sun Y, Mucke L(1999) Wild-type but not Alzheimer-mutant amyloid precursorprotein confers resistance against p53-mediated apoptosis.Proc. Natl. Acad. Sci. USA 96:7547–7552.

52. Akiyama H, Barger S, Barnum S, Bradt B, Bauer J, Cole GM,Cooper NR, Eikelenboom P, Emmerling M, Fiebich BL, FinchCE, Frautschy S, Griffin WST, Hampel H, Hull M, LandrethG, Lue L-F, Mrak R, Mackenzie IR, McGeer PL, O’BanionMK, Pachter J, Pasinetti G, Plata-Salaman C, Rogers J, RydelR, Shen Y, Streit W, Strohmeyer R, Tooyoma I, VanMuiswinkel FL, Veerhuis R, Walker D, Webster S,Wegrzyniak B, Wenk G, Wyss-Coray T (2000) Inflammationand Alzheimer’s disease. Neurobiol. Aging 21:383–421.

53. Buttini M, Akeefe H, Lin C, Mahley RW, Pitas RE, Wyss-Coray T, Mucke L (2000) Dominant negative effects of

Publications48

2002 ANNUAL REPORT

apolipoprotein E4 revealed in transgenic models of neurode-generative disease. Neuroscience 97:207–210.

54. Finkbeiner S (2000) Calcium regulation of the brain-derivedneurotrophic factor gene. Cell. Mol. Life Sci. 57:394–401.

55. Finkbeiner S (2000) CREB couples neurotrophin signals tosurvival messages. Neuron 25:11–14.

56. Mahley RW, Rall SC Jr (2000) Apolipoprotein E: Far morethan a lipid transport protein. Annu. Rev. Genomics Hum.Genet. 1:507–537.

57. Masliah E, Rockenstein E, Veinbergs I, Mallory M,Hashimoto M, Takeda A, Sagara Y, Sisk A, Mucke L (2000)Dopaminergic loss and inclusion body formation in α-synu-clein mice: Implications for neurodegenerative disorders.Science 287:1265–1269.

58. Mucke L, Buttini M, Mahley RW, Pitas RE, Raber J, Wyss-Coray T (2000) Contributions of the glial injury response tothe multifactorial pathogenesis of Alzheimer’s disease. In:Neuro-immune Interactions in Neurologic and PsychiatricDisorders (Patterson P, Kordon C, Christen Y, eds) Springer-Verlag, Berlin, pp 19–33.

59. Mucke L, Masliah E, Yu G-Q, Mallory M, Rockenstein EM,Tatsuno G, Hu K, Kholodenko D, Johnson-Wood K,McConlogue L (2000) High-level neuronal expression ofAβ1–42 in wild-type human amyloid protein precursor trans-genic mice: Synaptotoxicity without plaque formation. J.Neurosci. 20:4050–4058.

60. Mucke L, Yu G-Q, McConlogue L, Rockenstein EM,Abraham CR, Masliah E (2000) Astroglial expression ofhuman α1-antichymotrypsin enhances Alzheimer-like pathol-ogy in amyloid protein precursor transgenic mice. Am. J.Pathol. 157:2003–2010.

61. Raber J, Akana SF, Bhatnagar S, Dallman MF, Wong D,Mucke L (2000) Hypothalamic–pituitary–adrenal dysfunctionin Apoe–/– mice: Possible role in behavioral and metabolicalterations. J. Neurosci. 20:2064–2071.

62. Raber J, Wong D, Yu G-Q, Buttini M, Mahley RW, Pitas RE,Mucke L (2000) Apolipoprotein E and cognitive performance.Nature 404:352–354.

63. Redfern CH, Degtyarev MY, Kwa AT, Salomonis N, Cotte N,Nanevicz T, Fidelman N, Desai K, Vranizan K, Lee EK,Coward P, Shah N, Warrington JA, Fishman GI, Bernstein D,Baker AJ, Conklin BR (2000) Conditional expression of a Gi-coupled receptor causes ventricular conduction delay and alethal cardiomyopathy. Proc. Natl. Acad. Sci. USA97:4826–4831.

64. Smith SJ, Cases S, Jensen DR, Chen HC, Sande E, Tow B,Sanan DA, Raber J, Eckel RH, Farese RV Jr (2000) Obesityresistance and multiple mechanisms of triglyceride synthesisin mice lacking Dgat. Nat. Genet. 25:87–90.

65. Wyss-Coray T, Lin C, Sanan DA, Mucke L, Masliah E (2000)Chronic overproduction of transforming growth factor-β1 byastrocytes promotes Alzheimer’s disease-like microvasculardegeneration in transgenic mice. Am. J. Pathol. 156:139–150.

66. Wyss-Coray T, Lin C, von Euw D, Masliah E, Mucke L,Lacombe P (2000) Alzheimer’s disease–like cerebrovascularpathology in transforming growth factor-β1 transgenic miceand functional metabolic correlates. Ann. N.Y. Acad. Sci.903:317–323.

67. Wyss-Coray T, Mucke L (2000) Ibuprofen, inflammation andAlzheimer disease. Nat. Med. 6:973–974.

68. Finkbeiner S (2001) New roles for introns: Sites of combinato-rial regulation of Ca2+- and cyclic AMP-dependent gene tran-scription. Science’s STKE (http://stke.sciencemag.org/cgi/content/full/OC_sigtrans;2001/94/pe1).

69. Huang Y, Lin XQ, Wyss-Coray T, Brecht WJ, Sanan DA,Mahley RW (2001) Apolipoprotein E fragments present inAlzheimer’s disease brains induce neurofibrillary tangle-likeintracellular inclusions in neurons. Proc. Natl. Acad. Sci. USA98:8838–8843.

70. Masliah E, Ho G, Wyss-Coray T (2001) Functional role ofTGFβ in Alzheimer’s disease microvascular injury: Lessonsfrom transgenic mice. Neurochem. Int. 39:393–400.

71. Masliah E, Rockenstein E, Veinbergs I, Sagara Y, Mallory M,Hashimoto M, Mucke L (2001) β-Amyloid peptides enhance α-synuclein accumulation and neuronal deficits in a transgenicmouse model linking Alzheimer’s disease and Parkinson’s dis-ease. Proc. Natl. Acad. Sci. USA 98:12245–12250.

72. Raber J, LeFevour A, Mucke L (2001) Androgen treatmentreduces cognitive deficits in female apoE4 transgenic mice. In:Alzheimer’s Disease: Advances in Etiology, Pathogenesis andTherapeutics (Iqbal K, Sisodia SS, Winblad B, eds) John Wiley& Sons, Chichester, West Sussex, England, pp 747–757.

73. Raffaï RL, Dong L-M, Farese RV Jr, Weisgraber KH (2001)Introduction of human apolipoprotein E4 “domain interaction”into mouse apolipoprotein E. Proc. Natl. Acad. Sci. USA98:11587–11591.

74. Santiago-García J, Mas-Oliva J, Innerarity TL, Pitas RE (2001)Secreted forms of the amyloid-β precursor protein are ligandsfor the class A scavenger receptor. J. Biol. Chem.276:30655–30661.

75. Scearce-Levie K, Coward P, Redfern CH, Conklin BR (2001)Engineering receptors activated solely by synthetic ligands(RASSLs). Trends Pharmacol. Sci. 22:414–420.

76. Wyss-Coray T, Lin C, Yan F, Yu G-Q, Rohde M, McConlogueL, Masliah E, Mucke L (2001) TGF-β1 promotes microglialamyloid-β clearance and reduces plaque burden in transgenicmice. Nat. Med. 7:612–618.

77. Wyss-Coray T, McConlogue L, Kindy M, Schmidt AM, Yan SD,Stern DM (2001) Key signaling pathways regulate the biologicalactivities and accumulation of amyloid-β. Neurobiol. Aging22:967–973.

78. Bradley J, Finkbeiner S (2002) An evaluation of specificity inactivity-dependent gene expression in neurons. Prog.Neurobiol. 67:469–477.

79. Buckwalter M, Pepper J-P, Gaertner RF, Von Euw D, LacombeP, Wyss-Coray T (2002) Molecular and functional dissection ofTGF-β1-induced cerebrovascular abnormalities in transgenicmice. Ann. N.Y. Acad. Sci. 977:87–95.

80. Buttini M, Yu G-Q, Shockley K, Huang Y, Jones B, Masliah E,Mallory M, Yeo T, Longo FM, Mucke L (2002) Modulation ofAlzheimer-like synaptic and cholinergic deficits in transgenicmice by human apolipoprotein E depends on isoform, aging,and overexpression of amyloid β peptides but not on plaque for-mation. J. Neurosci. 22:10539–10548.

81. Gao F-B (2002) Understanding fragile X syndrome: Insightsfrom retarded flies. Neuron 34:859–862.

Publications 49


82. Humbert S, Bryson EA, Cordelières FP, Connors NC, Datta SR,Finkbeiner S, Greenberg ME, Saudou F (2002) The IGF-1/Aktpathway is neuroprotective in Huntington’s disease andinvolves huntingtin phosphorylation by Akt. Dev. Cell2:831–837.

83. Ji Z-S, Miranda RD, Newhouse YM, Weisgraber KH, Huang Y,Mahley RW (2002) Apolipoprotein E4 potentiates amyloid βpeptide-induced lysosomal leakage and apoptosis in neuronalcells. J. Biol. Chem. 277:21821–21828.

84. Morrow JA, Hatters DM, Lu B, Höchtl P, Oberg KA, Rupp B,Weisgraber KH (2002) Apolipoprotein E4 forms a molten glob-ule: A potential basis for its association with disease. J. Biol.Chem. 277:50380–50385.

85. Raber J, Bongers G, LeFevour A, Buttini M, Mucke L (2002)Androgens protect against apolipoprotein E4-induced cognitivedeficits. J. Neurosci. 22:5204–5209.

86. Raffaï RL, Weisgraber KH (2002) Hypomorphic apolipoproteinE mice. A new model of conditional gene repair to examineapolipoprotein E-mediated metabolism. J. Biol. Chem.277:11064–11068.

87. Scearce-Levie K, Coward P, Redfern CH, Conklin BR (2002)Tools for dissecting signaling pathways in vivo: Receptors acti-vated solely by synthetic ligands. Methods Enzymol.343:232–248.

88. Sweeney NT, Li W, Gao F-B (2002) Genetic manipulation ofsingle neurons in vivo reveals specific roles of Flamingo in neu-ronal morphogenesis. Dev. Biol. 247:76–88.

89. Wyss-Coray T, Mucke L (2002) Inflammation in neurodegener-ative disease—a double-edged sword. Neuron 35:419–432.

90. Wyss-Coray T, Yan F, Lin AH-T, Lambris JD, Alexander JJ,Quigg RJ, Masliah E (2002) Prominent neurodegeneration andincreased plaque formation in complement-inhibitedAlzheimer’s mice. Proc. Natl. Acad. Sci. USA 99:10837–10842.

91. Raffaï RL, Hasty AH, Wang Y, Mettler SE, Sanan DA, LintonMF, Fazio S, Weisgraber KH (2003) Hepatocyte-derived apoEis more effective than non-hepatocyte-derived apoE in remnantlipoprotein clearance. J. Biol. Chem. 278:11670–11675.

92. Santiago-García J, Kodama T, Pitas RE (2003) The class Ascavenger receptor binds to proteoglycans and mediates adhe-sion of macrophages to the extracellular matrix. J. Biol. Chem.278:6942–6946.

93. Gao F-B, Bogert BA (2003) Genetic control of dendritic mor-phogenesis in Drosophila. Trends Neurosci. 26:262–268.

94. Palop JJ, Jones B, Kekonius L, Chin J, Yu G-Q, Raber J, MasliahE, Mucke L (2003) Neuronal depletion of calcium-dependent pro-teins in the dentate gyrus is tightly linked to Alzheimer’s disease-related cognitive deficits. Proc. Natl. Acad. Sci. USA. In press.

Publications50

2002 ANNUAL REPORT

Seminars 51

SEMINARS

The GladstoneDistinguished Lectures

GIND Seminar Series

Other Seminar Seriesat Gladstone, SFGH,and UCSF

Seminars52

November 22, 1993Gerald R. Fink, Ph.D.DirectorWhitehead Institute for Biomedical ResearchCambridge, MADimorphism in yeast: A model for fungal pathogenesis

January 10, 1995Eric S. Lander, Ph.D.MemberWhitehead Institute for Biomedical ResearchProfessor of BiologyMassachusetts Institute of TechnologyDirector, Whitehead/MIT Center for Genome ResearchCambridge, MAMapping genes and genomes

March 7, 1995Nobel LaureateMichael S. Brown, M.D.Paul J. Thomas Professor of Medicine and GeneticsDirector, Center for Genetic DiseasesRegental Professor of the University of TexasDistinguished Chair in Biomedical SciencesUniversity of Texas Southwestern Medical SchoolDallas, TX

Nobel LaureateJoseph L. Goldstein, M.D.Professor and ChairmanDepartment of Molecular GeneticsPaul J. Thomas Professor of Medicine and GeneticsRegental Professor of the University of TexasLouis A. Beecherl, Jr., Chair in Biomedical SciencesUniversity of Texas Southwestern Medical SchoolDallas, TXMembrane-bound SREBP:Sterol sensor and transcriptional regulator

January 26, 1996Robert J. Lefkowitz, M.D.Investigator, Howard Hughes Medical InstituteJames B. Duke Professor of MedicineProfessor of BiochemistryDuke University Medical CenterDurham, NCG protein–coupled receptors and their regulation

November 21, 1996Nobel LaureateGünter Blobel, M.D., Ph.D.Investigator, Howard Hughes Medical Institute John D. Rockefeller, Jr., ProfessorHead of the Laboratory of Cell BiologyThe Rockefeller UniversityNew York, NYProtein traffic into and out of the nucleus

December 11, 1997Richard Axel, M.D.Investigator, Howard Hughes Medical InstituteHiggins Professor of Biochemistry and Molecular BiophysicsProfessor of PathologyColumbia UniversityNew York, NYThe molecular biology of smell

June 1, 1999Richard D. Klausner, M.D.Director, National Cancer InstituteBethesda, MDThe VHL tumor suppressor gene

October 7, 1999Joan A. Steitz, Ph.D.Investigator, Howard Hughes Medical InstituteHenry Ford II Professor of MolecularBiophysics and Biochemistry and ChemistryDirector, Molecular Genetics ProgramYale UniversityNew Haven, CTThe cell nucleolus: An RNA machine

April 11, 2000Judah Folkman, M.D.Julia Dyckman Andrus Professor of SurgeryProfessor of Cell BiologyHarvard Medical SchoolBoston, MAAngiogenesis research: From laboratory to clinic

December 20, 2000Nobel LaureateEric R. Kandel, M.D.University ProfessorSenior Investigator, Howard Hughes Medical InstituteCenter for Neurobiology and BehaviorCollege of Physicians and SurgeonsColumbia UniversityNew York, NYGenes, memory storage, and the search for new typesof synaptic actions

January 29, 2002Elaine Fuchs, Ph.D.Amgen Professor of Basic SciencesInvestigator, Howard Hughes Medical InstituteUniversity of ChicagoChicago, ILGenetic disorders of the cytoskeleton

November 13, 2002Huda Y. Zoghbi, M.D.Baylor College of MedicineProfessor of Pediatrics,Molecular and Human Genetics, and NeurologyInvestigator, Howard Hughes Medical InstituteHouston, TXCells, flies, and mice: A triune approach to triplet repeats

The Gladstone Distinguished Lectures

Seminars 53


GIND Seminar Series Guest Lectures

January 17, 2002Mark D. Linder, Ph.D.Neurocrine Biosciences, Inc.San Diego, CABehavioral models for drug discovery/development

February 21, 2002Cori Bargmann, Ph.D.Howard Hughes Medical InstituteUniversity of California, San FranciscoSan Francisco, CASignaling pathways in neuronal development in C. elegans

March 28, 2002Ted Dawson, M.D., Ph.D.Department of NeurologyJohns Hopkins University School of MedicineBaltimore, MDAnimal models, genes, oxidative stress and protein mishandling:Insights into the pathogenesis of Parkinson’s disease

April 10, 2002 William H. Frey II, Ph.D.Department of PharmaceuticsUniversity of Minnesota College of PharmacyMinneapolis, MNTreating stroke and Alzheimer’s disease with neurotrophins andantioxidants: Intranasal delivery to bypass the blood-brain barrier

April 25, 2002Jacqueline Crawley, Ph.D.National Institute of Mental HealthGeorgetown University School of MedicineBethesda, MDStrategies for behavioral phenotyping of transgenicand knockout mice

May 9, 2002Guojun Bu, Ph.D.Department of PediatricsWashington University School of MedicineSt. Louis, MOTale of LRP tail

May 23, 2002Christopher Ross, M.D., Ph.D.Department of Psychiatry & Division of NeurobiologyJohns Hopkins University School of MedicineBaltimore, MDPathogenesis of Huntington’s disease and related disorders

May 30, 2002Thomas Wisniewski, M.D.Department of NeurologyNew York University School of MedicineNew York, NYTherapeutic approach for Alzheimer and prion diseases

November 14, 2002Marcy MacDonald, Ph.D.Department of NeurologyMassachusetts General HospitalHarvard UniversityCharlestown, MAHuntington’s disease

December 12, 2002Huntington Potter, Ph.D.Department of Biochemistry and Molecular Biologyand Suncoast Gerontology CenterUniversity of South FloridaTampa, FLBeyond beta protein: The essential role of pathological chaperonesin Alzheimer amyloid formation and cognitive decline

December 19, 2002Alison Goate, Ph.D.Department of PsychiatryWashington UniversitySt. Louis, MOGenetics and the pathogenesis of Alzheimer’s disease

Seminars54

Bay Area RNA Club

Members of nearly 40 laboratories working on RNA research gath-er to hear three informal talks by participating labs; held the firstThursday of every month from 6:30–9:30 p.m. in Genentech HallAuditorium, Room 106, on the UCSF Mission Bay campus.Information is available at http://www.ucsf.edu/frankel/Frankel%20website/RNA_club/RNA_Club_ 2001-2002.html. Contact JudeHawley (Tel: 514-2072; email: jhawley@ biochem. ucsf.edu).

Biochemistry and Biophysics Seminar

Formal presentations by faculty or guest speakers organized by theUCSF Department of Biochemistry and Biophysics; held everyTuesday from 4:00–5:00 p.m. in Genentech Hall Auditorium, Room106, on the UCSF Mission Bay campus. Information is available athttp://biochemistry.ucsf.edu/B&B_seminars03.html. Contact: JudyPiccini (Tel: 476-1515; email: [email protected]).

BMS Journal Club

Journal club of the UCSF Biomedical Sciences (BMS) Program; heldevery Wednesday from 12:30–1:30 p.m. in the 4th floor conferenceroom of SFGH Building 40. Articles from any area of biomedicine arediscussed in two half-hour presentations by graduate students, post-doctoral fellows, and faculty. Contact: Naima Contos (Tel: 695-3729;email: [email protected]).

CVRI Lecture

Formal presentations by faculty or guest speakers organized by theUCSF Cardiovascular Research Institute (CVRI); held on irregulardates from 4:00–5:30 p.m. on the UCSF Parnassus campus (roomvaries). Contact: Julie Tom (Tel: 476-1310; email: [email protected]).

Frontiers in Neurology and Neuroscience

Lectures by faculty or guest speakers focusing on neurological dis-eases and their treatment; organized by the UCSF Department ofNeurology. Lectures are held every other Wednesday from 5:00–6:00p.m. in room N-225 of the School of Nursing on the UCSF Parnassuscampus. Contact: Laura Alexander (Tel: 476-1489; email: [email protected]).

GICD Scientists Meeting

Informal seminars focusing on research progress in cardiovasculardisease given by graduate students, postdoctoral fellows, or investiga-tors of the Gladstone Institute of Cadiovascular Disease (GICD); heldevery Friday from 9:00–10:00 a.m. in the 4th floor conference roomof SFGH Building 40. Contact: Aileen Santos (Tel: 695-3770; email:[email protected]).

GICD Seminar

Formal presentations focusing on topics related to cardiovascular dis-ease given by guest speakers and candidates for postdoctoral fellow-ships at the GICD; held from 12:00–1:00 p.m. in SFGH Building 40conference room (irregular dates). Contact: Aileen Santos (Tel: 695-3770; email: [email protected]).

GIND Seminar

Presentations focusing on research in disease-related neurosciencegiven by graduate students, postdoctoral fellows, or investigators ofthe Gladstone Institute of Neurological Disease (GIND) or guestspeakers; held every Thursday from 9:00–10:00 a.m. in the 5th floorlibrary of SFGH Building 3. Contact: Kelley Nelson (Tel: 695-3885;email: [email protected]).

GIVI Seminar

Presentations focusing on research progress in virology and immunol-ogy, including HIV and AIDS, given by national and internationalspeakers, alternating with presentations by members of the GladstoneInstitute of Virology and Immunology (GIVI); held every Thursdayfrom 12:00–1:00 p.m. in the 5th floor library of SFGH Building 3.Contact: Robin Givens (Tel: 695-3801; email: [email protected]).

Microbiology and Immunology Seminar

Formal presentations by faculty or guest speakers organized by theUCSF Department of Microbiology and Immunology; normally heldevery Monday from 5:00–6:00 p.m. in HSW-301 on the UCSFParnassus campus and broadcast to the 5th floor conference room ofSFGH Building 3. Information is available at http://itsa.ucsf.edu/~micro/immunology/seminarseries.html. Contact: EmmaSandoc (Tel: 502-1961; email: [email protected]).

Neuroscience Journal Club

Journal club of the UCSF Neuroscience Program; held every Fridayfrom 4:00–5:00 p.m. in N-217 on the UCSF Parnassus campus.Articles from any area of neuroscience are discussed in two half-hourpresentations by graduate students and faculty. Refreshments areserved after the meeting. Information is available at http://www.ucsf.edu/neurosc/jclub2002-2003.htm. Contact: Deb Rosenberg (Tel:476-1947; email: [email protected]).

Neuroscience Seminar

Formal presentations by guest speakers focusing on basic neuro-science, organized by the UCSF Neuroscience Program; held everyThursday from 4:00–5:00 p.m. in HSW-301 on the UCSF Parnassuscampus. Information is available at http://www.ucsf.edu/neurosc/seminars02_03.html. Contact: Deb Rosenberg (Tel: 476-1947;email: [email protected]).

Other Seminar Series at Gladstone, SFGH, and UCSF

Seminars 55


9:00–10:00

10:00–11:00

11:00–12:00

12:00–1:00

1:00–2:00

2:00–3:00

3:00–4:00

4:00–5:00

5:00–6:00

6:00–7:00

Monday Tuesday Wednesday Thursday Friday

GICD Seminar(day varies)SFGH B40

CVRI Lecture(not held regularly)(4:00–5:30)UCSF (room varies)

Microbiology andImmunology Seminar HSW-301

Signaling Club(monthly)L-1361

Biochemistry andBiophysics SeminarGenentech Hall

BMS Journal Club(12:30)SFGH B40

PIBS Journal ClubGenentech Hall

Seminar in Biomedical Sciences HSW-300

Frontiers inNeurology and Neuro-science (2 x/month)N-225

GIND SeminarSFGH B3

GIVI SeminarSFGH B3

NeuroscienceSeminar HSW-301

Bay Area RNA Club(6:30; monthly)Genentech Hall

GICD Scientists MeetingSFGH B40

NeuroscienceJournal Club S-217

PIBS Journal Club

Journal club of the UCSF Program in Biological Sciences (PIBS);held every Wednesday from 2:00–3:30 p.m. in Genentech HallAuditorium on the UCSF Mission Bay campus. Articles from anyarea of biomedicine are discussed in two half-hour presentations bygraduate students and faculty. Information is available athttp://www.ucsf.edu/pibs/pibs_seminars.html. Contact: MariaRealubin (Tel: 476-6178; email: [email protected]).

Seminar in Biomedical Sciences

Formal presentations by faculty or guest speakers from any area ofbiomedicine, organized by the BMS Program; held every

Wednesday from 4:00–5:00 p.m. in HSW-300 on the UCSFParnassus campus. Information is available at http://www.ucsf.edu/bms/activities.html. Contact: Monique Piazza (Tel: 476-2189;email: [email protected]).

Signaling Club

Informal presentations attended by people from 15–20 laboratories atUCSF; held on the first Tuesday of every month from 12:00–1:00 p.m.in room L-1361 of Long Hospital on the UCSF Parnassus campus.Research on signaling is discussed in two half-hour presentations bygraduate students or postdoctoral fellows. Contact: Mark Von Zastrow(Tel: 476-7855; email: [email protected]).

Calendar of Gladstone, SFGH, and UCSF Seminars

Documents

GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE