gladstone institute of neurological disease

2001 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

2001 ANNUAL REPORT

Copyright 2002by 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) 826-6541

TABLE OF CONTENTS

DIRECTOR’S REPORT.........5

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

MEMBERS OF THE INSTITUTE.........25

REPORTS FROM THE LABORATORIES.........31

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

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

Huang Laboratory.........47

Mahley Laboratory.........55

Mucke Laboratory.........63

Pitas Laboratory.........73

Weisgraber Laboratory.........79

Wyss-Coray Laboratory.........87

Behavioral Core Laboratory.........95

Gladstone Genomics Core.........103

OUTREACH.........109

PUBLICATIONS.........115

SEMINARS.........123

Director’s Report 5

Lennart Mucke, M.D.

DIRECTOR’S REPORT


Neurodegenerative disordersrob people of their ability toremember, speak, write, ambu-late, and control their lives.These conditions are on the risebecause people are livinglonger, and aging strongly

increases the risk of being afflicted by these condi-tions. The enormous cost of caring for individualswith these conditions threatens our health care system.A medical breakthrough is clearly needed, and thesurest way to such a breakthrough is to determineexactly how these diseases result in the dysfunctionand degeneration of nerve cells. In addition, neurolog-ical diseases raise a range of fascinating questions thatare of fundamental scientific interest. While the inves-tigation of neurological diseases has promoted basicneuroscientific discoveries for over a century, therehas never been a more promising and exciting conver-gence of basic and disease-related neuroscience thannow. Investigators at the Gladstone Institute ofNeurological Disease (GIND) have continued tounravel the molecular processes that trigger and cul-minate in neurodegenerative diseases, as well as thosethat underlie normal functions of the nervous system.

Several projects this year have examined the possibili-ty that many, if not all, neurodegenerative disorders arecaused by the abnormal folding or aggregation of pro-teins. Although different proteins accumulate in differ-ent neurodegenerative disorders, the ways in whichthey damage nerve cells appear to overlap. This possi-bility raises hope that it will be feasible to developtreatments that can prevent, stall, or even reverse morethan one of these conditions. Toward this goal, wehave investigated the conformational states of diversemisfolded proteins and the mechanisms by which they

impair neuronal function and survival. Interestingly,the pace and severity of neurodegenerative processescan be modified by endogenous factors secreted fromastrocytes and microglia, injury-responsive cells thatclosely interact with neurons in the brain and spinalcord. Among these modulatory factors are theapolipoproteins and their receptors, which play impor-tant roles in the nervous system in normal develop-ment, aging, and disease. Because of its intriguingrole in Alzheimer’s disease (AD) and other neurolog-ical conditions, apolipoprotein (apo) E has remained amajor research target for several GIND laboratories.Other studies have focused on the molecular mecha-nisms of neural plasticity, which is critical for braindevelopment and adaptations of the nervous system toenvironmental stimuli and challenges.

Scientific Discoveries

The laboratory of Dr. Steven Finkbeiner focuses onthe pathogenesis of Huntington’s disease. This fatalinherited neurodegenerative disorder is associatedwith increasingly disruptive involuntary movementsand a progressive loss of motor control. It is caused byabnormal glutamine expansions in the protein hunt-ingtin, which affect protein folding. Antibodies gener-ated by the Finkbeiner laboratory recognize only dis-ease-causing forms of mutant huntingtin. With theseantibodies, a specific region was identified in mutanthuntingtin that appears to be critical for its pathologi-cal activities. Interestingly, similar disease-associatedregions were detected in mutant proteins associatedwith other inherited polyglutamine diseases. To facil-itate the identification of strategies that can block theformation of the disease-associated regions or theireffects, Dr. Finkbeiner developed a sophisticatedrobotic microscope for the large-scale analysis of cell

Director’s Report

2001 ANNUAL REPORT

8 Director’s Report

cultures. This powerful new device has the potential torevolutionize the cell biological investigation of neu-rodegenerative pathways and other processes. TheFinkbeiner laboratory also studied the molecularmechanisms by which the entry of calcium throughchannels in neuronal membranes affects gene expres-sion and synaptic plasticity. Their findings suggestthat intracellular proteins located near the openings ofthe channels interact with the calcium ions, linkingcalcium influx to the activation of specific genes inthe neuronal nucleus.

The laboratory of Dr. Fen-Biao Gao focuses on thegenes and molecular pathways that regulate the devel-opment and maintenance of neuronal dendrites.Dendrites are tree-like extensions of neurons thatreceive signals and participate in information process-ing and storage. These highly branched structuresaccount for more than 90% of the surface of some neu-rons. In many neurological disorders, including ADand fragile X syndrome, the number of dendriticbranches and the density of dendritic spines arealtered. However, very little is known about the mech-anisms that control dendritic branching in vivo. Dr.Gao developed a method to express mutant proteinsselectively in individual neurons and to study the con-sequences of this manipulation in living fruit flies. Thisnew approach has allowed him to demonstrate that thedendritic branching patterns of individual neurons areextremely diverse but well conserved among flies, sug-gesting that they are of great physiological importance.Some of the mutations Dr. Gao identified in previousgenetic screens turned out to have surprisingly specif-ic effects on the outgrowth of dendrites or axons orboth. Since neural networks are established throughconnections between these neuronal structures, thesestudies are shedding light on one of the most funda-mental design principles of the nervous system. In thefuture, they may also help us understand how neuralcircuits are broken down by neurological diseases andhow broken circuits might be repaired.

The laboratory of Dr. Yadong Huang has continued itsinvestigation of intracellular apoE fragmentation andpathogenic interactions of apoE fragments with themicrotubule-associated protein tau. Human apoEexists in three major isoforms that differentially affect

the risk of developing AD (E4 > E3 > E2). ApoE4 isthe main known inherited risk factor for the most fre-quent form of AD. This year, lead findings obtained incell cultures were extended to transgenic mouse mod-els and human AD cases, underlining the significanceof the original cell-culture data. A much greater accu-mulation of apoE fragments was detected in brains ofhumans and transgenic mice expressing apoE4 than inthose expressing apoE3. Both in cultured neurons andin transgenic brains, the accumulation of apoE4 frag-ments was associated with abnormal phosphorylationof tau, cytoskeletal derangements, and neurodegener-ation. Abnormally phosphorylated tau is the majorconstituent of neurofibrillary tangles, a pathologicalhallmark of AD. The differential propensity of apoEisoforms to be broken down into potentially pathogen-ic fragments (E4 > E3) may relate to their effects on ADrisk (E4 > E3) and age of onset (E4 < E3). Inhibiting theprocesses that result in intraneuronal fragmentation ofapoE could be of therapeutic benefit, particularly inpeople with apoE4. Identifying the protease that mightbe involved in apoE cleavage has become a majorresearch target for the Huang laboratory.

The laboratory of Dr. Robert W. Mahley has continuedits cell biological investigation of the mechanisms thatunderlie the neuroprotective functions of apoE3 andthe pathogenic functions of apoE4 in AD and otherconditions. Previous studies revealed that apoE3 pro-motes neurite outgrowth, protects the nervous systemagainst diverse injuries, and facilitates neuroregenera-tion after trauma. In contrast, apoE4 was not protectiveand in many instances even worsened the outcome ofneural injuries. This year, studies of apoE isoformswere extended to the interaction of apoE with amyloidβ peptides (Aβ), which accumulate to abnormally highlevels in AD brains. Forms of Aβ with a strong ten-dency to aggregat can be taken up by brain cellsand are known to disrupt lysosomal membranes.Lysosomes are intracellular vesicles that contain manyprotein-degrading enzymes and other highly reactivemolecules. It has been postulated that the destabiliza-tion of lysosomal membranes may be an importantmechanism by which Aβ elicits neuronal degenerationin AD. The Mahley laboratory demonstrated that Aβ-induced lysosomal leakage was strongly enhanced byapoE4, but not by apoE3, and that the enhanced leak-

GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE


age was associated with increased neuronal death. Theidentified effects of apoE isoforms on the destabiliza-tion of lipid membranes (E4 > E3), Aβ-induced lyso-somal leakage (E4 > E3), and associated neuronaldeath (E4 > E3) may relate closely to their effects onAD risk (E4 > E3) and age of onset (E4 < E3). Notably,pharmacological inhibition of a specific cell deathmediator completely prevented the potentiation of neu-ronal death by apoE4.

My own laboratory has continued to investigate themolecular pathways that link genetic determinants orrisk factors of AD to neurodegeneration and cognitivedecline. In previous studies, we generated transgenicmouse models with neuronal expression of mutanthuman amyloid protein precursors (APP) that causeoverproduction of Aβ and early-onset familial AD inhumans. The AD-like pathology that develops in thesemice with aging was inhibited or enhanced by glia-derived factors, such as α1-antichymotrypsin (↑),apoE3 (↓), apoE4 (↑), and TGF-β1 (↓), raising hopethat AD pathogenesis might be modifiable by diversetherapeutic interventions. It is widely believed thatapoE4 increases AD risk and lowers the age of diseaseonset by increasing the deposition of Aβ into plaques.However, cognitive decline in AD correlates much bet-ter with synaptic and cholinergic deficits than withplaque load. We therefore analyzed these parameters inmice expressing human Aβ together with apoE3 orapoE4 in the brain. ApoE3, but not apoE4, delayed Aβ-dependent synaptic deficits independently of plaqueformation. These findings underline the importance ofnondeposited, prefibrillar Aβ species and suggest thatthe differential effects of apoE isoforms on the risk ofAD relate to differences in their neuroprotective poten-tial rather than to differences in their effect on plaqueformation. This year, we also expanded our studies intwo new directions: DNA microarrays and Aβ vaccina-tion. While there is evidence that Aβ promotes andimmunization against Aβ inhibits AD-like pathology,the underlying molecular mechanisms remain to bedefined. Because at least some of the critical cellularand molecular processes may be reflected in changes ofgene expression, we embarked on a large-scale analysisof gene expression in treated and untreated transgenicmice expressing human APP and Aβ in the brain.Among the most interesting preliminary findings from

this analysis was the identification of gene products thatshowed opposite responses to overexpression of Aβversus Aβ vaccination and were relatively unaffected byhuman APP or sham vaccination. Some of the identifiedmolecules may be effectors of Aβ-induced neurotoxici-ty. Others may mediate beneficial vaccination effects,constitute new therapeutic targets, or serve as useful endpoint measures for vaccination trials.

The laboratory of Dr. Robert E. Pitas has followed upon their discovery of a novel brain protein that inter-acts with apoE. They have begun to characterize thegene that encodes the apoE-binding protein anddemonstrated that it is expressed in neurons, but not inother brain cells such as astrocytes. Analyses of data-bases containing information on diverse nucleic acidand protein sequences revealed that the apoE-bindingprotein belongs to a family of proteins that have sever-al features in common. The existence of a whole fam-ily of related proteins and the similarities between fam-ily members identified in different species underscorethe potential physiological importance of the apoE-binding protein. This year, research efforts in the Pitaslaboratory were extended in an interesting new direc-tion through a collaboration with the Gao laboratory,which specializes in fruit fly research. The fruit fly sys-tem will facilitate the rapid molecular manipulationand physiological characterization of the apoE-bindingprotein and its relatives. Good progress has also beenmade toward inactivating the gene that encodes theapoE-binding protein in mice, which will help assessits function in the mammalian brain.

The laboratory of Dr. Karl H. Weisgraber has contin-ued to investigate how amino acid substitutions affectthe conformation, stability, and biological function ofapoE isoforms. Previous studies revealed that the sin-gle amino acid difference between apoE3 and apoE4has profound effects on the conformation and stabili-ty of these molecules. The biological consequences ofthese biophysical differences are beginning to beunraveled. This year, the Weisgraber laboratoryproved the biological relevance of an interactionbetween different domains within apoE that occurs inapoE4 but not in apoE3. Introduction of an apoE4-likedomain interaction into mouse apoE changed the lipidbinding profile of apoE in the plasma of the genetically

modified mice. In previous studies, the Weisgraberlaboratory identified small chemical compounds that,based on computer simulations, might be able to dis-rupt the domain interaction in apoE4. In vitro and cellculture assays have provided preliminary proof thatsome of these compounds can indeed convert apoE4into a molecule with apoE3-like properties. Thesefindings bode well for the development of apoE4-tar-geted drug treatments to prevent AD in apoE4 carri-ers. Lastly, in vitro studies revealed that apoE4 has agreater propensity than apoE3 to assume an unstableconformational state that might promote the degrada-tion of apoE4 or its pathogenic interactions with othermolecules, such as Aβ and tau. The further character-ization of this conformational state could shed light onthe role of apoE4 in AD and has become an importantobjective of the Weisgraber laboratory.

The laboratory of Dr. Tony Wyss-Coray has continuedto characterize the physiological functions of thecytokine transforming growth factor β1 (TGF-β1) inthe central nervous system. They also investigated therole of inflammation and, in particular, of complementactivation in the pathogenesis of AD. TGF-β1 wasshown to promote the activation of microglia and com-plement components in the brains of transgenic mousemodels overexpressing human APP. This effect wasassociated with a decrease in the deposition of Aβ asamyloid plaques. In contrast, inhibition of complementactivation in the brain increased the plaque load andtriggered neuronal degeneration in the mice. Thesefindings have important therapeutic implications, asthey challenge the widely held belief that activation ofmicroglia and complement promotes AD. The resultsobtained by the Wyss-Coray laboratory suggest thatgeneral inhibition of inflammatory responses in thebrain, including inhibition of complement components,may enhance rather than inhibit the development orprogression of AD. Conversely, the specific enhance-ment of protective inflammatory responses could be oftherapeutic benefit.

Education, Special Initiatives,and Recognition

The Gladstone Institutes and UCSF provide state-of-the-art research facilities and a highly interactive acade-

mic environment that is ideal for training in neuro-science and biomedical research. GIND investigatorshave continued to participate actively in the training ofstudents and residents from various UCSF departmentsand interdepartmental programs, including theDepartments of Neurology and Physiology, the Neuro-science Program, the Biomedical Sciences Program, thePharmaceutical Sciences and PharmacogenomicsProgram, and the Medical Scientist Training Program,as well as from graduate and undergraduate programs atUC Berkeley and other institutions. Many members ofour institute have collaborated this year to make ourtraining environment even more inspiring and reward-ing for students of all biomedical disciplines. Drs.Finkbeiner and Gao deserve particular mention for theoutstanding efforts they have made in this regard. Inthis context, I would also like to highlight the continuedsuccess of our weekly GIND seminar series, which isorganized by Dr. Wyss-Coray. It remains a popular andstimulating forum for education in disease-related neu-roscience and scientific exchange among members ofthe institutes and colleagues from the greater UCSFcommunity.

GIND investigators have extended their efforts to pro-mote education and scientific exchange in disease-related neuroscience far beyond the boundaries of ourinstitute. They have organized and participated in anumber of national and international conferences thathave advanced our field of research in various ways. Iam delighted that their research accomplishments andother contributions to the scientific community havenot gone unnoticed, as reflected by the honors andawards institute members received this year (seeOutreach section for details).

Despite their busy schedules and expanding researchefforts, GIND members have continued to devote timeto community outreach. As described in the Outreachsection of this report, these efforts included participa-tion in activities aimed at educating the public aboutAD and neuroscientific research in general. Our syner-gism with the UCSF Memory and Aging Center, thelocal chapter of the Alzheimer’s Association, and theHereditary Disease Foundation has allowed us to main-tain and expand fruitful links between our research andthe patients afflicted by the diseases we study.

2001 ANNUAL REPORT

10 Director’s Report

Acknowledgements

I would like to thank the participants of this year’sScientific Advisory Board meeting (Drs. Dale E.Bredesen, Buck Institute for Age Research; EricShooter, Stanford University; Sidney Strickland,Rockefeller University; and Marc Tessier-Lavigne,UCSF) for their outstanding input. It contributedgreatly to our success.

The progress we made in 2001 reflects the work of allthe researchers at the GIND and of the administrativestaff at the Gladstone Institutes. As outlined in thisreport, we have advanced our understanding of someof the most devastating diseases known to man. I amconfident that this knowledge will contribute to thedevelopment of better strategies for their treatmentand prevention.

Lennart Mucke, M.D.Director



Description of theTHE J. DAVID GLADSTONEINSTITUTES

Trustees

Richard S. Brawerman

Albert A. Dorman

Richard D. Jones

President

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

Executive Director

Richard Hille

Chief Financial Officer

Hal Orr, C.M.A.

Description 13

Although autonomous in their areas of specialization,the institutes share a common approach. Each instituteis organized around research units consisting of scien-tists, postdoctoral researchers, research associates, andstudents. This structure is designed to accommodatesmall groups of scientists who work together closelybut who also benefit from collegial interactions withother research groups. Collaborations among staffmembers with various areas of expertise create a stim-ulating environment that fortifies the scientificlifeblood of the organization.

Each institute receives expert input on the progress ofits science from an advisory board of distinguished sci-entists. The scientific advisory boards provide atwofold service in reviewing the quality of the researchand in advising the president, directors, and trustees.

The work of the scientific staff at all three institutesalso extends beyond the laboratory to the wider com-munity. The mission of the institutes includes the edu-cation of graduate and medical students, postdoctoralfellows, and visiting scientists; specialized trainingfor practicing physicians; and educational outreach tothe local and extended community.

The J. David Gladstone Institutes are the product ofthe wisdom and hard work of many individuals. Thefirst was J. David Gladstone himself, a Los Angelesreal-estate entrepreneur. Others are the trustees. Theoriginal trustees, all of whom had known or workedclosely with Mr. Gladstone, were Richard S.Brawerman, his attorney and executor of his estate;Richard D. Jones, his real-estate attorney; and DavidOrgell, his cousin and confidant. When Mr. Orgelldied in 1987, he was succeeded on the board by AlbertA. Dorman, a southern California executive withexperience in managing large organizations.

Description 15

Primary research efforts at the J. DavidGladstone Institutes focus on three of the mostimportant clinical problems of modern times:

cardiovascular disease, AIDS, and neurodegenerativedisorders. Cardiovascular disease, the nation’s lead-ing killer, claims the lives of over one millionAmericans each year. Despite more effective treat-ments, AIDS remains a leading cause of death in theUnited States. Worldwide, more than 40 million peo-ple are living with HIV/AIDS, and more than 21 mil-lion have died as a direct result of HIV infection.Alzheimer’s disease, the most recent focus of investi-gation by Gladstone scientists, is the fourth leadingcause of death in adults, affecting four millionAmericans. The realization of the impact of these dis-eases on world health infuses Gladstone scientistswith a sense of purpose and urgency.

Gladstone is composed of three institutes, each ofwhich issues its own annual report. The GladstoneInstitute of Cardiovascular Disease (GICD), whichopened in 1979, focuses on atherosclerosis and itscomplications. In 1992, the Gladstone Institute ofVirology and Immunology (GIVI) was established tostudy HIV, the causative agent of AIDS. The 1993 dis-covery that apolipoprotein (apo) E—long studied atGICD for its role in heart disease—plays a role inAlzheimer’s disease as well led to the establishmentof the Gladstone Institute of Neurological Disease(GIND) in 1998. The three institutes are located at theSan Francisco General Hospital (SFGH) campus ofthe University of California, San Francisco (UCSF).While independent, Gladstone is formally affiliatedwith UCSF, and Gladstone investigators hold univer-sity appointments and participate in many universityactivities, including the teaching and training of grad-uate students.

Description of the Institutes

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 SouthwesternMedical Center

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

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

Donald M. Small, M.D.Chairman, Department of BiophysicsProfessor of Biophysics, Medicine andBiochemistryBoston 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 Physiciansand Surgeons

2001 ANNUAL REPORT

16 Description

At the time of Mr. Gladstone’s death in 1971, thesouthern California real-estate market was just begin-ning to flourish. His estate, left almost entirely formedical education and research, was relatively mod-est by later standards. However, the trustees recog-nized the estate’s potential for growth and, throughtheir inspired management, increased its worth sever-alfold within the first decade. From the beginning, thedecisions of the trustees had profound and positiveeffects on the research organization that evolved.They continue to manage and enlarge the assets of theJ. David Gladstone Institutes and to oversee their use.

Gladstone Instituteof Cardiovascular Disease

Close ties already existed between the UCSF Schoolof Medicine and SFGH, the hospital of the City andCounty of San Francisco, when the trustees leasedvacant space from the City in 1977 in which to createlaboratories and offices. The partnership has flour-ished. Gladstone scientists collaborate with their col-leagues at UCSF and SFGH and provide service tothose organizations as professors and staff physicians.The mutually beneficial association between theGladstone, UCSF, and SFGH has created a productiveand supportive environment in which scientists con-duct basic research while availing themselves of clin-ical and academic opportunities.

To choose a director for the developing research facil-ity, the trustees sought guidance from the scientificcommunity. The choice was Robert W. Mahley, M.D.,Ph.D. At the time of Mr. Gladstone’s death, he wasjust completing his internship. However, by 1979,when he was appointed director, Dr. Mahley hadestablished himself as a leading researcher in the fieldof lipoprotein metabolism and atherosclerosis. Hecame to the Gladstone from the National Institutes ofHealth, where he headed the Laboratory of Experi-mental Atherosclerosis. Less than a year after hisappointment, Dr. Mahley had assembled a staff of 25,and the new organization, then called the GladstoneFoundation Laboratories for Cardiovascu-lar Disease,officially opened on September 1, 1979. Dr. Mahley isprofessor of pathology and medicine at UCSF and isa member of the National Academy of Sciences andthe Institute of Medicine.

By the end of 2001, the research staff of the GICDhad grown to more than 100 scientists, postdoctoralfellows, students, and research associates, occupyingabout 48,000 square feet of laboratory and officespace in buildings 9 and 40 on the SFGH campus. In21 years of operation, the institute has attained aninternational reputation for excellence. Its productivi-ty is documented in the more than 850 scientificpapers published by GICD scientists.

Research at the GICD is conducted in five areas andis supported by three core laboratories.


Description 17

Lipoprotein Biochemistry and Metabolism. Amajor focus of research in this area is to correlate thestructure and function of the apolipoproteins involvedin cholesterol transport, with particular emphasis onapoE. One of the structural tools that scientists in thisunit use is x-ray crystallography to determine thethree-dimensional structures of proteins. The investi-gators in this unit are Dr. Mahley, Karl H. Weisgraber,Ph.D., and Yadong Huang, M.D., Ph.D.

Cell Biology. Studies in this unit examine how thebody’s various cells regulate the storage and use ofcholesterol as it relates to the development of athero-sclerosis. The focus is on the roles of apoB, apoE, andclass A scavenger receptors in cellular cholesterolmetabolism and atherogenesis. The investigators inthis unit are Robert E. Pitas, Ph.D., and Thomas L.Innerarity, Ph.D. (retired 3/01).

Molecular Biology. Scientists in this unit apply thelatest DNA techniques to understand the regulationof genes important in controlling cholesterol, triglyc-erides, and apolipoprotein production. Studies focuson apoE and apoB, which mediate the interaction oflipoproteins with cell-surface receptors. Enzymescontrolling cholesteryl ester and triglyceride produc-tion represent a new area of research. This has led tostudies of adipose tissue metabolism and obesity. Inaddition, transgenes and homologous recombinationare used to create animal models of human diseases.The investigators in this unit 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 howmonocytes/macrophages are attracted to sites of ather-osclerotic lesion formation and to delineate the role ofplatelets in forming the occlusive thrombus that leadsto myocardial infarction. Another research goal is toelucidate cell-signaling pathways that can be used toconfer proliferative advantages to genetically modi-fied cells. The investigators in this unit are Israel F.Charo, M.D., Ph.D., and Bruce R. Conklin, M.D.

Clinical Molecular Genetics. Patient studies andnational and international population screening pro-jects conducted by Gladstone researchers aim to iden-

tify unique genetic abnormalities that cause hypercho-lesterolemia and premature myocardial infarction.Researchers in this unit operate the Lipid DisordersTraining Center, which trains medical personnel tomanage dyslipidemic patients, and the Lipid Clinic,which provides consultation on disease managementto SFGH patients and to private, referring physicians.This unit also conducts the Turkish Heart Study, whichinvestigates cardiovascular risk factors in a developingnation with a high incidence of heart disease. Theinvestigators in this unit are Dr. Mahley and Thomas P.Bersot, M.D., Ph.D.

Gladstone Genomics Core. The Genomics Coreassists scientists with the unprecedented researchopportunities presented by the decoding of the mouseand human genomes. Directed by Christopher S.Barker, Ph.D., this laboratory provides state-of-the-arttechnologies in the area of functional genomics forGladstone scientists and other investigators at SFGH.The core focuses on DNA microarray technology,including the preparation of custom oligonucleotidemicroarrays and customized microarray hybridiza-tion, array scanning, and data analysis.

Gladstone Transgenics Core. The GICD also main-tains a sophisticated transgenic core facility that isheavily used by investigators of all three institutes.The core’s activities are coordinated by John M.Taylor, Ph.D.

Gladstone Microscopy Core. The Microscopy Core,under the direction of David A. Sanan, Ph.D., pro-vides expertise, instrumentation, service, and trainingfor the generation and capture of research data in theform of microscopic images and for the quantitation,analysis, and interpretation of those images to allthree institutes.

Gladstone Instituteof Virology and Immunology

The GIVI resulted from the convergence of severalfactors. On the forefront of the battle against AIDSsince the beginning of the pandemic, SFGH is widelyrecognized as one of the world’s leading clinicalresearch centers for the study of HIV disease. The

2001 ANNUAL REPORT

18 Description

State of California provided funding to build an AIDSresearch center at SFGH under the auspices of theUCSF School of Medicine. Additional funds wereneeded to finish and equip the center and to undertakethe research. The success of the established relation-ship of UCSF and the City with the Gladstone formedthe foundation for a unique agreement by which theGladstone would lease the center, establish theresearch program, and manage the ongoing studies.

Gladstone and UCSF were able to attract an outstand-ing physician-scientist to direct the new institute.

Warner C. Greene, M.D., Ph.D., an internationallyrecognized immunologist and virologist, officiallytook the helm in September 1991. Before coming toGladstone, Dr. Greene was professor of medicine andinvestigator in the Howard Hughes Medical Instituteat Duke University Medical Center. Currently, Dr.Greene is also a professor of medicine and of micro-biology and immunology at UCSF, co-director of theUCSF Center for AIDS Research, and a member ofthe executive committees of the UCSF AIDSResearch Institute and UCSF Biomedical SciencesGraduate Program.

Formally dedicated on April 19, 1993, the GIVI occu-pies 27,000 square feet of space on the top two floorsof SFGH’s building 3. Studies at the GIVI are con-ducted under the direction of an outstanding group ofphysician-scientists in six state-of-the-art laboratoriesand three supporting laboratories.

Laboratory of Molecular Immunology. TheLaboratory of Molecular Immunology studies themechanisms by which proteins within the immunecells harboring HIV may act to trigger the growth ofthe virus and how the virus’s own proteins subse-quently amplify its replication and pathogenic effectsin primary T cells and macrophages. This work, head-ed by the director of the institute, Dr. Warner C.Greene, specifically focuses on the HIV proteins Vprand Nef and select host factors, including the NF-κB/Rel family of transcription factors.

Laboratory of Molecular Evolution. TheLaboratory of Molecular Evolution focuses on evolu-tion and its implications for medicine and epidemiol-ogy. Genetic variations in host susceptibility and inmicrobial replication capacity, virulence, and drugsusceptibility typically determine who develops dis-ease and who remains healthy. The laboratory exam-ines several consequences of molecular evolution,including HIV-1 drug resistance, selection pressuresbearing on HIV-1 populations during transmissionand in tissues, and nonpathogenic simian immunode-ficiency virus infection in natural host species. Thislaboratory is directed by Robert M. Grant, M.D.,M.P.H., an assistant investigator in GIVI and assistantprofessor of medicine at UCSF.

Gladstone Institute of Virologyand Immunology


Elizabeth H. Blackburn, Ph.D.Professor of Microbiologyand ImmunologyUniversity of California, San Francisco

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

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

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

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

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


Description 19

for analysis. The laboratory is directed by MartinBigos, M.S., a staff research scientist in GIVI.

Gladstone-UCSF Laboratory of Clinical Virology.The Gladstone-UCSF Laboratory of Clinical Virology,directed by Dr. Grant, provides key virological testingin support of HIV-related clinical research projects atUCSF. Established in collaboration with the UCSFAIDS Research Institute, this laboratory evaluatespatients who are failing combination antiviral therapy,studies HIV replication in the central nervous system,and investigates mechanisms of primary HIV infectionand sexual transmission. This laboratory is also devel-oping state-of-the-art assays for genotypic and pheno-typic drug resistance and assessment of viral loadsusing ultrasensitive techniques to further enhance clin-ical AIDS research at SFGH and UCSF.

Antiviral Drug Research Division. The AntiviralDrug Research Division evaluates potential newantiviral drugs. Using a novel animal model system,the SCID-hu mouse, the group is developing newmethods for drug evaluation and extending its work tothe field of viral pathogenesis. The laboratory isdirected by Cheryl A. Stoddart, Ph.D., a staff researchscientist in GIVI.

Gladstone Instituteof Neurological Disease

The GIND resulted from the natural expansion ofhighly successful research programs. Its predecessor,the Gladstone Molecular Neurobiology Program, wascreated in 1996 as a joint venture of the GladstoneInstitutes and the UCSF Department of Neurology.Lennart Mucke, M.D., recruited to head the new pro-gram, brought with him a group of researchers withexpertise in diverse areas of disease-related neuro-science. With its establishment, neuroscientists in thenew program could leverage Gladstone’s wealth ofexperience with apoE by applying it to the burgeoningfield of neurodegenerative diseases. These efforts werecomplemented with research on amyloid proteins,which play a seminal role in Alzheimer’s disease.

Significant findings were rapidly made in a broadrange of research areas, including molecular biology,

Laboratory of Receptor Biology. The Laboratory ofReceptor Biology investigates the molecular basis oftransmembrane signaling by cell-surface receptors onhematopoietic cells and the interplay between lym-photropic viruses and normal intracellular pathways ofsignal transduction. The laboratory is headed by MarkA. Goldsmith, M.D., Ph.D., an associate investigatorin GIVI and associate professor of medicine at UCSF.

Laboratory of Viral Pathogenesis. The Laboratoryof Viral Pathogenesis focuses on the pathogenicmechanisms of HIV in vivo, with the specific intent offinding better ways to prevent or suppress HIV-induced disease. The work falls into two areas: effectsof HIV on the central hematopoietic system and trans-mission of HIV across mucosal and placental barriers.Research in this laboratory is directed by Joseph M.McCune, M.D., Ph.D., a senior investigator in GIVIand professor of medicine at UCSF.

Laboratory of Molecular Virology. The Laboratoryof Molecular Virology studies how HIV transcriptionis controlled by host chromatin structure and by viralproteins such as HIV Tat. More recently, this labora-tory has also investigated the molecular basis for HIV-induced T-cell death, focusing on the role of apopto-sis. This laboratory is directed by Eric M. Verdin,M.D., a senior investigator in GIVI and professor ofmedicine at UCSF.

Laboratory of Cellular Immunology. TheLaboratory of Cellular Immunology investigatesinnate and adaptive cellular immune responsesagainst HIV and simian immunodeficiency virus atmucosal and systemic sites. The work focuses onunderstanding host immune/pathogen interactionsthat might be manipulated by vaccination or thera-peutic drugs. The laboratory is headed by Douglas F.Nixon, M.D., Ph.D., an associate investigator in GIVIand associate professor of medicine at UCSF.

Flow Cytometry Core Laboratory. The FlowCytometry Core Laboratory is dedicated to providingcutting-edge techniques in fluorescence-based cellsorting and analysis to Gladstone and UCSF scien-tists. This laboratory operates both a Becton-Dickinson FACS Vantage for sorting and a FACScan

2001 ANNUAL REPORT

20 Description

cell biology, physical structure, signal transduction,experimental pathology, and behavioral neurobiology.In 1998, the trustees expanded the program to createthe GIND. Its goal is to increase understanding of themolecular pathogenesis of neurological deficitsresulting from neurodegenerative disorders such asAlzheimer’s disease, from cerebrovascular disease, orfrom HIV infection. Productive synergies exist withscientists studying cardiovascular disease and AIDSin the other institutes.

As was the case with the two other institutes,Gladstone and UCSF were able to attract an out-standing physician-scientist to head the new institute.Dr. Mucke was educated at the Max-Planck-Institutefor Biophysical Chemistry in Germany, the

Massachusetts General Hospital, and HarvardMedical School. He came to Gladstone from TheScripps Research Institute to expand Gladstone’sresearch efforts in disease-oriented neuroscience inthe context of the Molecular Neurobiology Programand then to direct the new institute. Dr. Mucke is alsothe Joseph B. Martin Distinguished Professor ofNeuroscience at UCSF.

The GIND was formally dedicated on September 11,1998. Its laboratories are housed in buildings 1, 9, and40 of the SFGH campus. Studies at the GIND are con-ducted in eight state-of-the-art laboratories and abehavioral core laboratory. The research focuses onsix major areas relating to neurodegenerative disor-ders, cognitive function, and brain inflammation asoutlined below.

Physiological and Pathophysiological Roles ofAmyloidogenic Proteins in the Brain. While amy-loidogenic molecules, such as the amyloid β proteinprecursor and α-synuclein, may facilitate learning andmemory, they can be broken down into peptides oraltered in their conformation to form neurotoxicaggregates in cells and tissues. Understanding howthese toxic proteins form and act could facilitate thedesign of better treatments for Alzheimer’s diseaseand other neurodegenerative disorders. Defining thenormal function of the amyloidogenic precursor mol-ecules is of fundamental neuroscientific interest.Investigators involved in research on this topic are Dr.Mucke, Tony Wyss-Coray, Ph.D., Robert W. Mahley,M.D., Ph.D., Yadong Huang, M.D., Ph.D., and KarlH. Weisgraber, Ph.D.

Role of ApoE in Neurodegeneration and CognitiveImpairment. The apoE4 allele is the main knowngenetic risk factor for the most common form ofAlzheimer’s disease and for poor neurological out-come after head injury and cardiac bypass surgery.Defining the effects of the three main human apoEisoforms (E2, E3, and E4) on the structure and func-tion of the brain should provide crucial insights intothe contribution of the apoE4 variant to neurologicaldisease. Characterizing how changes in the x-raycrystallographic three-dimensional structure of apoEaffect its activity may result in the development of

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 Medicineand 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.Professor of Anatomyand of Biochemistry and BiophysicsUniversity of California, San Francisco

novel apoE-targeted drug treatments for Alzheimer’sdisease and other neurological conditions.Investigators involved in research on this topic areDrs. Mahley, Weisgraber, Huang, Mucke, and RobertE. Pitas, Ph.D.

Huntingtin and Other Polyglutamine-RepeatProteins. Huntington’s disease, the most commoninherited neurodegenerative disorder, is caused by anabnormally long stretch of the amino acid glutaminewithin the protein called huntingtin. Abnormal polyg-lutamine stretches within other proteins are responsi-ble for several other midlife neurodegenerative disor-ders. Determining how abnormal polyglutaminestretches cause neurons to die may make it possible todevelop specific therapies for these disorders. It mayalso reveal general mechanisms of neurodegenerationthat are relevant to other neurological diseases. Thistopic is a major focus of Steven M. Finkbeiner, Ph.D.

Neural Plasticity. Plasticity is a property of the ner-vous system that enables it to undergo long-lasting,sometimes permanent adaptive responses to briefstimuli. Plasticity is believed to be important forestablishing precise patterns of synaptic connectionsduring early neuronal development and for learningand memory in adults. Disturbances in plasticity andsynaptic function could contribute significantly tomemory disorders characteristic of many neurodegen-erative diseases, such as Alzheimer’s disease andHuntington’s disease. An understanding of the molec-ular mechanisms that regulate the formation, activity,degeneration, and regeneration of synapses and neu-ronal dendrites could form the basis for therapeuticstrategies to prevent memory loss and cognitive

decline in diverse diseases. Investigators involved inresearch on this topic include Drs. Finkbeiner, Mucke,and Fen-Biao Gao, Ph.D.

Neurobiological Function of Glial Cells and TheirRole in Neurological Disease. Glial cells are special-ized brain cells that support the health and function ofneurons. In response to brain injuries, these cells pro-duce a large number of molecules that participate ininflammatory and immune responses. While acuteglial responses may help prevent neuronal damageand facilitate the removal of toxic amyloid proteins,abnormal activation of these cells could contribute toneurological disease. Genetic and pharmacologicalstrategies are used to characterize the beneficial anddetrimental roles of glial cells in cerebral amyloido-sis, neurodegeneration, and HIV-associated dementia.Investigators involved in research on this topicinclude Drs. Wyss-Coray, Mucke, and Pitas.

Behavioral Core Laboratory. Because many of ourmouse models are designed to simulate aspects ofhuman diseases resulting in memory deficits, behav-ioral disturbances, or movement disorders, thedetailed behavioral characterization of these modelsplays an important role in the assessment of their clin-ical relevance. Behavioral alterations in transgenicmodels can shed light on the central nervous systemeffects of diverse molecules and are used to assessnovel therapeutic strategies at the preclinical level.Established by Jacob Raber, Ph.D., and Dr. Mucke,this core is now engaged in collaborative studies withinvestigators at all three Gladstone Institutes, as wellas with scientists in various UCSF departments andother institutions.


Description 21

Mission Bay 23

The vision of Gladstone’s new research build-ing at Mission Bay is becoming clearer as theplanners make good progress in hammering

out the details. This vision represents Gladstone’soptimistic and progressive spirit for growth over thenext 10 years.

Most recently, the trustees—Richard S. Brawerman,Albert A. Dorman, and Richard D. Jones—completedthe bond financing necessary to fund the project. Theyworked hard to secure the highest ratings and the low-est interest rate possible for this 30+ year investment.The interest rate of the $145 million loan is the lowestsecured in the past 20 years.

New Gladstone Laboratories at the UCSF Mission Bay Campus

15A

18B

21A

21B

24C

25A 25B

23B

23A

20A 20B

17B17A16B

17A16B

24A/B

18A

15B 16A

19A 19B

The Green

16th Street

3rd S

treet

The Courts

The Commons

The Plaza

Gladstone

InstitutesParking

Structure

ProposedBiotech Labs

Owens Street

Location of the Gladstone building at the new UCSF Mission Bay campus. The new laboratories will be located across from building24A/B, the initial UCSF building, currently under construction as part of Phase I, and in close proximity to the Student/CommunityCenter to be located in building 21A.

2001 ANNUAL REPORT

24 Mission Bay

The architects have also been hard at work. They haveprepared a preliminary blueprint of the six-floor build-ing. The first floor will hold the offices for administra-tion and building operations, a 150-person lecture hall,four seminar rooms, and an eating facility. The secondfloor will be left as shell space, which can be leased outand/or developed into laboratories in the future. Floorsthree, four, and five will house the offices and labora-tories of the three institutes, while the animal quarterswill be located on the sixth floor. The architects arecurrently working with the design team—composed ofRobert Mahley, Lennart Mucke, Warner Greene, KarlWeisgraber, Robert Pitas, Israel Charo, Debbie Addad,Todd Sklar, and designers—to refine the plans for thelaboratories and the building’s layout. The plannershave also benefited significantly from the input ofother Gladstone scientists and staff members.

With about 200,000 square feet of space, the newbuilding will provide sufficient room for Gladstone to

grow from the current 265 employees to more than500. This will help Gladstone achieve its goal of beinghome to 27–30 investigators by 2010. Gladstone willalso enjoy the synergy that comes from having allthree institutes, the core laboratories, and central facil-ities integrated in a single research building.

Scheduled for completion in 2004, the Gladstonebuilding will be located at Owens and 16th Streets atUCSF’s new 43-acre Mission Bay campus. Whencompleted, this campus will host about 9000 scien-tists and support staff and will house many of UCSF’sbasic biomedical research programs. Initial researchprograms include structural and chemical biology,molecular and cell biology, neuroscience, develop-mental biology, and genetics. The new buildingpromises to strengthen Gladstone’s excellent rela-tionship with UCSF and provide an opportunity forincreasingly productive collaborations with universi-ty colleagues.

Courtesy of NBBJ Architects

Members of the Institute 25

MEMBERS OF THE INSTITUTE


Director

Lennart Mucke, M.D.

Joseph B. Martin Distinguished Professor

of Neuroscience

Investigators

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

Assistant Professor of Neurology

and Physiology

Fen-Biao Gao, Ph.D.

Assistant Professor of Physiology

and Neurology


Professor of Pathology and Medicine

Robert E. Pitas, Ph.D.

Professor of Pathology

Karl H. Weisgraber, Ph.D.

Professor of Pathology

Staff Research Investigators

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

Assistant Professor of Pathology

Tony Wyss-Coray, Ph.D.


Staff Research Scientists

Christopher S. Barker, Ph.D.

Jacob Raber, Ph.D.


Research Scientists

Manuel J. Buttini, Ph.D.

Robert L. Raffaï, Ph.D.

Visiting Scientists

Andrea Barczak

Marion Buckwalter, M.D., Ph.D.

Toru Kawamura, Ph.D.

Yvonne Kew

Xiao Xu, M.D., Ph.D.

Shirley Zhu, Ph.D.

Members of the Gladstone Institute of Neurological Disease

Postdoctoral Fellows

Montserrat Arrasate, Ph.D.

John Bradley, Ph.D.

Jeannie Chin, Ph.D.

Luke A. Esposito, Ph.D.

Christian Essrich, Ph.D.

Paul C. R. Hopkins, Ph.D.

Amy Hsiu-Ti Lin, Ph.D.

Jorge J. Palop-Esteban, Ph.D.

Juan Santiago-Garcia, Ph.D.

Kimberly Scearce-Levie, Ph.D.

Ina Tesseur, Ph.D.

Zheng Wang, Ph.D.

Shiming Ye, Ph.D.

Students

Duane M. Allen

Gerold Bongers

Sarah Carter

Patrick Chang

Ammon Corl

Jennifer Fu

Richard J. Han

Sharon E. Haynes

Jason Held

Marian L. Logrip

Siddhartha Mitra

Linda Ngo

Ryan P. Owen

Amina A. Qutub

Hélène Rangone

Vikram Rao

Neal T. Sweeney

Senior Research Associates

Maureen E. Balestra

Walter J. Brecht

Zhong-Sheng Ji, Ph.D.

Yvonne M. Newhouse

Gui-Qiu Yu, M.S.

Research Associates

Thomas C. Brionne

Elizabeth S. Brooks, M.S.

Anita Chow

Jacob Corn

Jessica J. Curtis

Nhue Do

Sarah E. Goulding, Ph.D.

Kristina Hanspers, M.S.

Yanxia Hao, M.D.

Faith M. Harris

Shyamal G. Kapadia

Lisa N. Kekonius

Anthony D. LeFevour

Wenjun Li, Ph.D.

Xiao Qin Liu, M.D.

Rene D. Miranda

Lauren Mondshein

Hilda C. Ordanza

Jon-Paul Pepper

Michelle E. Rohde

Nathan G. Salomonis

Kristina P. Shockley

Richard M. Stewart

Fenrong Yan

Yuhua Zheng

Lab Aide

John A. Gray

2001 ANNUAL REPORT

28 Members of the Institute

Administrative Staff

of the J. David Gladstone Institutes

President


Administrative Assistants

Catharine H. Evans

Karina G. Fantillo

Mariena D. Gardner

Marlette A. Marasigan

Kelley S. Nelson

Nannette I. Nemenzo

September C. Plumlee

Emily K. O’Keeffe

Aileen C. Santos

Bethany J. Taylor

Executive Assistants

Brian Auerbach

Denise Murray McPherson

Sylvia A. Richmond

Finance and Accounting

Marc E. Minardi, M. Div., Officer

Richard S. Melenchuk, M.S., Manager

Emilia M. Herrera

Kai Yun W. Sun

Kenneth J. Weiner

Grants and Contracts

Rex F. Jones, Ph.D., Officer

Frank T. Chargualaf, M.B.A.

Lynne M. Coulson

Marga R. Guillén, M.P.A.

Marya Pezzano

Martin C. Rios

Michael S. Whitman

Yvonne L. Young

Human Resources

Migdalia Martinez, M.S., Officer

John R. LeViathan, M.A., Manager

Wendy M. Foster

Anthony R. Gomez

Chad E. Popham

Alyssa S. Uchimura

Information Services and Communications

Reginald L. Drakeford, Sr., Officer

Susan H. Dan

Iris Newsum, M.B.A., M.S.

Sylvia A. Richmond

Teresa R. Roberts

Editorial

Gary C. Howard, Ph.D.

Stephen B. Ordway

Graphics and Photography

John C. W. Carroll, Manager

Stephen Gonzales

Christopher A. Goodfellow

John R. Hull, M.F.A.

Information Services

Jon W. Kilcrease, Manager

Theodore E. Doke

Matthew L. Lyon

Joseph R. Solanoy

Public Affairs

Laura Lane, M.S., Manager



2001 ANNUAL REPORT

30 Members of the Institute

Intellectual Property/Technology Transfer

Joan V. Bruland, J.D., Officer

Erin Madden

Anne Scott, M.A., M.S.

Office of the President

Susan H. Dan

Teresa R. Roberts

Operations

Deborah S. Addad, Officer

Facilities

Vincent J. McGovern, M.S., Manager

David R. Bourassa

Randy A. Damron

George R. Leeds

Roger A. Shore

Purchasing

P. J. Spangenberg, Manager

Tyler G. Campos

Judy H. Cho

P. Sidney Oduah

Alberto L. Reynoso

Benjamin V. Young

Receptionist

Hope S. Williams

Student Assistants

Shannon P. Chi

April D. Hughes

Christina N. Luna

Luvy C. Vanegas

Reports from the Laboratories 31

REPORTS FROMTHE LABORATORIES

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

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

Huang Laboratory.........47

Mahley Laboratory.........55

Mucke Laboratory.........63

Pitas Laboratory.........73

Weisgraber Laboratory.........79

Wyss-Coray Laboratory.........87

Behavioral Core Laboratory.........95

Gladstone Genomics Core.........103


FINKBEINER LABORATORY

Assistant Investigator



Montserrat Arrasate, Ph.D.

John Bradley, Ph.D.

Students

Sarah Carter

Patrick Chang

Ammon Corl

Jennifer Fu

Siddhartha Mitra

Hélène Rangone

Vikram Rao

Research Associates

Elizabeth Brooks

Jessica Curtis

Shyamal Kapadia

Administrative Assistant

Nannette Nemenzo


Our laboratory is interested in two biologicalquestions. First, how does the nervous systemadapt to brief experiences with long-lasting

changes in its structure and function? The molecularmechanisms that underlie this process, collectivelyknown as plasticity, are important for the properdevelopment of the nervous system and for formingmemories. We are especially interested in the role thatnew gene expression plays in coupling transient neu-ronal activity to long-term changes in synaptic func-tion. The second question that we are pursuing is howa genetic mutation leads to an adult-onset progressiveneurodegenerative disease. We have focused onunderstanding the molecular mechanisms that medi-ate the inherited neurological disorder Huntington’sdisease (HD).

A Robotic Microscope

The molecular mechanisms that mediate geneexpression–dependent synaptic plasticity and muta-tion-dependent neurodegeneration have at least onefeature in common. Significant time must elapsebetween the initiating process (e.g., transient synap-tic activity or the inheritance of a genetic mutation)and the final outcome (lasting changes in synapticfunction or neurodegeneration). That these processesoccur over a significant time span has complicatedour efforts to study them.

We have developed a cellular model of HD by intro-ducing versions of the causative gene into neuronsgrown from regions of the brain that are most vulner-able in people. We fix the neurons days after intro-ducing the gene and then use immunocytochemistryto assay for neurodegeneration. The model recapitu-lates three key features of HD: mutation-dependent

Molecular Mechanisms of Plasticity and Neurodegeneration


neurodegeneration, cell-specific death, and formationof abnormal deposits of aggregated huntingtin knownas intranuclear inclusions.

However, this immunocytochemical approach hassignificant limitations. First, it is relatively insensi-tive. Only a fraction of the neurons that undergo neu-rodegeneration are caught—some have not begun todegenerate at the time the cells are fixed, whereas oth-ers have degenerated so completely that they are dif-ficult to identify or are absent altogether. Second,manual scoring is extremely time consuming.Because the assay relies on a microscopic assessmentof morphology, it is inherently user-dependent.Criteria may not be applied uniformly, and if neuronsdegenerate by multiple effector pathways, the rangeof morphological changes may not be fully encom-passed in a single definition. Third, the static nature ofimmunocytochemistry limits its potential for address-ing mechanistic questions, for unraveling thesequence of events that are responsible for the processunder study, and for determining whether an observedchange is a cause or an effect.

In the last year, we have developed a new platform forstudying mechanisms of plasticity and neurodegener-ation. Central to this effort is a device we call a robot-ic microscope. The core of the device is an invertedmicroscope body equipped with special optics tomake automated image collection and analysis feasi-ble. Computer-controlled motors move the stage, thefocus knob, multiple fluorescence filters, and shutters.We programmed the instrument to automaticallyfocus itself by brief 20-millisecond pulses of light andfast Fourier transform analysis. Once focused, thecomputer collects fluorescence images of differentwavelengths and then precisely moves the stage to an

2001 ANNUAL REPORT

36 Reports from the Laboratories

adjacent but nonoverlapping field. These steps arerepeated until selected fields of each well of a multi-well plate are automatically imaged. We have writtenprograms that automatically analyze the stacks ofimages and quantify features of interest.

This platform has many new capabilities. For thefirst time, we have the ability to follow the fates ofspecific neurons over any time interval. We canimage a particular living neuron or field of livingneurons, return the tissue-culture plate to the incu-bator, and at any time mount the plate back on themicroscope and quickly find the same neuron andcollect another image (Figure 1). This is a key capa-bility for elucidating mechanisms of plasticity andneurodegeneration that develop over days to weeks.Second, the system is very fast, making possibleexperiments that were previously unfeasible. Forexample, in one typical experiment, we collectedimages and counted approximately 300,000 trans-

fected neurons from a single 24-well plate in about15 minutes. In our previous assay, this would havetaken us nearly 6 weeks. Third, the criteria for scor-ing are defined explicitly in the automated analysisprogram. This removes a component of user bias,ensures that each neuron is quantified similarly, andmakes it possible to communicate the exact criteriato other labs. Fourth, the assay is much more sensi-tive than our previous assay. By collecting imageswithin an hour of transfection and periodicallythereafter, we can accurately measure the initialtransfected population and precisely follow the rateof neurodegeneration. This method accounts for allthe cells and has the capability to detect neuron-specific patterns of degeneration. The system hasmany other capabilities and is useful for a widevariety of different applications that would benefitfrom high-throughput analysis, including our ownstudies on gene expression–dependent mechanismsof synaptic plasticity.

Figure 1. The robotic microscope facilitates high-throughput cellbiology. Striatal neurons have been cultured in multiwell dishes andtransfected with the reporter gene, green fluorescent protein (GFP).The microscope has been programmed to scan the whole dish, col-lecting stacks of images representing nonoverlapping fields fromeach well of the entire dish. The plate of neurons is returned to theincubator for a period of time and then remounted on the micro-scope for repeated imaging. In this experiment, imaging was per-

formed 15, 39, 48, 72, 96, and 136 hours after transfection with GFP.After the experiment was complete, a field was chosen from thestack of images collected on the first day and then the correspond-ing image was chosen from the stacks collected on the subsequentfive days. The arrows point to individual neurons whose fate can befollowed over the course of the six days. The arrowheads point to aneuron that survives the experiment; the arrows point to a neuronthat dies between the third and fourth days.



subtype of glutamate receptor, plays an essential role ininitiating activity-dependent plasticity at many synaps-es. Our laboratory is interested in understanding howcalcium signals are encoded into the activity of specificsignal transduction pathways, how the activity of thesepathways regulates the transcription of certain gene tar-gets that are important for synaptic plasticity, and hownewly transcribed or translated genes regulate the spe-cific subsets of synapses whose strength has been ini-tially and transiently changed.

In the last year, we have especially focused on howcalcium influx through the NMDA receptor regulatesneuronal gene expression. Our previous work hadsuggested that calcium influx through the NMDAreceptor couples to signal transduction pathways nearthe cytoplasmic mouth (within ~1 µm of the plasmamembrane) to control gene expression in the nucleus.Thus, accessory proteins near the cytoplasmic mouthof the channel, possibly directly associated with the

Molecular Mechanismsof Synaptic Plasticity

Although new gene transcription and protein expres-sion are believed to play a critical role in the processof learning and memory formation, delineating thatrole poses a number of difficult cell biological ques-tions. The pattern of synaptic activity determineswhether the strength of a synapse grows, diminishes,or remains unchanged. How are these distinct patternsof synaptic activity related to the gene expression pro-grams that regulate synaptic strength? If the influx ofextracellular calcium is crucial for initiating the bio-chemical processes that strengthen or weaken asynapse as well as the processes that control activity-dependent gene transcription, how does calcium spec-ify these distinct responses?

Although multiple neuronal calcium channels are sensi-tive to activity, one, the N-methyl D-asparate (NMDA)

Figure 2. (A) The domain structure of the cytoplasmic tail of wild-type NR1 subunit (NR1A), a normal splice variant (NR1C), and adeletion mutant (NR1∆). M4 designates a portion of the fourth trans-membrane-spanning region, and C0, C1, and C2 are the namesgiven to cassette domains of the carboxyl terminus. The numbersalong the top correspond to the amino acid positions of the bound-aries, counting from the amino terminus. (B) NMDA receptor–medi-ated gene expression reconstituted in NR1–/– neurons. NR1–/– andNR1+/– neurons were transfected with a reporter gene (CaRE-luciferase) and stimulated to activate NMDA receptors (100 µM,NMDA) or L-type voltage-sensitive calcium channels (L-VSCCs) (55mM, KCl). NR1+/– neurons responded to all stimuli (left set of bars);similar responses were seen with NR1+/+ (wild-type) neurons (notshown). NR1–/– neurons responded normally to depolarization butnot to NMDA (middle set of bars) until the NR1A subunit wasrestored by transfection (pCMV-NR1A, right set of bars) (n = 5, *p <0.005). (C) Variants of the NR1 subunit flux Ca2+ but differ in theirability to regulate gene expression. NR1–/– neurons were transfectedwith NR1A, NR1C, or NR1∆ and the transfection marker GFP-NLS.Ca2+ responses to NMDA (100 µM, arrow) measured by imagingFura-2, were comparable. Scale bars: time (tim) = 20 seconds; [Ca2+]= 5% ∆F/F for NR1A and NR1C and 4% for NR1∆. ∆F/F is propor-tional to intracellular Ca2+ concentration. It is calculated by measur-ing the average fluorescence intensity before and after stimulation,subtracting the former from the latter, and then dividing the result bythe fluorescence intensity before stimulation. (D) NR1–/– neuronswere transfected with NR1A, NR1C, or NR1∆, the reporter geneCaRE-luciferase, and the control gene Ren-luciferase. NMDA recep-tors reconstituted with NR1C or NR1∆ mediated significantly lessNMDA-stimulated CaRE-dependent gene expression than NR1A (n= 5, *p < 0.005). Similarly transfected neurons mediated compara-ble responses to L-VSCC stimulation, indicating that the effect of theNR1 subunit is specific to the ability of the reconstituted NMDAreceptor to regulate gene expression. Control fold induction = 1.

2001 ANNUAL REPORT


NMDA receptor itself, might play a role in couplingcalcium influx to nuclear gene expression. The ideathat channel-associated proteins could play a role inlong-range signal transduction emanating from theNMDA receptor could explain how a pleiotropic sec-ond messenger, the calcium ion, could nonethelesssend a “channel-specific” signal.

To explore this hypothesis further, we have developeda reconstitution system that enables us to determinehow channel-associated proteins couple calcium influxthrough the NMDA receptor to new gene transcription.The NMDA receptor is believed to be a heteromericchannel, possibly a tetramer composed of two proteinsubunits encoded by the NR1 gene and two subunitsencoded by members of the NR2 gene family. Withoutthe NR1 gene product, no functional NMDA receptoris synthesized, and animals that lack the NR1 gene failto live beyond their first postnatal day.

If we stimulate cultured neurons from NR1–/– mice withNMDA, they fail to show calcium or gene expressionresponses. However, when we transfect NR1–/– neuronswith an expression plasmid that encodes NR1, we res-cue both responses (Figure 2A–C). Since the bindingsite for NMDA is on the NR2 subunit and channels thatlack the NR1 subunit fail to function, the rescuedresponses must be mediated by reconstituted het-eromeric NMDA receptors that have incorporated thetransfected NR1 subunit. Thus, the system has given uscontrol of the cytoplasmic tail of the NR1 subunit with-in every functional NMDA receptor.

To test whether the cytoplasmic tail of the NMDAreceptor or the proteins that associate with it areimportant for NMDA-dependent gene expression, wecompared the abilities of carboxyl-terminal truncationmutants of NR1 to mediate NMDA-induced calciumand gene expression responses in our reconstitutionsystem (Figure 2A). We found that specific deletionsin the carboxyl terminus of the NMDA receptor didnot measurably affect the ability of the channel to pro-duce cytoplasmic calcium increases, as measuredwith the calcium indicator Fura-2 (Figure 2C).However, some of these same deletions substantiallydiminished the ability of the NMDA receptor to acti-vate neuronal gene transcription (Figure 2D).

The fact that deletions in the carboxyl terminus ofNR1 can dissociate bulk cytoplasmic calciumresponses from the ability of the NMDA receptor toregulate adaptive gene expression suggests twoimportant conclusions. First, a critical component ofcalcium-dependent signal transduction specificitymay be conferred by the molecules that the calciumion engages near its site of entry. Second, some of thelong-range signals that a calcium channel sends maybe determined by the repertoire of proteins locatednear its cytoplasmic mouth. Future work will focus onrefining our understanding of the sequences withinNR1 and associated proteins that are required to cou-ple calcium influx to gene transcription and on ana-lyzing the promoters of NMDA-induced genes todetermine the extent to which a channel-specific sig-nal is perceived by the nucleus.

Molecular Mechanisms of HD

From initial work with our cellular model of HD, wehad found that the addition of caspase inhibitors, thecotransfection of the anti-apoptosis gene BClXL, orthe addition of extracellular factors such as brain-derived neurotrophic factor, ciliary neurotrophic factor,or insulin growth factor 1 (IGF-1) delayed or sup-pressed neurodegeneration in response to mutanthuntingtin. Surprisingly, the manipulations had differenteffects on the abnormal deposition of mutant hunt-ingtin within neurons to form intranuclear inclusions.IGF-1 suppressed the fraction of neurons with visibleinclusion bodies, whereas all the other manipulationsdoubled or tripled the fraction of neurons withintranuclear inclusions. In the last year, we and ourcollaborators have focused on the signal transductionmechanisms by which IGF-1 suppresses degenerationand inclusion body formation by mutant huntingtin.

We have discovered that the pro-survival kinase, Akt,is responsible for many of the effects of IGF-1 in ourcellular model of HD. IGF-1 activates Akt in the stri-atal neurons that we study, and active forms of Aktintroduced into these neurons recapitulate the effectsof IGF-1 on huntingtin-induced neurodegenerationand inclusion body formation. Surprisingly, we havediscovered a highly conserved Akt phosphorylationsite within the huntingtin protein itself and have



demonstrated that huntingtin is an Akt substrate. Wehave generated antibodies that specifically recognizethe Akt-phosphorylated form of huntingtin and havefound that IGF-1 stimulation leads to huntingtinphosphorylation in neurons. Blocking phosphoryla-tion by site-directed mutation (S421A) rendersmutant huntingtin partly resistant to the protectiveproperties of IGF-1. Conversely, mutation of this siteto mimic tonic phosphorylation (S421D) renders

mutant huntingtin nontoxic. This is the first evidencethat huntingtin is a phosphoprotein and that its abili-ty to induce neurodegeneration can be regulated byphosphorylation. These results also suggest that hunt-ingtin may be an in vivo target of Akt. Finally, sinceAkt activity can be regulated by small-moleculedrugs, these findings suggest that Akt may be anattractive therapeutic target for HD and other neu-rodegenerative disorders.

Selected References

Saudou F, Finkbeiner S, Devys D, Greenberg, ME(1998) Huntingtin acts in the nucleus to induce apop-tosis but death does not correlate with the formationof intranuclear inclusions. Cell 95:55–66.

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

Bradley J, Curtis J, Finkbeiner S (2001) The C-termi-nus of the NMDA receptor NR1 subunit is requiredfor NMDA-dependent gene expression. Soc.Neurosci. 27 (Part 2):2451 (abstract).

Finkbeiner S (2001) Disease-associated polyglutamineexpansions as protein epitopes involved in neurode-generation. Soc. Neurosci. 27 (Part 2):674.5 (abstract).

Finkbeiner S (2001) New roles for introns: Sites ofcombinatorial regulation of Ca2+- and cyclic AMP-dependent gene transcription. Science’s STKE( h t t p : / / s t k e . s c i e n c e m a g . o r g / c g i / c o n t e n t /full/OC_sigtrans;2001/94/pe1).

GAO LABORATORY

Assistant Investigator

Fen-Biao Gao, Ph.D.


Sarah Goulding, Ph.D.

Wen-Jun Li, Ph.D.

Students

Richard Han

Linda Ngo

Neal T. Sweeney

Research Associates

Nhue L. Do

Yu-Hua Zheng





Molecular Mechanisms of Dendritic Morphogenesisand Their Involvement in Neurological Diseases

Fen-Biao Gao, Ph.D.

Signaling between neurons requires specializedsubcellular structures, including axons and den-drites. Dendrites can be highly branched and

may account for more than 90% of the postsynapticsurface of some neurons. Only recently have den-drites been appreciated as having much more activeroles in neuronal function. In addition, the number ofdendritic branches and dendritic spines is altered inmany neurological disorders, including Alzheimer’sdisease and fragile X syndrome. Despite the impor-tance of dendrites in neuronal function and dysfunc-tion, the molecular mechanisms underlying dendriticmorphogenesis in vivo remain elusive.

Using Drosophila as a Model Systemto Study Dendrite Development

Systematic genetic analysis in Drosophila offers apowerful approach to unravel complex biologicalprocesses. To understand the molecular mechanismsthat control dendritic morphogenesis during devel-opment and in neurological disorders, our laboratoryuses the Drosophila peripheral nervous system(PNS) as a genetic model system. Each abdominalhemisegment contains only 44 PNS sensory neu-rons, which can be grouped into dorsal, lateral, andventral clusters. In the dorsal cluster, there are sixmutiple dendritic (MD) neurons, four external sen-sory (ES) neurons, one bipolar dendritic (BD) neu-ron, and one internal sensory neuron. Using theUAS-GAL4 system to express green fluorescentprotein (GFP) in a cell type–specific manner, we candirectly visualize dendritic morphogenesis of dorsalMD neurons in living Drosophila embryos and lar-vae and follow their growth, branching, and remod-eling in real time. These neurons elaborate their den-drites just underneath the epidermal cell layer in a

two-dimensional plane. The dendritic branching pat-tern of the MD neurons is fairly invariant fromembryo to embryo, suggesting that a genetic pro-gram controls dendritic morphogenesis. Using thisassay system, we have begun to identify the molec-ular components of the genetic program, includingthe flamingo and sequoia genes.

GFP Labeling of Single Neuronsin Living Drosophila Larvae

To study how neuronal morphogenesis is controlledby intracellular factors during development, our labo-ratory uses the mosaic analysis with a repressiblemarker technique to visualize single wildtype andmutant PNS neurons in living Drosophila larvae.Briefly, with the UAS-GAL4-targeted expression sys-tem, all of the larval neurons are labeled by mCD8-GFP. When GAL80, a yeast repressor that can bind toGAL4, is ubiquitously expressed, mCD8-GFP expres-sion is suppressed in all neurons. When FLP recombi-nase–mediated recombination occurs in precursorcells, one daughter cell loses GAL80 and is labeled byGFP. Using this approach, we could label differentindividual MD neurons with GFP in livingDrosophila larvae.

Morphological Diversityof PNS Sensory Neurons

We obtained images of the dendritic branching pat-terns of each subtype of PNS neuron in the dorsalcluster, as well as images of other PNS sensory neu-rons in the lateral and ventral clusters. Our geneticstudies mainly focused on the dorsal cluster.Therefore, only the development of dendritic fields ofdorsal cluster MD neurons is described here in detail.

We found that even in the relatively simpleDrosophila nervous system, different neurons devel-oped strikingly diverse dendritic morphologies. Thecentral nervous system (CNS) neuron in Figure 1Asends its axon across the ventral midline and elabo-rates neuronal processes in a three-dimensional man-ner, which makes it difficult to reconstruct the neu-ron’s morphology. This complex morphology alsoprevents clear differentiation of the dendritic andaxonal processes. In contrast, the morphologicalpolarity of PNS sensory neurons is much easier todefine. For example, ES neurons extend a single den-

drite in the direction opposite to that of its axon(Figure 1B). Dorsal cluster BD neurons extend twounbranched dendrites along the anteroposterior axisand an axon ventrally toward the CNS (Figure 1C).Most MD neurons develop highly diverse dendriticbranching patterns (Figure 1D). The complexity of thedendritic morphologies of some MD neurons inDrosophila is comparable to that of many mammalianCNS neurons. In addition, some MD neurons (Figure1D) have processes similar to the “headless” spineson many developing mammalian neurons. Theseprocesses are typically 5–10 µm long in third instarlarvae and are more numerous on dendrites distal tothe cell body. Similar to the previously described den-dritic filopodia of embryonic MD neurons, thesespine-like processes are not labeled by tau-GFP, sug-gesting that they lack microtubules.

Development and Organizationof MD Neuron Dendritic Fieldsin the Dorsal Cluster

Labeling single PNS neurons in living larvae allowed usto study how the dendritic fields of different MD neu-rons are formed and organized in the same dorsal clus-ter, even though their cell bodies are close to each other.

Our single-neuron analysis revealed that each MDneuron has a defined dendritic field that is similar indifferent abdominal segments of the same larva.Particular MD neurons vary little among larvae at thesame stage. Individual MD neurons in the same dorsalcluster have distinctive dendritic fields. For instance,the ddaC neuron sends its primary dendrite dorsally.This dendrite soon branches into secondary and ter-tiary branches that cover the whole hemisegment fromthe anterior segment boundary to the posterior seg-ment boundary and from the dorsal midline to the lat-eral cluster of PNS neurons (Figure 2A). The ddaCneuron also extends many smaller dendritic processesmore or less parallel to the dorsal midline.Interestingly, these dendritic processes from the sameddaC neuron never overlap, suggesting a “self-avoid-ance” mechanism, as previously described for axonalbranches of a single mechanosensory neuron in leech-es. Like the ddaC neuron, the ddaD neuron in the dor-sal cluster sends dendrites covering the area between

2001 ANNUAL REPORT


Figure 1. Neuronal polarity and diversity of neuronal morphologyin Drosophila. (A) A CNS neuron sends its axon past the ventralmidline (dashed line). (B) An ES neuron in a dorsal cluster. (C) ABD neuron in a dorsal cluster. (D) An MD neuron with numerousspine-like protrusions in a lateral cluster. Arrows indicate theaxons of mCD8-GFP-labeled neurons. The bar represents 10 µmfor A and 40 µm for B–D.

segment boundaries (Figure 2B). However, it onlysends out four or five major branches to cover the areabetween the dorsal midline and its cell body and hasmany fewer smaller branches. Unlike the ddaC andddaD neurons, the four other MD neurons in the dor-sal cluster have much smaller dendritic fields. TheddaF and ddaE neurons extend only a few branchestoward either the anterior or the posterior segmentboundaries; their most dorsal dendritic branches fallshort of the dorsal midline (Figure 2C and D). In con-trast, the ddaB neuron has one or two dendritic branch-es that reach the dorsal midline and a few branchesthat extend toward the anterior segment boundary(Figure 2E). Like the ddaD neuron, the ddaA neuroncovers the area between segment boundaries (Figure2F). The dendritic fields of the ddaC and ddaD neu-rons overlap with each other and with the dendriticfields of the four other MD neurons. However, the

dendritic fields of ddaE and ddaF neurons have mini-mal overlap. The presence of different dendritic fieldsin the same dorsal cluster raises the possibility thateach MD neuron has a defined physiological functionand that the dendritic morphology of each MD neuronis mainly determined by its intrinsic properties.

Flamingo Primarily Controls the Extensionof Dorsal Dendrites in MD Neurons

The flamingo gene encodes a protein resembling a Gprotein–coupled receptor, with seven transmembranesegments and a large amino-terminal domain contain-ing nine cadherin repeats. The molecule was identifiedbased on its function in planar polarity determinationand in dendrite development. To further understandhow Flamingo controls neuronal morphogenesis andto assess the extent to which Flamingo functions in a



Figure 2. Dendritic fields of the six MD neurons in the dorsal clusterin wildtype larvae. (A) A ddaC neuron sends out dendrites that covera large area from the anterior segment boundary to the posterior seg-ment boundary. (B) A ddaD neuron has fewer dendritic branches thanthe ddaC neuron. (C) Dendrites of ddaF neuron cover only the ante-rior half of the hemisegment. GAL4C155 also drives low-level expres-

sion of mCD8-GFP in epithelial cells. Therefore, mCD8-GFP-labeledsingle epithelial cells can be seen in some larvae (asterisk). (D)Dendrites of a ddaE neuron only cover the posterior half of thehemisegment. (E) A ddaB neuron extends dendrites to the anteriorsegment boundary and the dorsal midline. (F) A ddaA neuron extendsdendrites along the anteroposterior axis.White arrows indicate axons.

cell-autonomous fashion, we generated larvae in whichthe flamingo mutation was restricted to single neurons.We found that when dorsal cluster MD neurons weredevoid of flamingo gene activity, one or more dendrit-ic processes overextended toward the dorsal midline.Surprisingly, the basic architecture of the dendriticbranching patterns was not obviously altered. Forinstance, the ddaC neuron had a normal dendriticbranching pattern except that one dorsal dendriteoverextended across the dorsal midline. The numberand total length of the lateral branches of flamingomutant ddaF or ddaE neurons and the dendritic fieldscovered by these branches also appeared to be normal.For instance, the average total length of the lateralbranches of wildtype ddaE neurons is 1.2 ± 0.2 mm (n= 10), which is similar to that of flamingo mutant ddaEneurons (1.3 ± 0.2 mm, n = 4). However, in theseflamingo mutant cells, one or more dendrites with fewside branches overextended toward the dorsal midline.These data demonstrate that Flamingo does not controlthe general dendritic branching patterns of MD neu-rons. Instead, Flamingo has a cell-autonomous functionin limiting the extension of dorsal dendrites withoutgrossly affecting lateral branches.

Cell-Autonomous Function of Flamingoin Axonal Growth and Guidance

Flamingo is expressed on both dendrites and axons ofMD neurons. This finding suggests that Flamingo alsohas a role in axon development. To investigatewhether the flamingo mutations that affect dendriticgrowth also affect axonal growth, we examined theaxons of single MD neurons containing flamingomutations in wildtype larvae. In about 10% of morethan 100 mutant neurons examined, their axons didnot extend all the way to the CNS. This finding was

consistent with the axonal phenotype that weobserved in living flamingo mutant embryos, in whichaxonal break points could be found in the axon bun-dles of dorsal cluster MD neurons. Interestingly, 70%of the axons that stopped prematurely also branchedat their termini, whereas PNS neurons of wildtype lar-vae never branched before reaching the CNS. Thesestudies demonstrate that Flamingo has a cell-autonomous function in promoting axonal elongationand in preventing premature branching of axonsbefore they reach their synaptic targets.

Next, we asked whether Flamingo also affects axonguidance. We found flamingo mutant ES neuronswhose axons veered dramatically from the normalpath and ended at the wrong location, whereas thewildtype neuron extended its axon to the ventral nervecord. Similar pathfinding defects were also found foraxons of single MD neurons. These findings suggestthat Flamingo has a cell-autonomous function in con-trolling both axon guidance and axon elongation dur-ing development.

Using Drosophila as a Model Systemto Study Neurological Disorders

Reductions in the number and length of dendriticbranches and spines have been observed in a numberof neurological disorders, including Alzheimer’s dis-ease. We have begun to use Drosophila as a geneticmodel system to study the molecular mechanismsunderlying human neurological disorders. We are par-ticularly interested in the effects of human diseasegenes, such as APP and presenilin, on dendritic mor-phogenesis at single-neuron resolution. These studieswill provide a solid basis for genetic screens to identi-fy and characterize modifiers of human disease genes.

2001 ANNUAL REPORT


Selected References

Gao F-B (1998) mRNAs in dendrites: Localization,stability, and implications for neuronal function.Bioessays 20:70–78.

Gao F-B, Brenman JE, Jan LY, Jan YN (1999) Genesregulating dendritic outgrowth, branching and routingin Drosophila. Genes Dev. 13:2549–2561.

Gao F-B, Kohwi M, Brenman JE, Jan LY, Jan YN(2000) Control of dendritic field formation inDrosophila: The roles of Flamingo and competitionbetween homologous neurons. Neuron 28:91–101.

Brenman JE, Gao F-B, Jan LY, Jan YN (2001)Sequoia, a Tramtrack-related zinc finger protein func-tions as a pan-neural regulator for dendrite and axonmorphogenesis in Drosophila. Dev. Cell 1:667–677.


HUANG LABORATORY

Staff Research Investigator


Visiting Scientist


Senior Research Associate

Walter J. Brecht

Research Associates

Faith M. Harris

Xiao Qin Liu, M.D.


Karina Fantillo

Apolipoprotein E Proteolysis and Alzheimer’s Disease


Human apolipoprotein (apo) E has three majorisoforms, apoE2, apoE3, and apoE4. ApoE4has been identified as a major risk factor or

susceptibility gene for the development of Alzheimer’sdisease. ApoE is found in both neuritic amyloid plaquesand neurofibrillary tangles (NFTs)—the neuropatholog-ical hallmarks of Alzheimer’s disease—but its role inthe pathogenesis of these two lesions is largely un-known. A long-term research goal in this laboratory is tounderstand the molecular and cellular mechanisms bywhich apoE4 increases the risk for Alzheimer’s diseaseand other neurodegenerative disorders.

We have demonstrated that intracellular apoE under-goes proteolytic processing to generate carboxyl-ter-minal-truncated fragments, with apoE4 being moresusceptible than apoE3 to the cleavage. The carboxyl-terminal-truncated fragments of apoE enter thecytosol and interact with the cytoskeletal components,resulting in the formation of NFT-like inclusions inneurons. Truncated apoE4, which can be generated incultured neurons and in the brains of Alzheimer’s dis-ease patients, has a greater ability to induce NFT-likeinclusions than truncated apoE3.

In the past year, we expanded these studies to determine


Figure 1. ApoE fragmentation in brains of NSE-apoE or GFAP-apoEmice and humans. ApoE in brain lysates of NSE-apoE mice (A),GFAP-apoE mice (B), or humans (C) was detected by western blot-

ting with antibodies against full-length apoE or carboxyl-terminalapoE. ApoE fragmentation similar to that in human brains occurs inNSE-apoE mouse brains but not in GFAP-apoE mouse brains.

the relationship of apoE proteolysis and Alzheimer’sdisease in transgenic mice expressing apoE3 or apoE4specifically in neurons or astrocytes.

ApoE Fragmentation Occurs in NSE-ApoE Mice, but Not in GFAP-ApoE Mice

Brain tissues from transgenic mice expressing apoE3or apoE4 specifically in neurons (NSE-apoE) or inastrocytes (GFAP-apoE) were homogenized and ana-lyzed by western blotting with antibodies against full-length apoE or carboxyl-terminal apoE (amino acids273–299). Polyclonal anti-apoE revealed the full-length 34-kDa apoE band in both NSE-apoE3 andNSE-apoE4 mouse brains (Figure 1A). In addition, aprominent 29-kDa fragment of apoE was found pri-marily in NSE-apoE3 mouse brains. ApoE fragmentswith molecular masses of 14–20 kDa were muchmore abundant in NSE-apoE4 mouse brains than inNSE-apoE3 mouse brains. Anti-carboxyl-terminalapoE only reacted with full-length apoE but not withthe fragments of apoE, suggesting that these are thecarboxyl-terminal-truncated forms of apoE. However,in sharp contrast, no apoE fragmentation was found inGFAP-apoE3 or GFAP-apoE4 mouse brains, in whichapoE was expressed specifically in astrocytes at alevel sixfold higher than in NSE-apoE mice (Figure

1B). These results suggest a neuron-specific prote-olytic processing of apoE, with apoE4 being moresusceptible than apoE3 to the cleavage.

The Pattern of ApoE Fragmentation in NSE-ApoE Mice Is Similar to That in Humans

Importantly, the pattern of apoE fragmentation in NSE-apoE mice is similar to that in humans (compare Figure1A and C). A 29-kDa apoE fragment was found in bothNSE-apoE3 mice and nondemented (control) humansubjects with an apoE3/3 genotype. ApoE fragments of14–20 kDa were found in both NSE-apoE4 mice andAlzheimer’s disease patients with an apoE4/3 geno-type. These apoE fragments in human brains were notdetected by anti-carboxyl-terminal apoE, indicatingthat they are carboxyl-terminal-truncated forms. Theseresults suggest that the NSE-apoE mouse is a bettermodel than the GFAP-apoE mouse for studying therole of apoE fragmentation in neurodegeneration.

Age-Dependent Accumulation ofCarboxyl-Terminal-Truncated ApoEin Brains of NSE-ApoE4 Mice

Anti-apoE immunoblotting revealed an age-dependentaccumulation of carboxyl-terminal-truncated apoE in

2001 ANNUAL REPORT


Figure 2. Age-dependent accumulation of carboxyl-terminal-trun-cated apoE in brains of NSE-apoE4 mice. (A) ApoE in brain lysatesof NSE-apoE3 or NSE-apoE4 mice was detected by western blotting

with antibodies against full-length apoE. (B) Quantitative analysis ofthe ratios of carboxyl-terminal-truncated apoE to full-length apoE inbrain lysates of NSE-apoE3 or NSE-apoE4 mice.

brains of NSE-apoE4 mice 1–9 months of age (Figure2A). Many fewer apoE fragments were found in brainsof NSE-apoE3 transgenic mice with similar ages(Figure 2A). Quantitative analysis demonstrated thatthe ratios of the apoE fragments to full-length apoEwere much higher in NSE-apoE4 mice than in NSE-apoE3 mice at any age (Figure 2B). These results sug-gest that apoE4 is much more susceptible than apoE3to proteolytic cleavage in the brains of transgenic miceexpressing apoE specifically in neurons.

Age-Dependent Accumulationof Phosphorylated Tau in Brains of NSE-ApoE4 Mice

Since increased phosphorylation of tau is one of themajor biochemical changes in Alzheimer’s disease,we next determined whether there is an age-depen-dent accumulation of phosphorylated tau (p-tau) inbrains of NSE-apoE4 mice. Brain lysates of 3–9-month-old NSE-apoE4 or NSE-apoE3 mice wereanalyzed by western blotting with the monoclonalantibody AT8, which recognizes the phosphorylatedepitope of tau in NFTs. As demonstrated in Figure3A, the amount of p-tau with a molecular mass of55–60 kDa increased gradually with age in bothNSE-apoE4 and NSE-apoE3 mice, but the increasewas much greater in NSE-apoE4 mice. Note that asmall amount of p-tau aggregates resistant to sodiumdodecyl sulfate (SDS) was found in NSE-apoE4 mice5–9 months of age, but not in NSE-apoE3 mice ofsimilar age (Figure 3A). In 18-month-old mice, SDS-resistant p-tau aggregates, which remained at the topof the gel, accumulated to a much greater extent inNSE-apoE4 mice than in NSE-apoE3 or apoE knock-out mice (Figure 3B). Quantitative analysis demon-strated that there was an age-dependent accumulationof SDS-resistant p-tau aggregates in both NSE-apoE3 and NSE-apoE4 mice, but it was much greaterin NSE-apoE4 mice, especially in the aged mice(Figure 3C). Other antibodies against p-tau (AT100and AT270) also recognized the SDS-resistant p-tauaggregates, suggesting that they are hyperphosphory-lated at many sites.

p-Tau Is Not Increased in Brainsof GFAP-ApoE Mice

We next determined tau phosphorylation in brains ofGFAP-apoE mice. Anti-p-tau immunoblots demon-strated that the amount of p-tau was not increased inbrains of GFAP-apoE3 or GFAP-apoE4 mice up to 20months old (data not shown). Since there was no apoEfragmentation in the GFAP-apoE mice (Figure 1B),these results suggest that the apoE fragments, whichare generated in NSE-apoE4 mice and in Alzheimer’sdisease brains, may accelerate tau phosphorylationand NFT formation.



Figure 3. Age-dependent accumulation of p-tau in brains of NSE-apoE4 mice. In brain lysates of NSE-apoE3 or NSE-apoE4 mice, p-tau was detected by western blotting with monoclonal antibody AT8 (Aand B). More SDS-resistant p-tau aggregates, which remained at thetop of the gel, were detected in aged NSE-apoE4 mice than in agedNSE-apoE3 mice. (C) Quantitative analysis of the SDS-resistant p-tauaggregates in brain lysates of NSE-apoE3 or NSE-apoE4 mice.

Formation of Intraneuronalp-Tau-Containing Inclusions in theHippocampus of Aged NSE-ApoE4 Mice

Consistent with the above conclusion, we found intra-neuronal p-tau-containing inclusions in the hippocam-pus of 21-month-old NSE-apoE4 mice. Anti-AT8immunostaining of brain sections revealed p-tau-posi-tive intraneuronal inclusions in the CA3 region andhilus of the dentate gyrus of the hippocampus in 21-month-old NSE-apoE4 mice (Figure 4A). These inclu-sions were also stained with monoclonal antibodiesAT100 and AT270, suggesting that the tau in the inclu-sions was hyperphosphorylated at different sites.However, such p-tau-positive intraneuronal inclusionswere barely detectable in the same region of the hip-pocampus in NSE-apoE3 mice of similar age (Figure4B). Quantitative analysis demonstrated fourfold moreneurons containing p-tau-positive inclusions in agedNSE-apoE4 mice than in aged NSE-apoE3 mice(Figure 4C). Double immunostaining with anti-apoEand anti-p-tau revealed the colocalization of p-tau andapoE in the inclusions (data not shown).

In summary, our findings suggest that, after beingsynthesized in neurons, apoE undergoes proteolyticprocessing, probably in the secretory pathway, to gen-erate carboxyl-terminal-truncated fragments of apoE,with apoE4 being more susceptible than apoE3 to thecleavage. The fragments of apoE probably enter thecytosol, stimulate tau phosphorylation, and may con-tribute to the formation of NFTs. This process maynot occur to a significant extent under physiologicalconditions because little apoE is normally expressedin neurons. However, increasing evidence suggeststhat neurons, at least human neurons, do express lowlevels of apoE. We hypothesize that, in response toaging, oxidative stress, brain injuries, or amyloid-βdeposition, neurons may turn on or increase apoEexpression to repair or remodel the damaged neurons,thereby activating this proteolytic process, especiallyin apoE4 carriers. We are now testing this hypothesis.In addition, it will also be of interest to identify theputative protease that cleaves apoE at its carboxyl ter-minus, as it may serve as a therapeutic target for pre-vention and treatment of neurodegenerative diseasesassociated with apoE4.

2001 ANNUAL REPORT


Figure 4. Formation of intraneuronal inclusions containing p-tau inthe hippocampus of aged NSE-apoE4 mice. Brain sections from21-month-old NSE-apoE4 (A) or NSE-apoE3 (B) mice wereimmunostained with the monoclonal antibody AT8. p-tau-positiveintraneuronal inclusions were found in cell bodies (arrows) and

neuronal processes (arrowheads) in the CA3 region (A) of NSE-apoE4 mice but were barely detected in the same region of NSE-apoE3 mice (B). (C) Quantitative analysis of neurons containing p-tau-positive inclusions in the hippocampus of 21-month-old NSE-apoE4 and NSE-apoE3 mice.

Selected References

Huang Y, Mahley RW (1999) Apolipoprotein E andhuman disease. In: Plasma Lipids and Their Role inDisease (Barter PJ, Rye K-A, eds) HarwoodAcademic Publishers, Amsterdam, pp 257–284.

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

Huang Y, Liu XQ, Wyss-Coray T, Brecht WJ, SananDA, Mahley RW (2000) Bioactive fragments of

apolipoprotein E induce neurofibrillary tangles in cul-tured neurons. Soc. Neurosci. 26 (Part 1):540(abstract).

Huang Y, Liu XQ, Wyss-Coray T, Brecht WJ, SananDA, Mahley RW (2001) Apolipoprotein E fragmentspresent in Alzheimer’s disease brains induce neu-rofibrillary tangle-like intracellular inclusions in neu-rons. Proc. Natl. Acad. Sci. USA 98:8838–8843.



MAHLEY LABORATORY

Senior Investigator


Visiting Scientist


Postdoctoral Fellow

Shiming Ye, Ph.D.

Senior Research Associates

Maureen E. Balestra

Walter J. Brecht

Zhong-Sheng Ji, Ph.D.

Research Associate

Rene D. Miranda

Executive Assistant

Sylvia A. Richmond


Catharine H. Evans



Apolipoprotein E: Impact on Neurodegenerationand Alzheimer’s Disease Pathobiology


Along-term major focus of this laboratoryrelates to understanding the function ofapolipoprotein (apo) E in neurobiology. By

the mid-1980s, clues indicating that apoE played animportant role in neurological diseases had begun tosurface. ApoE was produced in abundance in the brainand served as the principal lipid transport vehicle incerebrospinal fluid. It was induced at high concentra-tions in peripheral nerve injury, appeared to play a keyrole in repair by redistributing lipids to regeneratingaxons and to Schwann cells during remyelinization,and modulated neurite outgrowth in cultured rabbitdorsal root ganglion cells or Neuro-2a cells. The stagewas set for the discovery by Roses and associates(Duke University) that apoE4 is a major susceptibili-ty gene associated with approximately 40–65% ofcases of sporadic and familial Alzheimer’s diseaseand increases the occurrence and lowers the age ofonset of the disease. Furthermore, the apoE4 allele isassociated with poor clinical outcome in patients withacute head trauma, whereas apoE2 may be protectiveagainst neurodegenerative diseases.

A key to understanding the role of apoE in neurologi-cal diseases resides in determining how apoE modu-lates neuronal repair, remodeling, or protection(Figure 1). Injurious agents can cause neuronal dam-age, requiring neuronal repair of synaptodendriticconnections. We would suggest that apoE3 and apoE2may be effective in mediating the repair process andin protecting neurons from excessive damage, where-as apoE4 may be relatively ineffective. The mecha-nism responsible for the isoform-specific effects is thefocus of the laboratory.

Isoform-Specific Effects on NeuriteExtension and Cytoskeletal Functionin Neurons

In in vitro studies, apoE3 induces neurons to producelong neurites, whereas apoE4 inhibits neurite out-growth. The isoform-specific effects of apoE on neu-rite outgrowth are associated with changes in thecytoskeleton. ApoE3 stimulates the polymerization ofβ-tubulin and stabilizes the formation of microtubulesin cultured neurons, whereas apoE4 apparently desta-bilizes microtubule assembly. While it is clear that theapoE isoforms have differential effects on thecytoskeleton, the mechanism is unclear. ApoE mightmodulate the cytoskeleton by interacting withcytoskeletal components, a process that would requireinternalized apoE to enter the cytosol.

In fact, we now have evidence suggesting that a bioac-tive fragment of apoE occurs in the cytosol and altersthe structure and function of the cytoskeleton. Ourworking hypothesis is that a fraction of apoE escapesan intracellular membrane compartment and enters thecytosol, where apoE3 and apoE4 may be processedproteolytically and then differentially modulate

Figure 1. Hypothesized role of apoE in the pathogenesis of neu-rodegeneration and Alzheimer’s disease.

cytoskeletal structure and function. As described indetail in the report by Dr. Yadong Huang, our mostrecent studies demonstrate that carboxyl-terminal-truncated forms of apoE lacking the final 25–30amino acids, which occur in cultured neurons and inAlzheimer’s disease brains, induce intracellular neu-rofibrillary tangle–like inclusions in neurons in vitro.These inclusions were composed of phosphorylatedtau (p-tau), phosphorylated neurofilaments of highmolecular weight (p-NF-H), and truncated apoE.Truncated apoE4, especially apoE4(∆272–299),induced neurofibrillary tangle–like inclusions intransfected neuronal cells, but not in transfected non-neuronal cells. ApoE4 was more susceptible to trun-cation than apoE3 and resulted in much greater intra-cellular inclusion formation. These results suggestthat apoE4 preferentially undergoes intracellular pro-cessing, creating a bioactive fragment that interactswith cytoskeletal components and induces neurofib-rillary tangle–like structures in neurons.

The mechanism responsible for the escape of apoE ora bioactive fragment of apoE into the cytosol is ofcritical importance. We hypothesize that a reactiveintermediate (a molten globular state of the protein) isformed within a cellular membrane compartment, asshown in Figure 2. One of the properties of reactiveprotein intermediates is their ability to translocateacross cell membranes. Studies are in progress to

investigate the reactivity of apoE and its fragments(also see the report by Dr. Karl Weisgraber).

Differential Intracellular Handlingor Processing of ApoE3 and ApoE4in Neuro-2a Cells

The differential effects of apoE3 and apoE4 on neu-rons may be associated with a differential intracellu-lar accumulation of apoE3 and apoE4 (apoE3 >apoE4), as we previously reported. These differencesin intracellular accumulation may reflect differencesin how apoE3 and apoE4 are handled by specificuptake pathways in neurons. In the past year, we havetested this hypothesis further.

Murine neuroblastoma (Neuro-2a) cells were incubat-ed with apoE3 or apoE4 complexed with β-very lowdensity lipoproteins (β-VLDL) (1.5:1 by protein) fordifferent times, and intracellular apoE was detectedby anti-apoE immunofluorescence staining. After a 1-hour incubation at 4°C, similar amounts of apoE3(Figure 3A) and apoE4 (Figure 3D) bound on the cellsurface without significant internalization. After a 20-minute incubation at 37°C, a portion of apoE3 wasinternalized into the cell bodies and neurites in apunctate distribution pattern, although significantamounts of apoE3 were still bound to the cell mem-branes (Figure 3B). However, at the same time inter-val (20 minutes), apoE4 was distributed largely in thecell bodies, with much less on cell membranes andnone in the neurites (Figure 3E). After a 60-minutechase incubation at 37°C, the differences in intracel-lular localization of apoE3 and apoE4 were even moredramatic (Figure 3C and F). Some apoE3 stillremained on the cell membranes and in the neuriteswith a punctate distribution pattern (Figure 3C),whereas apoE4 localized only in the cell bodies(Figure 3F). Double-immunofluorescence stainingwith antibodies against apoE and against subcellularorganelle markers revealed that the majority of theintracellular apoE3 or apoE4 in the cell bodies was inthe lysosomes.

To determine whether heparan sulfate proteoglycans(HSPG) mediate the differential intracellular handlingor processing of apoE3 and apoE4, we pretreated

2001 ANNUAL REPORT


Figure 2. Model suggesting how the reactive intermediates of apoE4may modulate cell processes by modifying the intracellular compart-ment and membrane stability. APP, amyloid protein precursor.

Neuro-2a cells with heparinase for 1 hour at 37°C toremove cell-surface HSPG and incubated them withapoE3/β-VLDL or apoE4/β-VLDL for an additional 1hour at 37°C. Anti-apoE immunofluorescence stainingrevealed that heparinase treatment of the cells totallyabolished the differences in the intracellular distribu-tion of apoE3 and apoE4. Both apoE3 and apoE4 weredistributed only in the cell bodies. Again, double-immunofluorescence staining demonstrated that themajority of the intracellular apoE3 or apoE4 was in thelysosomes. Presumably, the internalization directingthe apoE-containing lipoproteins to the lysosomes ismediated by the low density lipoprotein receptor.

Taken together, these results demonstrate that a frac-tion of apoE3 internalized by an HSPG-dependentpathway enters an intracellular compartment that istargeted to neurites, whereas most of the internalizedapoE4 enters an endosomal pathway that is targeted tolysosomes. These studies were conducted in collabo-ration with Drs. Yadong Huang and Toru Kawamura.

Effects of ApoE on Lysosomal Leakageand Apoptosis Induced by Amyloid-βPeptide in Neuronal Cells

The neurotoxicity of the amyloid β peptide (Aβ)appears to be an important factor in the pathogenesisof Alzheimer’s disease. Recently, we have begun toexamine the interaction between apoE and Aβ anddetermine if apoE3 and apoE4 have different effectson lysosomal stability, cell death, and apoptosis incultured neuronal cells. We have shown that apoE4, inconcert with Aβ1– 42, stimulates lysosomal leakage andpotentiates cell death and apoptosis in neurons.

ApoE4-transfected cells showed a greater tendencytoward lysosomal leakage than neo- and apoE3-trans-fected cells, as determined by the Lucifer Yellowintracellular staining pattern and release of the lyso-somal enzyme β-hexosaminidase into the cytosol.Furthermore, in response to aggregated Aβ1– 42, theNeuro-2a cells secreting apoE4 showed a significant



Figure 3. Differential intracellular handling or processing ofapoE3 and apoE4 by Neuro-2a cells. Neuro-2a cells were pulse-incubated with apoE3 or apoE4 (7.5 µg/ml) plus rabbit β-VLDL (5µg/ml) at 4°C for 1 hour (A and D). The cells were washed threetimes with serum-free minimal essential medium and chase-incu-

bated at 37°C for 20 (B and E) or 60 (C and F) minutes. The cellswere washed three times with phosphate-buffered saline, fixedwith 3% paraformaldehyde, permeabilized with 0.5% Triton X-100, and immunofluorescently stained with polyclonal anti-human apoE.

increase in cell death and more than twofold greaterDNA fragmentation than either the apoE3-secretingor control cells (apoE3-secreting cells and controlswere not significantly different) (Figure 4). On theother hand, when treated with H2O2 or staurosporin,apoE4-transfected cells showed enhanced cell deathand apoptosis, whereas apoE3-transfected cells wereprotected against the apoptotic effects of theseagents. The apoE4 potentiation of Aβ1–42-inducedapoptosis was abolished by a caspase-9 inhibitor(Figure 4). Conditioned medium from cells secretingapoE3 or apoE4 gave results similar to thoseobtained with apoE-transfected Neuro-2a cells:apoE4 potentiated and apoE3 had little effect onAβ1–42-induced apoptosis. Importantly, cells preincu-bated for 4 hours with a source of apoE3 or apoE4,followed by removal of apoE from the medium andfrom the cell surface, still demonstrated the isoform-specific response when Aβ1–42 was added subse-quently (i.e., the apoE4 potentiation of the Aβ-induced apoptosis required apoE to be intracellular,presumably in the endosomes or lysosomes).

The mechanism for the apoE4-associated lysosomalleakage and apoptosis was explored with phospholipid

(dimyristoylphosphatidylcholine) bilayer vesiclesencapsulating the fluorescent dye carboxyfluorescein.ApoE4 remodeled and disrupted the phospholipidvesicles to a greater extent than did apoE3, and whenAβ1–42 was added to the phospholipid bilayer vesi-cles possessing apoE3 or apoE4, the apoE4 vesicleswere disrupted to a greater extent than those contain-ing apoE3. We speculate that apoE4 forms a reactivemolecular intermediate that avidly binds phospho-lipid, may insert into the lysosomal membrane, anddestabilizes the membranes, causing lysosomal leak-age and inducing apoptosis in response to Aβ1–42(Figure 2). These studies are conducted with Dr.Zhong-Sheng Ji.

Insights into Isoform-Specific EffectsDerived from ApoE Structure/Function

The single amino acid change at residue 112 in apoE4that distinguishes it from either apoE3 or apoE2appears to alter the overall conformation of the mole-cule. Specifically, in apoE4, Arg-61 in the amino ter-minus is oriented in such a way that it can interact withGlu-255 in the carboxyl terminus. We have hypothe-sized that this unique domain interaction, which does

2001 ANNUAL REPORT


Figure 4. Effect of caspase inhibitors on Aβ1–42-induced DNA frag-mentation in Neuro-2a cells. (A) Neo-, apoE3-, and apoE4-transfect-ed cells were incubated with or without caspase inhibitors at 37°C for2 hours. Aβ1–42 was added to the medium and incubation continuedfor 18 hours. Apoptotic cells were measured by the DNA fragmenta-

tion assay. Values are the mean ± SD of two separate experimentsperformed in quadruplicate. (B) Additional study demonstrating thatthe apoE4-potentiated increase of Aβ1–42-induced apoptosis is abol-ished with a caspase-9 inhibitor. Values are the mean ± SD of twoexperiments (each with six separate wells for each condition).

not occur in apoE3 or apoE2, is responsible for the iso-form-specific effects of apoE in the nervous system. Aformal test of this hypothesis may be possible if asmall molecule can be identified that prevents theinteraction between the amino-terminal and carboxyl-

terminal domains. This would convert apoE4 to an“apoE3-like” molecule and might prevent the detri-mental effects of apoE4 on the structure and functionof the central nervous system. These studies are con-ducted in collaboration with Dr. Karl Weisgraber.



Selected References

Ji Z-S, Pitas RE, Mahley RW (1998) Differential cel-lular accumulation/retention of apolipoprotein Emediated by cell surface heparan sulfate proteogly-cans. Apolipoproteins E3 and E2 greater than E4. J.Biol. Chem. 273:13452–13460.

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

Buttini M, Orth M, Bellosta S, Akeefe H, Pitas RE,Wyss-Coray T, Mucke L, Mahley RW (1999)Expression of human apolipoprotein E3 or E4 in thebrains 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 E4 revealed in transgenicmodels of neurodegenerative disease. Neuroscience97:207–210.

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

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

Huang Y, Liu XQ, Wyss-Coray T, Brecht WJ, SananDA, Mahley RW (2001) Apolipoprotein E fragmentspresent in Alzheimer’s disease brains induce neu-rofibrillary tangle-like intracellular inclusions in neu-rons. Proc. Natl. Acad. Sci. USA 98:8838–8843.

MUCKE LABORATORY

Director and Senior Investigator

Lennart Mucke, M.D.

Research Scientist

Manuel J. Buttini, Ph.D.

Visiting Scientist

Yvonne Kew


Jeannie Chin, Ph.D.

Luke A. Esposito, Ph.D.

Christian Essrich, Ph.D.

Jorge J. Palop-Esteban, Ph.D.

Kimberly A. Scearce-Levie, Ph.D.

Zheng Wang, Ph.D.

Students

Duane M. Allen

Sharon E. Haynes

Jason Held

Marian L. Logrip


Gui-Qiu Yu, M.S.

Research Associates

Hilda C. Ordanza

Kristina P. Shockley

Lab Aide

John A. Gray

Executive Assistant

Denise Murray McPherson


Mariena D. Gardner



Neurobiology of Dementia

Lennart Mucke, M.D.

Neurological diseases that result in the degen-eration of neuronal circuits in the braincause severe impairments of cognitive func-

tions and behavioral disturbances. These dementingillnesses represent increasing medical and socioeco-nomic problems and raise a wide range of fundamen-tal neuroscientific questions. Research in this labora-tory focuses on the molecular and cellular pathogene-sis of Alzheimer’s disease (AD) and related condi-tions. Our major long-term goal is to advance theunderstanding of the central nervous system (CNS) toa level where these conditions can be effectively pre-vented and cured.

Transgenic Mouse ModelsSimulating Key Aspects of AD

The pathogenesis of AD appears to involve diversegenetic and epigenetic determinants and risk fac-tors. While this implies equally diverse opportuni-ties for prevention and therapeutic modulation ofthe disease, it also makes it extremely difficult tostudy the roles of specific factors in the human con-dition. The strength of AD-related transgenic mousemodels lies not so much in an ability to faithfullyreplicate AD in all its complexity (an unrealisticexpectation) but rather in the ability to isolate spe-cific factors from copathogens and to study them invivo with reasonable mechanistic resolving power.Molecules whose expression is increased in ADbrains have turned out to either promote or counter-act AD-like pathologies in transgenic models. Thishas helped to identify them as either pathogenic orneuroprotective and allowed the careful differentia-tion of secondary associations from cause-effectrelationships. In addition, AD-related transgenicmodels have contributed greatly to the development

and preclinical evaluation of novel treatments forAD and other neurological disorders.

PDGF-hAPP Mice

Amyloid β (Aβ) peptides accumulate to abnormallyhigh levels in the brains of people with AD, and thisprocess is presumed to play a key role in the pathogen-esis of AD-related neuronal deficits. Aβ peptides arederived by proteolytic cleavage from the amyloid pro-tein precursor (APP), and most of them end at residue40 (Aβ40) or 42 (Aβ42) (Figure 1). Alternativelyspliced minigene constructs directed by the platelet-derived growth factor (PDGF) β chain promoter wereused to express wildtype (WT) or familial AD (FAD)-mutant human APP (hAPP) in neurons of transgenicmice (Figure 1). The FAD mutations increased Aβ pro-duction without increasing the expression of hAPP. Byscreening a large number of lines, we identified linesmatched in hAPP levels but differing in Aβ levels andlines matched in Aβ levels but differing in hAPP levels.We also identified lines with equivalent levels of hAPPand Aβ42 that did or did not bear FAD mutations. Thisset of carefully matched lines has made it possible toassess the roles of hAPP and Aβ in the development ofAD-like pathologies in a manner that was previouslyimpossible.

Since progress in this field was hampered in the pastby an inability of the scientific community to readilyobtain high-expresser hAPP transgenic models, wehave distributed our PDGF-hAPP mice to variousacademic institutions, including Columbia University,Harvard University, Sun Health Research Institute,University of Kentucky, University of Paris, Univer-sity of California at San Diego, University of SouthernCalifornia, McGill University, Buck Institute for Age

Research, Rockefeller University, University ofZürich, and Keio University.

Amyloid Deposition

Our studies of PDGF-hAPP mice have revealed thatthe cerebral accumulation of amyloid plaques is criti-cally influenced by interactions between the highlyfibrillogenic Aβ42 and the less fibrillogenic Aβ40, aswell as by a range of glia-derived factors. We foundthat mice with high levels of Aβ42 and a highAβ42/Aβ40 ratio developed an abundance of amyloidplaques, whereas mice with similar levels of Aβ42 buthigher levels of Aβ40 (lower Aβ42/Aβ40 ratio) neverdeveloped plaques. These results suggest that Aβ40may be antifibrillogenic in vivo, consistent with

results obtained by others in vitro. Ablation of theclass A scavenger receptor had no effect on amyloiddeposition in these models. In contrast, ablation ofapolipoprotein (apo) E strongly inhibited amyloiddeposition, consistent with results reported by others.Compared with apoE3, apoE4 augmented plaquedeposition in hAPP/apoE doubly transgenic mice, butthis effect was observed only in very old mice andmay not explain the effect of these apoE isoforms onAD (see below). Astroglial expression of the humanserine protease inhibitor α1-antichymotrypsin (ACT)strongly enhanced Aβ deposition in hAPP/ACT dou-bly transgenic mice, a finding that was confirmed byanother group in a different hAPP transgenic model.As outlined in Dr. Tony Wyss-Coray’s report, thecytokine transforming growth factor β1 had more

2001 ANNUAL REPORT


Figure 1. Aβ1–42 levels in brains of PDGF-hAPP mice are deter-mined by transgene expression levels and the presence of FADmutations. (A) Diagram of hAPP indicating the mutations expressedin some of the transgenic lines. FAD-linked mutations are common-ly referred to by place of discovery or residence of affected kindred.The 670/671KM→NL double mutation affects a large pedigree inSweden, and the 717V→F mutation was identified in Indiana (num-bers refer to residues in the 770–amino acid form of APP). Thesequence of Aβ is indicated in bold in single-letter amino acid code.KPI, Kunitz-type protease inhibitor domain. Elements are not drawn

to scale. (B) Wildtype and FAD-mutant hAPP mice with matchinglevels of cerebral transgene expression were identified by RNaseprotection assay (n = 4 mice/line). Total RNA was extracted fromentire hemibrains. hAPP mRNA signals were quantitated by phos-phorimager and divided by actin mRNA signals to correct for anyvariations in RNA content or loading. Values represent mean ± SD.(C) Comparison of human Aβ levels in hippocampi of mice express-ing wildtype or FAD-mutant hAPP. Aβ1–42 was quantitated by ELISA(n = 6–9 mice/line) at 2–4 months of age (before plaque depositionoccurs in hAPPFAD lines). Values represent mean ± SD.

complex effects on Aβ deposition, promoting cere-brovascular amyloidosis while inhibiting the forma-tion of amyloid plaques in the brain parenchyma.These results demonstrate that plaque formation is aprocess that can be enhanced and inhibited by multi-ple factors. Determining whether the inhibition ofplaque formation also blocks synaptic and functionalneuronal deficits is an important objective.

Molecular Dissection of Plaque-Dependent and Plaque-IndependentComponents of AD Pathology

While many current efforts to develop better ADtreatments focus on the inhibition of plaques, itremains unclear whether these structures are actuallyresponsible for the degeneration and dysfunction ofbrain cells that underlie the cognitive decline in AD.Our analysis of PDGF-hAPP mice indicates thatplaques contribute to some aspects of AD-related neu-ronal damage but not others (Figure 2). We focused

our analysis on morphological alterations that corre-late well with cognitive decline in AD, namely, loss ofcholine acetyltransferase–immunoreactive (choliner-gic) neurons and of synaptophysin-immunoreactive(SYN-IR) presynaptic terminals.

Degeneration of cholinergic neurons in the nucleusbasalis results in major learning deficits in the watermaze test in rodents. It is also a potentially importantdeterminant of cognitive decline in AD. Comparedwith nontransgenic controls, PDGF-hAPP miceshowed a significant age-dependent loss of cholinergicneurons in the nucleus basalis. However, the loss ofthese neurons was found primarily in old mice andclearly followed the loss of SYN-IR presynaptic termi-nals (described below), consistent with the asynchro-nous decline in SYN-IR presynaptic terminals andcholinergic neurons others have observed in AD.

In mice from lines with different levels of hAPP andAβ, the density of SYN-IR presynaptic terminals



Figure 2. Plaque-dependent and plaque-independent aspects ofAD pathology. Conditions that promote the misfolding and assem-bly of Aβ increase the concentration of prefibrillar Aβ species.These Aβ species appear to be able to elicit plaque-independentneurotoxic effects, resulting in synaptic and behavioral deficits.Under appropriate conditions, the prefibrillar Aβ species matureinto fibrils that form larger aggregates and culminate in the forma-tion of plaques. While plaques may result in some local damage inthe form of neuritic dystrophy, it is possible that their formation rep-resents a protective attempt to inactivate highly reactive prefibrillar

Aβ species by focal sequestration. Plaque formation can be mod-ulated by diverse proteins and therapeutic interventions, asdemonstrated by us and other groups. Fewer studies haveaddressed plaque-independent Aβ toxicity. We have demonstratedthat, in hAPP/α-synuclein doubly transgenic mice, α-synucleinenhances neurodegeneration and behavioral deficits independent-ly of plaque formation. ApoE3, but not apoE4, prevented ordelayed neuronal deficits in hAPP/apoE doubly transgenic mice(see text). NSAIDs, nonsteroidal antiinflammatory drugs; TGF,transforming growth factor.

correlated inversely with Aβ levels but not with hAPPlevels or plaque load. Despite their lack of amyloidplaques, hAPPWT mice with high Aβ levels also haddecreases in SYN-IR presynaptic terminals. Althoughthe decreases in SYN-IR presynaptic terminals weidentified in PDGF-hAPP mice were more subtle thanthose observed in late stages of AD, they were accom-panied by major (up to 80%) decreases in synaptictransmission strength, underlining their pathophysio-logical relevance. Like cognitive impairments in AD,synaptic transmission deficits in PDGF-hAPP miceincreased with age. Notably, these functional neuronalimpairments were seen well before the formation ofamyloid plaques. Decreasing hAPP expression levelswhile increasing Aβ production by mutagenesis ofhAPP further augmented the synaptic transmissiondeficits, consistent with an insidious role of Aβ in thedisease process.

Neural circuits in the hippocampus play a key role inthe formation of memories and are severely disruptedin AD. Our results suggest an important role for non-deposited forms of Aβ in the pathogenesis of synapticdeficits in this brain region. That Aβ can be synapto-toxic even in the absence of plaques provides a cir-cuit-level explanation for the discrepancies betweenplaque load and functional deficits others have report-ed in AD. These results also have important therapeu-tic implications. Treatments that block Aβ productionand lower nondeposited Aβ toxins may diminish bothplaque-dependent and plaque-independent neuronaldeficits, whereas treatments that prevent primarily thesequestration of Aβ into plaques will probably be lesseffective and might even augment plaque-indepen-dent Aβ toxicity.

Modulation of Aβ Toxicityby ApoE Isoforms

The most frequent human apoE isoforms, E3 and E4,differentially affect AD risk (E4 > E3) and age of onset(E4 < E3). Compared with apoE3, apoE4 promotes thecerebral deposition of Aβ (see above). However, it isuncertain whether Aβ deposition into plaques is themain mechanism by which apoE isoforms affect AD.To address this issue, we studied murine apoE–defi-cient transgenic mice expressing hAPP/Aβ together

with apoE3 or apoE4 in neurons. Three age rangeswere analyzed: 6–7, 12–15, and 19–24 months. Brainaging in the context of high levels of nondepositedhuman Aβ resulted in progressive synaptic/cholinergicdeficits. ApoE3 delayed these deficits until old age,whereas apoE4 was not protective at any of the agesanalyzed. Old hAPP/apoE4 mice had more plaquesthan old hAPP/apoE3 mice, but synaptic/cholinergicdeficits preceded plaque formation in hAPP/apoE4mice. Furthermore, old hAPP/apoE3 and hAPP/apoE4mice had comparable synaptic/cholinergic deficitsdespite their different plaque loads, and cholinergicdeficits were found not only in the hippocampus butalso in the neocortex, which was mostly devoid ofplaques. Thus, apoE3, but not apoE4, delays age- andAβ-dependent synaptic/cholinergic deficits through aplaque-independent neuroprotective function. Thisdifference could contribute to the differential effects ofthese isoforms on AD risk and onset.

Impact of hAPP/Aβ and ApoEon Behavior

The clinical hallmarks of AD are progressive cogni-tive decline and behavioral alterations. It is thereforecritical to evaluate the behavioral impact of morpho-logical and molecular alterations or novel treatmentstrategies identified in AD-related transgenic modelsin order to assess their potential clinical relevance.While it is, of course, impossible to assess all aspectsof human dementia in rodent models, there are multi-ple aspects of AD-related functional neurologicalimpairments that can be examined in mice. For exam-ple, the “Safe Return” program of the Alzheimer’sAssociation was established to assist people with ADwho wander off and become lost. Its existence high-lights the severe deficit in spatial memory in AD andthe distress it causes patients and their families.Visuospatial deficits are an important determinant offunctional disability in this illness and can be testedreadily in transgenic mouse models.

To examine the roles of hAPP/Aβ, apoE, and α-synu-clein in the development of AD-related behavioraldeficits, we studied the behavior of PDGF-hAPP mice,of transgenic mice in which expression of humanapoE3 or apoE4 is directed by the neuron-specific eno-

2001 ANNUAL REPORT


lase (NSE) or the glial fibrillary acidic protein pro-moter, and of doubly transgenic mice coexpressingPDGF-hAPP/NSE-apoE or PDGF-hAPP/PDGF-α-synuclein constructs (see publications and BehavioralCore Laboratory for details). Like other hAPP trans-genic models, PDGF-hAPP mice develop age-depen-dent deficits in the water maze test, which providesputative measures of spatial learning and memory. Incollaborative experiments with Dr. Eliezer Masliah(University of California at San Diego), we discoveredthat intraneuronal accumulation of wildtype human α-synuclein worsens hAPP/Aβ-dependent water mazedeficits in doubly transgenic mice. This effect mayrelate to the severity of cognitive deficits in the Lewybody variant of AD, which combines pathologicalhallmarks of AD with the intraneuronal accumulationof α-synuclein in Lewy bodies. Pathogenic interac-tions between different misfolded proteins, such as Aβand α-synuclein, may expand not only the complexityof the associated neurodegenerative disorders but alsothe therapeutic spectrum of drugs that can inhibit theirproduction or misfolding.

Interestingly, apoE3 prevented or delayed hAPP/Aβ-dependent water maze deficits, whereas apoE4 did not.These results are most consistent with a loss of neuro-protective function in apoE4 and may relate closely tothe effects of these isoforms on AD risk and onset.However, there also is behavioral evidence for anadverse activity of apoE4 that may affect femalesmore than males. As described in the report of theBehavioral Core Laboratory, we have begun to unrav-el the molecular mechanisms underlying the patho-genic interaction between apoE4 and female genderand identified a therapeutic strategy to block it.

GeneChipping Away at AD

While there is good evidence that Aβ plays a criticalrole in AD, the exact molecular processes by which itcontributes to neuronal dysfunction and degenerationin vivo remain to be defined. In a similar vein, immu-nization of hAPP transgenic mice with Aβ42 wasrecently shown by others to prevent and even reverseAD-like pathologies, but the mechanisms underlyingthis promising therapeutic effect remain hotly debated.It is likely that at least some of the cellular and mole-

cular responses underlying the detrimental effects ofAβ or the beneficial effects of Aβ vaccination arereflected in changes of gene expression (Figure 3).

We therefore embarked on a large-scale gene expres-sion analysis in collaboration with the GladstoneGenomics Core. We analyzed five groups of 6–8-month-old mice (n = 4–6/group): untreated nontrans-genic mice and transgenic mice expressing hAPPWT orhAPPFAD at comparable levels and hAPPFAD mice thatwere vaccinated with Aβ or sham vaccinated. In addi-tional experiments, we compared hAPPWT andhAPPFAD mice at different ages or doubly transgenicmice expressing hAPP/Aβ in the context of apoE3 orapoE4. Total RNA was isolated from individual hip-pocampi and analyzed with high-density oligonu-cleotide arrays (Affymetrix GeneChips) representingroughly 13,000 mouse sequences. Our experimentaldesign carefully controlled for variables such as gen-der, background strain, environmental influences, andnonspecific interindividual variations.

Transgenic mice of any genotype differed from non-transgenic littermate controls with respect to manymRNAs, as did untreated mice from mice that hadreceived Aβ or sham vaccinations. Comparisonsbetween groups of mice that differed from eachother in select variables (e.g., single amino acid sub-stitutions) revealed a more limited number of geneexpression changes and turned out to be substantial-ly more informative. Transgenic lines that expressedhAPPWT or hAPPFAD at similar levels typically differ



Figure 3. hAPP/Aβ and apoE4 are associated with age-dependentalterations in cognition and neuronal integrity in AD and relatedtransgenic models. We have begun to test the hypothesis that thedevelopment of these alterations involves changes in gene expres-sion that can be detected by DNA microarray analysis.

markedly in their production of Aβ42 (Figure 4A).Some gene expression changes were seen inhAPPWT and hAPPFAD mice but not in nontransgeniccontrols, suggesting that they were caused by theoverexpression of hAPP per se but not specificallyby the FAD mutation or its effects on Aβ production(Figure 4B). In contrast, other gene products wereexpressed in hAPPFAD mice at levels significantlyhigher or lower than those in hAPPWT or nontrans-genic mice (Figure 4C), consistent with a more spe-cific relationship to the FAD mutation and its patho-physiological consequences.

Of equal or even greater interest were gene expressionchanges that showed such a FAD mutation/Aβ-depen-

dent pattern and that were reversed to wildtype levelsby Aβ vaccination but not by sham vaccination(Figure 4D). These gene expression changes may helpto identify mediators of therapeutic vaccinationeffects and provide useful end points for ongoing vac-cination trials. We are in the process of confirmingspecific gene expression changes by quantitative flu-orogenic reverse transcriptase–polymerase chainreaction, in situ hybridization, western blotting, andimmunohistochemistry. So far, we have been able tovalidate several, but not all, changes identified byDNA microarray analysis. Experiments are under wayto assess the most interesting and robust moleculartargets with respect to their roles as mediators or indi-cators of AD-related pathogenicity.

2001 ANNUAL REPORT


Figure 4. Select patterns of hippocampal gene expression elicit-ed by overexpression of hAPP, effects of FAD mutations, nonspe-cific immunization, or vaccination with Aβ. DNA microarrays wereused to detect gene expression profiles in the hippocampus of dif-ferent groups of mice as described in the text. The results are pre-liminary and the interpretation is speculative. (A) hAPPWT andhAPPFAD mice were matched for transgene expression but dif-fered strongly with respect to Aβ1–42 levels (data from Figure 1).(B) Example of an mRNA (encoding a nuclear transcription factor)whose expression is affected by hAPP but not by the FAD muta-tion. Other transcripts, such as those encoding a serpin (C) or

representing an unidentified expressed sequence tag (D),showed an expression pattern suggesting a dependence on Aβ orclosely related APP metabolites. Their expression was affectedmore by hAPPFAD than by hAPPWT (compared with nontransgenic[NTG] controls), which may reflect the greater production ofAβ1–42 from hAPPFAD than APPWT. Consistent with this interpre-tation, vaccination with Aβ1–42 (Vacc), which may induce theremoval of neurotoxic Aβ species, eliminated the differencebetween hAPPFAD and APPWT. Sham vaccination (Sham) had nosignificant effect on these transcripts but affected the expressionof many other genes (not shown).



Selected References

Hsia AY, Masliah E, McConlogue L, Yu G-Q, TatsunoG, Hu K, Kholodenko D, Malenka RC, Nicoll RA,Mucke L (1999) Plaque-independent disruption ofneural circuits in Alzheimer’s disease mouse models.Proc. Natl. Acad. Sci. USA 96:3228–3233.

Masliah E, Rockenstein E, Veinbergs I, Mallory M,Hashimoto M, Takeda A, Sagara Y, Sisk A, Mucke L(2000) Dopaminergic loss and inclusion body forma-tion in α-synuclein mice: Implications for neurode-generative disorders. Science 287:1265–1269.

Mucke L, Masliah E, Yu G-Q, Mallory M,Rockenstein EM, Tatsuno G, Hu K, Kholodenko D,Johnson-Wood K, McConlogue L (2000) High-levelneuronal expression of Aβ1-42 in wild-type humanamyloid protein precursor transgenic mice: Synap-totoxicity without plaque formation. J. Neurosci.20:4050–4058.

Mucke L, Yu G-Q, McConlogue L, Rockenstein EM,Abraham CR, Masliah E (2000) Astroglial expression

of human α1-antichymotrypsin enhances Alzheimer-like pathology in amyloid protein precursor trans-genic mice. Am. J. Pathol. 157:2003–2010.

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

Masliah E, Rockenstein E, Veinbergs I, Sagara Y,Mallory M, Hashimoto M, Mucke L (2001) β-Amyloid peptides enhance α-synuclein accumulationand 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-β1promotes microglial amyloid-β clearance and reducesplaque burden in transgenic mice. Nat. Med.7:612–618.

PITAS LABORATORY

Senior Investigator



Paul C. R. Hopkins, Ph.D.

Juan Santiago-Garcia, Ph.D.

Research Associate

Richard M. Stewart

Administrative Assistants

Emily K. O’Keeffe



Mechanisms for the Differential Effects of the Apolipoprotein EIsoforms on Neurite Outgrowth and Cognitive Impairment


Amajor focus of the laboratory continues to bethe mechanisms by which apolipoprotein(apo) E contributes to the development of

neurological disease. We showed previously thatapoE2 and apoE3 stimulated the outgrowth of neuritesfrom cultured neurons, whereas apoE4 inhibited neu-rite outgrowth and disrupted cellular microtubules.ApoE4 also exerted detrimental effects in the centralnervous system (CNS) of transgenic mice. HumanapoE3 or apoE4 expression in neurons of Apoe–/– miceresulted in age-related impairments in performing awater maze task and in vertical exploratory behavior inapoE4-expressing mice, but not in apoE3-expressingmice. The effects of apoE4 were dominant.

Work in our laboratory is currently centered on exam-ining mechanisms that may contribute to the differen-tial effects of the apoE isoforms on neurite outgrowthand cognitive impairment. We are particularly interest-ed in identifying proteins that interact with apoE in theCNS and in determining if there are differences in lipidmetabolism in the brain that are apoE isoform specific.

ApoE-Binding Proteins in the Brain

We have continued to investigate a CNS protein thatwe identified by its binding to apoE in a yeast two-hybrid screen of a human brain cDNA library. BLASTsearches showed that the protein is a member of a pre-viously uncharacterized family of proteins withunknown function. The proteins are encoded by fourhomologous genes in humans with equivalent genes inmice. There is one related gene in Drosophila, whichis predicted to produce a protein with 45% amino acididentity with the human protein. The mRNAs thatencode the human and mouse forms of this protein arepresent almost exclusively in the brain. The protein we

identified contains two predicted transmembranedomains near the carboxyl terminus. Expression ofFlag-tagged protein in Neuro-2a cells and detection byimmunocytochemistry suggest that it is expressed inthe endoplasmic reticulum. We are performing in vitrostudies with recombinant protein and transfected cellsto characterize its interaction with apoE.

Last year, we reported the results of in situ hybridiza-tion studies of mouse brain sections that revealed pan-neuronal expression of this apoE-binding protein. Wehave now confirmed and extended these findings.Using primary cultures derived from fetal mousebrain, we have shown by reverse transcriptase–poly-merase chain reaction (RT-PCR) that the mRNA forthis protein is expressed in neurons but not in astro-cytes (Figure 1). Northern blot analysis demonstrated

Figure 1. The mRNA for the apoE-binding protein is expressed inmouse primary neurons but not in astrocytes. Primary cortical neu-rons or astrocytes were obtained from embryonic (E15.5) mousebrains. After 1 week in culture, total RNA was isolated and cDNAwas synthesized. PCR was performed with two different primerpairs corresponding to the sequence of the murine cDNA.

predominant expression in the brain and only low-levelexpression in the heart, skeletal muscle, and testis.

We have begun to analyze the expression patterns ofthe three other family members in mouse tissues. Oneis expressed predominantly in the liver, and another ismainly expressed in testis. These homologs haveessentially no expression in the brain. The thirdmouse homolog is expressed in most mouse tissues,with the highest levels of mRNA in testis, ovary,spleen, and kidney.

Since the expression patterns of the apoE-bindingprotein in mice and humans are virtually identical, themouse is an ideal species in which to study its metab-olism and physiologic functions. We have, therefore,cloned and sequenced the cDNA that encodes themouse protein. This was accomplished by sequencingtwo mouse expressed sequence tag clones, sequenc-ing mouse genomic DNA fragments, and sequencingRT-PCR products. The full-length murine cDNAencodes a 628–amino acid protein, three amino acidsshorter than its human counterpart. The predictedamino acid sequences of the mouse and human pro-teins are 96% identical.

We have developed polyclonal antibodies inresponse to a mixture of three peptides correspond-ing to sequences near the amino terminus of the pro-tein. These rabbit antibodies detect both the humanand mouse proteins when they are expressed bytransfected cells. Immunoblot analysis showed thatthe mouse and human proteins occur in cell extractsas both the full-length protein of ~75 kDa and astwo smaller molecules of ~60–70 kDa (Figure 2).We are currently determining whether these lower-molecular-mass proteins represent biologically sig-nificant proteolytic processing of the protein. Withthese antibodies, we are studying the expression ofthe protein in murine primary neurons and brainsections.

Since the protein was previously uncharacterized, ishighly conserved throughout evolution, and isexpressed exclusively by neurons, we will determineits function in mice by gene knockout technologyand in Drosophila by double-stranded RNA-mediat-ed gene silencing, also called RNA interference. Wehave sequenced large portions of the gene thatencodes the mouse protein and determined the genestructure. The gene consists of five exons spanningmore than 50 kb. Exon 1 contains a 5′-untranslatedregion, and exons 2–5 contain the coding region. Wehave sequenced the entire coding region and 14 kbof intronic DNA sequence. Based on thesesequences, we have designed and are constructing aconditional knockout vector. The gene knockoutshould be relatively straightforward for our veryexperienced blastocyst core facility. The RNA inter-ference experiments are being performed in collabo-ration with Dr. Fen-Biao Gao, who has considerableexperience in the identification of genes responsiblefor neurite outgrowth in Drosophila and in geneticmanipulation in this model.

Effects of ApoE2, ApoE3, and ApoE4on Lipid Metabolism

As noted last year, we found that apoE2, E3, and E4differ in their effects on the cellular uptake of freefatty acids (FFA) and in their incorporation into cel-lular lipids. ApoE2 enhanced uptake, apoE4 inhibit-ed uptake, and apoE3 had an intermediate effect.

2001 ANNUAL REPORT


Figure 2. Immunodetection of themouse and human proteins in trans-fected CHO cells. Cell extracts wereprepared from control CHO cells orCHO cells transfected to expressthe human or mouse proteins.Protein extracts (40 µg) were sepa-rated by sodium dodecyl sulfate–polyacrylamide gel electrophoresisand transferred to nitrocellulose.Theprotein was detected with a poly-clonal antibody.

These effects were mediated by extracellular apoE.Using two independent techniques, we have nowdemonstrated that apoE interacts with FFA. ApoE2,E3, and E4 interacted to form complexes with FFAthat could be isolated by centrifugation. ApoE alsointeracted with FFA emulsions, resulting in reducedlight scattering. The kinetics of the interaction indi-cate that apoE2 interacts more rapidly with FFA thanapoE3 or apoE4. We have also examined the interac-tion of apoE with FFA under more physiologicalconditions. In blood and cerebrospinal fluid, FFAare transported bound to albumin. These albumin•FFA complexes are in equilibrium with low levels ofunbound FFA. It is the unbound fatty acid that istaken up by cells by a combination of passive andprotein-facilitated processes. We determined theconcentration of unbound fatty acid present whenthe albumin•FFA complexes were incubated withand without added apoE. ApoE reduced the amountof unbound fatty acid, thereby reducing the concen-tration of FFA available for uptake by cells. In vivoand in vitro studies are continuing in an effort to elu-cidate the physiological roles of these processes infatty acid metabolism in the CNS.

Essential fatty acids cross the blood-brain barrierand enter the brain tissue at capillaries, where astro-cyte foot processes terminate and secrete apoE. Ourdata suggest that the apoE isoforms might exert dif-ferential effects on the uptake of FFA from the bloodand on the redistribution of FFA to cells within thebrain. It is intriguing to note that apoE2, which mayprotect against the development of Alzheimer’s dis-ease, enhances fatty acid uptake by cells, whereasapoE4, which is a risk factor for Alzheimer’s dis-ease, inhibits fatty acid uptake by cells. The differ-ential uptake or impaired distribution of essentialfatty acids could have a significant effect on brainmetabolism and on membrane biosynthesis, which isrequired for synaptic plasticity and for neurite out-growth and remodeling, especially during periods ofstress. ApoE2 may support the delivery of essentialfatty acids required by cells for membrane produc-tion, thereby contributing to neuronal plasticity. Incontrast, apoE4 may not effectively support thisprocess, thereby contributing to the development ofneurological disease.

Class A Scavenger ReceptorBinds sAPP

Our laboratory has a long-standing interest in theclass A scavenger receptor (SR-A), which isexpressed by activated microglia. Recent studieshave shown that this receptor interacts with the amy-loid-β peptide (Aβ). We have determined that SR-Abinds, internalizes, and degrades sAPP, which is anα-secretase cleavage product of the amyloid precur-sor protein (APP). A mixture of sAPP751 andsAPP770 was isolated from the secreted products ofthrombin-activated human platelets. Platelet α-gran-ules contain sAPP, which is also called Nexin II. Theidentity of the protein isolated from platelets withsAPP was confirmed by amino-terminal sequenceanalysis, by amino acid composition, and by interac-tion with antibodies to sAPP. Chinese hamster ovary(CHO) cells transfected to stably express the SR-Ametabolized fourfold more sAPP751/770, and boththe binding and degradation were inhibited by theSR-A antagonist fucoidin. In addition, sAPP751/770competed as effectively as fucoidin for the SR-A-mediated binding of 125I-acetyl low density lipopro-teins (LDL), a well-characterized ligand for the SR-A.Taken together, these data demonstrate that sAPP isan SR-A ligand.

Both sAPP751 and sAPP770 contain a Kunitz-typeprotease inhibitor (KPI) domain. It was shown previ-ously that sAPP binds to the LDL receptor–relatedprotein, a member of the LDL receptor gene family,and that binding to that receptor requires the KPIdomain. We have established that the KPI domain isnot required for binding of sAPP to the SR-A. Weexpressed both sAPP695, which does not contain aKPI domain, and sAPP751 in transfected CHO cellsand purified them from the medium. Both wereequally effective in competing for the SR-A-mediat-ed cell association of 125I-acetyl LDL. Studies withdeletion mutants of sAPP indicated that a negativelycharged region of the protein (residues 191–264)contributes to binding to the SR-A. The SR-A inter-nalizes Aβ aggregates and contributes to the adhesionof microglia to Aβ fibrils. Our current findings sug-gest that the SR-A may play a prominent role in APPmetabolism and contribute to the turnover of sAPP.



2001 ANNUAL REPORT


Selected References

Pitas RE, Ji Z-S, Weisgraber KH, Mahley RW (1998)Role of apolipoprotein E in modulating neurite out-growth: Potential effect of intracellular apolipoproteinE. Biochem. Soc. Trans. 26:257–262.

Raber J, Wong D, Buttini M, Orth M, Bellosta S, PitasRE, Mahley RW, Mucke L (1998) Isoform-specificeffects of human apolipoprotein E on brain functionrevealed in ApoE knockout mice: Increased suscepti-bility of females. Proc. Natl. Acad. Sci. USA95:10914–10919.

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

Huang F, Buttini M, Wyss-Coray T, McConlogue L,Kodama T, Pitas RE, Mucke L (1999) Elimination of

the class A scavenger receptor does not affect amyloidplaque formation or neurodegeneration in transgenicmice expressing human amyloid protein precursors.Am. J. Pathol. 155:1741–1747.

Buttini M, Akeefe H, Lin C, Mahley RW, Pitas RE,Wyss-Coray T, Mucke L (2000) Dominant negativeeffects of apolipoprotein E4 revealed in transgenicmodels of neurodegenerative disease. Neuroscience97:207–210.


Santiago-Garcia J, Mas-Oliva J, Innerarity TL, PitasRE (2001) Secreted forms of the amyloid-β precursorprotein are ligands for the class A scavenger receptor.J. Biol. Chem. 276:30655–30661.

WEISGRABER LABORATORY

Senior Investigator


Research Scientist

Robert L. Raffaï, Ph.D.


Yvonne M. Newhouse

Executive Assistant

Brian Auerbach



Structure and Function of Apolipoprotein E


Our research focuses on the structural and func-tional relationships of apolipoprotein (apo) Ein lipoprotein metabolism, heart disease, and

neurodegenerative diseases, including Alzheimer’s dis-ease. ApoE is a 299–amino acid, single-chain proteinwith two structural domains that also define functionaldomains (Figure 1). The three common human iso-forms, apoE2, apoE3, and apoE4, differ at two posi-tions in the molecule and have very different metabol-ic properties and impacts on disease. ApoE3 (Cys-112,Arg-158) binds normally to low density lipoprotein(LDL) receptors and is associated with normal lipidmetabolism, whereas apoE2 (Cys-112, Cys-158) bindsdefectively to LDL receptors and, under certain cir-cumstances, is associated with the genetic disorder typeIII hyperlipoproteinemia. ApoE4 (Arg-112, Arg-158)binds normally to LDL receptors but is associated withelevated cholesterol levels and increased risk for car-diovascular disease. In addition, apoE4 is a major riskfactor for Alzheimer’s disease and for poor outcomefrom head injury and stroke. Our objective is to deter-mine how the structural and biophysical properties ofapoE influence its metabolic properties and contributeto its isoform-specific effects in disease and injury.

X-Ray Crystallography

The structures of the amino-terminal domains ofapoE2, apoE3, and apoE4 in lipid-free forms havebeen determined, and all three structures adopt a four-helix-bundle motif (Figure 1). However, subtle differ-ences in side-chain conformations and in salt-bridgearrangements among the isoforms affect their func-tions and characteristics. In addition, since apoE like-ly performs most, if not all, of its functions in a lipid-associated state, a major focus is to determine theinfluence of lipid binding on structure and function.

In collaboration with Dr. Bernhard Rupp (Macromo-lecular Crystallization Facility, Lawrence LivermoreNational Laboratory), we crystallized an amino-terminalfragment (residues 1–165) of apoE in three differentcrystal forms and solved their structures. Comparison

Figure 1. The two-domain structure of apoE. As determined by x-ray crystallography, the amino-terminal domain assumes a four-helix-bundle folding motif. The structure of the carboxyl-terminaldomain is not known and is depicted as a series of α-helices, con-sistent with circular dichroism measurements. The receptor-bindingregion of apoE is located in the amino-terminal domain on helix 4.The carboxyl-terminal domain contains the elements for binding tospherical lipoprotein particles (boxed area). Amino acid differencesat position 112 distinguish apoE3 (Cys) and apoE4 (Arg). ApoE3displays a lipoprotein preference for HDL, whereas apoE4 displaysa preference for VLDL.The concept of domain interaction was intro-duced to account for the influence of the polymorphic site at posi-tion 112 in the amino-terminal domain on the lipid-binding proper-ties of the carboxyl-terminal domain.

of the structures revealed a large degree of conforma-tional flexibility at the end of the molecule containingthe loop (residues 80–91) connecting helices 2 and 3and bends within helices 2 and 3. These bends, alongwith the flexible loop, suggest that this end of themolecule is flexible and likely represents the initiationsite for lipid binding and the opening of the four-helixbundle as it reorganizes to bind lipid.

To test this model, we introduced single, double, andtriple interhelical disulfide bonds to restrict the open-ing of the bundle in the amino-terminal domain frag-ment. Interaction of these mutants with dimyris-toylphosphatidylcholine (DMPC) was assessed byvesicle disruption, turbidimetric clearing, and gel fil-tration assays. The results indicate that apoE•DMPCdiscoidal particles form in a series of steps. A tripledisulfide mutant, in which all four helices were teth-ered, did not form complexes but could releaseencapsulated 5-(6)-carboxyfluorescein from DMPCvesicles, indicating that the initial interaction doesnot involve major reorganization of the helical bun-dle. After the initial interaction, the four-helix bundleopens to expose the hydrophobic faces of the amphi-pathic helices. In this step, helices 1 and 2 and helices3 and 4 preferentially remain paired, since thesedisulfide-linked mutants bound to DMPC in a man-ner similar to that of the nonlinked control. In con-trast, when helices 2 and 3 and/or helices 1 and 4were paired, they bound poorly. However, all singleand double helical pairings resulted in the formationof larger discs than were formed by the control, indi-cating that the helices undergo further reorganizationafter the initial opening of the four-helix bundle asthe protein assumes its final lipid-bound conforma-tion. In support of this rearrangement, reducing thedisulfide bonds converted the large disulfide mutantdiscs to normal size.

The x-ray crystal studies indicated that the end of thebundle containing the flexible loop (residues 80–91)might initiate lipid binding. This loop contains threenegatively charged residues, suggesting that an elec-trostatic interaction with lipid might be involved. Thispossibility was supported by three findings: (1) bind-ing to DMPC was reduced by increasing ionicstrength; (2) binding was reduced with a phospholipid

with a negatively charged headgroup, dimyris-toylphosphatidylglycerol (DMPG); and (3) neutraliz-ing the negative charges in the flexible loop produceda mutant that bound to DMPG. These studies providemajor new insights into how apoE binds lipid and thestructural changes that occur with lipid interaction.

A major breakthrough in studying the interaction ofapoE with lipid is the successful crystallization ofapoE complexed with DMPC. This very excitingresult opens for the first time the possibility ofobtaining detailed structural information on pro-tein–lipid complexes. This is important for apoEbecause high-affinity binding to LDL receptorsrequires lipid association.

ApoE4•DMPC crystals displayed a fiber-like diffrac-tion pattern with a unit cell spacing of 54 Å along thefiber axis and cell spacings of ~150 Å and ~300 Å,respectively, for the two axes approximately perpen-dicular to the fiber axis. These findings are consistentwith the idea that the apoE•DMPC discs (~150 Å indiameter and 55 Å thick) stack to form long, fiber-likerod structures. The stacking of the discs appears to bewell defined along the fiber axis, as indicated by the

2001 ANNUAL REPORT


Figure 2. Effect of apoE4 structure on function. To assess theimpact of apoE4 structure on function, our long-term objective is tointroduce each of the known isoform differences individually intothe mouse apoE gene by gene targeting. This approach will providemouse models that we can use to assess the relative contributionsof these differences to known isoform-specific effects in plasma(lipoprotein metabolism and atherosclerosis) and brain (amyloid-βmetabolism and fibrillogenesis, cognitive behavior, lipid transport,and neuronal repair).

resolution extending to about 7–9 Å along this axis.The connection between the resulting rod-like fibersis less defined and extends to only ~15 Å, indicatinga weaker stacking interaction along the long cell axeswhere the sides of the discs touch each other.Recently, improved crystals have been obtained bysubstituting dipalmitoylphosphatidylcholine forDMPC. These crystals diffract to approximately 8 Åalong all three axes.

ApoE Isoform Differences

We have identified three structural and biophysicaldifferences among the three apoE isoforms: (1) apoE4domain interaction; (2) protein stability and folding;and (3) cysteine content (Figure 2). Our workinghypothesis is that one or more of these differences areresponsible for the isoform-specific effects of apoE4on plasma lipoprotein metabolism, atherosclerosis,and neurodegeneration. To test this hypothesis, wewill engineer each of these isoform differences indi-vidually into the mouse Apoe gene by gene targeting.In this way we can determine the individual contribu-tion of each difference to isoform-specific effects thatare known to occur in plasma and the brain (Figure 2).Sorting out the relative contributions of these differ-ences has important implications in developing effec-tive, apoE4-based therapeutic strategies.

ApoE4 Domain Interaction. ApoE4 binds preferen-tially to very low density lipoproteins (VLDL),whereas apoE3 binds preferentially to high densitylipoproteins (HDL) (Figure 1). We determined thatthe two domains in apoE4 interact and that this inter-action is restricted to apoE4. The interaction is modu-lated by Arg-61 (amino-terminal domain) and Glu-255 (carboxyl-terminal domain) and is responsible forthe VLDL binding preference of apoE4 (Figure 3).

We are collaborating with Drs. Fred Cohen and IrwinKuntz (UCSF) and using their DOCK program toidentify small molecules that will bind to apoE4 in thevicinity of Arg-61 but not to apoE3 and thereby inter-fere with domain interaction. We expect that suchmolecules will represent a therapeutic approach byconverting apoE4 into an “apoE3-like” molecule(Figure 3).

In several species, including the mouse, apoE con-tains arginine and glutamic acid at positions equiva-lent to positions 112 and 255, respectively, in humanapoE. However, these species lack the critical humanArg-61 required for domain interaction. Their apoEcontains threonine and, like human apoE3, displays apreference for HDL. Based on these results, we useda “knock-in” gene targeting approach to introduce anarginine codon into the mouse gene to “humanize”



Figure 3. ApoE4 domain interaction. In apoE3, with cysteine at posi-tion 112, the Arg-61 side chain is positioned in a cleft between twohelices and cannot interact with the carboxyl-terminal domain. InapoE4, with arginine at position 112, however, the Arg-61 side chain

projects into the aqueous environment, where it can interact withGlu-255, thereby mediating domain interaction, resulting in a differ-ent overall structure for apoE4.

mouse apoE at position 61 and introduce domaininteraction.

In heterozygous targeted mice, as in human apoE4heterozygotes, the plasma level of the Arg-61 form is20–40% lower than the level of the wildtype apoE.This characteristic pattern reflects the more rapidclearance of apoE4 from plasma. The “Arg-61”mouse apoE also displays the expected preference forVLDL. These results demonstrate that domain inter-action was successfully introduced in vivo. With thismodel, we can determine the contribution of domaininteraction to the increased risk of cardiovascular dis-ease, neurodegeneration, and cognitive impairmentassociated with apoE4.

Protein Stability and Folding. In addition to struc-tural differences among protein isoforms, biophysicalproperties are important determinants of their func-tional properties. An emerging concept in proteinfolding is the stable folding intermediate, alsoreferred to as the molten globule state. The moltenglobule is a semirigid structure that is almost as com-pact as the native structure. It retains most of the sec-ondary structure of the native state and much of thetertiary structure; owing to the partial loss of tertiarystructure, it usually contains an exposed hydrophobicsurface. Until recently, it had been assumed that themolten globule was a relatively rare occurrence.However, there is a large body of experimental evi-dence that the molten globule state is a common fea-ture of most proteins, can exist in cells, and plays akey role in a wide variety of physiological processes,including translocation across membranes, increasedaffinity for membranes, binding to liposomes andphospholipids, protein trafficking, extracellular secre-tion, and the control and regulation of the cell cycle.

To compare the physical characteristics of the apoEisoforms, we conducted guanidine, urea, and thermaldenaturation studies of apoE2, apoE3, and apoE4 andtheir 22- and 10-kDa fragments. Guanidine and ureadenaturation demonstrated that the two domainsunfold independently in the three isoforms. However,all three denaturation methods showed differences instability among the amino-terminal fragments of the

apoE isoforms. ApoE4 denatured at the lowest con-centrations of guanidine and urea and at the lowesttemperature, while apoE2 denatured at the highestconcentrations and temperature. Furthermore, guani-dine and urea denaturation showed that apoE4, unlikeapoE2 and apoE3, did not fit a two-state denaturationequilibrium. The lack of cooperative unfolding sug-gests that apoE4 forms a stable, partially folded inter-mediate.

Since unfolding intermediates are often more stable atan acidic pH, we examined urea denaturation of thethree 22-kDa fragments at pH 4.0. The results demon-strated two-phase denaturation for apoE4 and a shoul-der in the denaturation curve of apoE3, similar to thatseen with apoE4 at pH 7.0. These findings suggestthat both apoE3 and apoE4, but not apoE2, displayfolding intermediates at pH 4.0 in urea. Analysis ofdenaturation curves using a three-stage modelrevealed that the intermediate in apoE4 representsapproximately 90% of the mixture at 3.75 M urea,whereas in apoE3 the intermediate represents approx-imately 30% of the total protein population at thisurea concentration and can be increased to approxi-mately 80% at 4.75 M urea.

In collaboration with Drs. Anthony Fink and KeithOberg (University of California, Santa Cruz), weexamined the apoE4 folding intermediate in theabsence and presence of urea at pH 4.0 with theirnovel method of Fourier transmittance reflectiveinfrared analysis. In the absence of urea, the sec-ondary structure of the apoE4 22-kDa fragment wasestimated to consist of 75% α-helix and 3% β-sheet,consistent with previous estimates. In 3.75 M urea,apoE4 consisted of 46% α-helix and 17% β-sheet.Thus, the apoE4 intermediate contained 61% of theoriginal helical content and had an increased β-sheetstructure. Pepsin proteolysis of apoE4 at pH 4.0 in 0M and 3.75 M urea showed cleavages between helices2 and 3, within helix 3, and between helices 3 and 4in the presence of urea. These results indicate thatthere is a conformational change in apoE4 at pH 4.0in the presence of urea. We speculate that the four-helix bundle is partially unfolded, similar to theunfolded structure when this fragment binds to lipid.

2001 ANNUAL REPORT


The results from the characterization of the apoE4 sta-ble folding intermediate indicate that this intermediateis a molten globule. Since molten globules have beenimplicated in a variety of physiological processes,including membrane binding and translocation, weexamined the ability of apoE4 and apoE3 to bind anddisrupt DMPC vesicles at pH 4.0, with and withouturea. Under both conditions, apoE4 was more effec-tive than apoE3, suggesting that the apoE4 moltenglobule may be involved in membrane translocation.

Cysteine Content. The three major isoforms differ incysteine content: apoE4, apoE3, and apoE2 contain 0,

1, and 2 cysteines, respectively. As a result, apoE2and apoE3 can form both disulfide-linked homo-dimers and heterodimers with apoA-II. In apoE3/3subjects, approximately 50% of the apoE3 exists inone of these disulfide-linked forms. In addition, theapoE3 homodimer is present in cerebrospinal fluid.These disulfide-linked dimers affect lipid-bindingproperties and interaction with LDL receptors, buttheir effect in neurobiology has not been systemati-cally assessed. Since mouse apoE also lacks cysteine,a future objective will be to use gene targeting tointroduce a cysteine codon to produce the functionalequivalent of apoE3 and determine its effect on neu-rodegeneration.



Selected References

Dong L-M, Weisgraber KH (1996) Humanapolipoprotein E4 domain interaction. Arginine 61and glutamic acid 255 interact to direct the preferencefor very low density lipoproteins. J. Biol. Chem.271:19053–19057.

Weisgraber KH, Mahley RW (1996) Humanapolipoprotein E: The Alzheimer’s disease connec-tion. FASEB J. 10:1485–1494.

Lu B, Morrow JA, Weisgraber KH (2000)Conformational reorganization of the four-helix bun-dle of human apolipoprotein E in binding to phospho-lipid. J. Biol. Chem. 275:20775–20781.

Morrow JA, Segall ML, Lund-Katz S, Phillips MC,Knapp M, Rupp B, Weisgraber KH (2000)Differences in stability among the human apolipopro-tein E isoforms determined by the amino-terminaldomain. Biochemistry 39:11657–11666.

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. USA 98:11587–11591.

WYSS-CORAY LABORATORY

Staff Research Investigator


Visiting Researcher

Marion Buckwalter, M.D., Ph.D.


Amy Hsiu-Ti Lin, Ph.D.

Ina Tesseur, Ph.D.

Research Associates

Thomas Brionne

Jacob Corn

Lauren Mondshein

Jon-Paul Pepper

Michelle Rohde

Fengrong Yan


Kelley Nelson



Molecular Mechanisms of Amyloid Clearance


Over the past year, a large effort in our labora-tory was directed toward the dissection ofproinflammatory activities that may reduce

Alzheimer’s-related neuropathology in human amy-loid precursor protein (hAPP) transgenic mice. Wehad previously observed that overexpression of thecytokine transforming growth factor-β1 (TGF-β1) inhAPP mice results in prominent activation ofmicroglial cells associated with a 50–60% reductionof amyloid deposition and plaque formation.Moreover, stimulation of microglial cells with TGF-β1 in culture resulted in more efficient degradation ofsynthetic β-amyloid (Aβ).

TGF-β1 Increases Microglial ComplementC3 Synthesis in Vivo

Alzheimer’s disease (AD) is characterized by the accu-mulation of protein deposits in the brain in the form ofextracellular Aβ deposits in amyloid plaques and cere-bral blood vessels and also as tangles inside neurons.The peptide Aβ (40–42 amino acids), which is contin-uously produced at low levels in the normal brain,accumulates in AD and appears key to disease patho-genesis. Understanding how Aβ is normally clearedand how it may promote neuronal dysfunction is likelyto reveal novel targets for therapeutic intervention.

Figure 1. Increased complement C3 expression in hAPP/TGF-β1and TGF-β1 transgenic mice. (A) Brain sections from 12-month-oldhAPP/TGF-β1 and TGF-β1 mice show C3 immunostaining (2/11antibody) in the CA3 region of the hippocampus in cells withmicroglial morphology. Less C3 staining is present in littermatehAPP and nontransgenic (Non-tg) mice; brains from C3 knockoutmice (C3 –/–) were not stained. Lower right panels show at highermagnification more C3 immunostaining around plaques in

hAPP/TGF-β1 (A/T) than in hAPP (A) mice. Scale bars: 50 µm (atboth magnifications). (B) Brains from 12–15-month-old hAPP (opencircles) and hAPP/TGF-β1 (filled circles) mice were divided sagittal-ly, and relative C3 mRNA levels were measured by RNase protec-tion assay in one hemibrain. In the opposite hemibrain, averagenumbers of thioflavin S–positive plaques per 40-µm brain section (n= 5–6 sections per mouse) in the hippocampus and neocortex werecounted. Each circle represents one mouse.

Aβ deposits and degenerating cells in AD are associ-ated with activated microglia and astrocytes and withincreased local production of inflammatory media-tors, in particular complement components. Thisinflammatory response may be detrimental and con-tribute to neurodegeneration, or it could reflect afailed attempt of the brain to serve regenerative func-tions by removing Aβ and degenerating cells. As men-tioned above, reduced cerebral Aβ accumulation andneurodegeneration were associated with a prominentmicroglial activation in hAPP mice overproducingTGF-β1, and TGF-β1 increased the clearance of Aβin cultured BV-2 microglial cells. Because the com-plement system, which is a key trigger of inflamma-tory responses, is activated in the AD brain and itscomponents are produced by central nervous systemcells, we examined whether complement levels areassociated with Aβ accumulation.

In brain sections of aged hAPP/TGF-β1 mice, cellswith microglial morphology were prominentlylabeled with antibodies against C3 (Figure 1A), thecentral component of complement. C3 immunoreac-tivity was often accentuated around plaques inhAPP/TGF-β1, but not in hAPP, mice (Figure 1A)and was generally less intense in the hippocampus ofhAPP singly transgenic or nontransgenic mice than inmice overexpressing TGF-β1 (Figure 1A). This effectof TGF-β1 on C3 immunostaining was accompaniedby increased levels of C3 protein by western blot inbrain homogenates of 10–12-month-old TGF-β1-overexpressing mice. To determine whether theincrease in C3 accumulation in TGF-β1 transgenicmice was associated with increased C3 synthesis, wedetermined relative TGF-β1 mRNA levels in hemi-brains of old and young mice. Aged hAPP mice hadclearly detectable levels of C3 mRNA in their brains,and coexpression of TGF-β1 led to an almost twofoldincrease in expression. This stimulatory effect ofTGF-β1 on C3 mRNA levels was even stronger inyoung hAPP/TGF-β1 transgenic mice at an agebefore Aβ accumulation could be detected.

To determine the possible relationship between cere-bral C3 expression levels and the extent of Aβ depo-sition in vivo, we compared C3 mRNA levels with Aβaccumulation in aged hAPP and hAPP/TGF-β1 mice.

Although only one dose of TGF-β1 transgene expres-sion was analyzed, there was an inverse relationship(R = –0.68, p = 0.0009 by logarithmic regressionanalysis) between C3 mRNA and numbers ofthioflavin S–positive plaques (e.g., plaques with fib-rillar Aβ in β-pleated sheet conformation) (Figure1B). In addition, hAPP/TGF-β1 mice had 50–60%less Aβ1–x (approximates total Aβ) and Aβ1–42 in theirbrains than hAPP controls. These data demonstratethat TGF-β1 overexpression in brains of transgenicmice increases C3 production while decreasing Aβaccumulation and plaque formation. Thus, higher lev-els of complement C3 expression in mouse brainsmay not necessarily be detrimental but can be associ-ated with reduced AD-type pathology.

Inhibition of Complement C3 Activationin hAPP Mice Results in Increased AβAccumulation and Plaque Formation.

Aggregated Aβ can activate the complement systemin vitro through the classical pathway by binding Clqand through the alternative pathway by binding C3b.Both pathways lead to the formation of multiproteinenzyme complexes, the C3 convertases, which cleaveC3 into C3a and C3b. C3a is released in the fluidphase and is involved in the chemotaxis of phago-cytes. C3b can bind covalently to acceptor moleculesin a process called opsonization and induce activationof the lytic pathway leading to the formation of themembrane attack complex (MAC) and possible celllysis. Alternatively, C3b-opsonized particles or cellsare phagocytosed via C3b-binding complementreceptors on specialized cells. It is, therefore, con-ceivable that Aβ–C3b complexes in the brain triggerthe formation of the MAC or mediate the phagocyto-sis of opsonized cells or Aβ deposits.

To determine the role of complement activation in Aβaccumulation and neurodegeneration more directly,we inhibited C3 activation genetically in the brains ofhAPP mice by expressing the C3-convertase inhibitorCrry under control of the metallothionein promoter.Crry is a rodent-specific functional homologue of twohuman regulators of complement activation (mem-brane cofactor protein and decay-accelerating factor)that inhibits complement activation and C3 deposition

2001 ANNUAL REPORT


by both the classical and the alternative pathways.While Crry is normally expressed in a membrane-bound form in astrocytes, microglia, and neurons, thetransgenic mice used here also express Crry in a solu-ble form (sCrry). Cerebral Crry mRNA levels weresixfold higher in sCrry and hAPP/sCrry mice than inhAPP mice and nontransgenic littermate controls, andimmunohistochemical analysis of C3 activation prod-ucts in the brain indicated that Crry overexpressioninhibits C3 activation in vivo. In contrast, hAPPexpression did not change Crry mRNA levels, andoverexpression of the sCrry transgene did not affectcerebral C3 protein levels in 3-month-old mice.Likewise, hAPP protein levels in brain homogenatesor hAPP immunoreactivity in hippocampal CA neu-rons were similar in hAPP and hAPP/sCrry mice.

This inhibition of complement was associated withsignificantly increased Aβ accumulation as deter-mined with various methods. At 10–12 months of age,hAPP/sCrry mice showed more Aβ immunostainingthan hAPP mice (Figure 2A and B) and a significant

increase in hippocampal Aβ-immunoreactive area(Figure 2C). Total Aβ (Aβ1–x) and Aβ1–42 levels wereon average threefold higher in the neocortex andalmost twofold higher in the hippocampus ofhAPP/sCrry mice than in hAPP littermate controls(Figure 2D). In addition, hAPP/sCrry mice had sig-nificantly more thioflavin S–positive plaques andCongo Red–positive plaques in the hippocampus thanhAPP littermate controls. Thus, inhibition of comple-ment in hAPP mice results in faster and greater accu-mulation of Aβ and increases the number of compactamyloid plaques. Given the two possible outcomes ofcomplement activation, cell lysis or phagocytosis, theabove findings suggest that complement may have arole in Aβ phagocytosis and clearance.

Crry Overexpression and ComplementInhibition in hAPP Mice Resultsin Prominent Neurodegeneration

In collaboration with Dr. Eliezer Masliah (Universityof California at San Diego), we found that along with



Figure 2. C3 inhibition in vivo promotes Aβ accumulation and amy-loid formation. Brains from 10–12-month-old hAPP (n = 6) andhAPP/sCrry (n = 8) mice were dissected and analyzed for Aβ accu-mulation and amyloid formation. Aβ immunostaining in the hip-pocampus and neocortex of an hAPP (A) and an hAPP/sCrry (B)mouse. (C) The area occupied by Aβ immunoreactivity (IR) was sig-

nificantly larger in hAPP/sCrry than in hAPP mice. Values are mean± SEM. **p < 0.01 by Mann-Whitney U test. (D) Total Aβ (Aβ1–x, black+ gray bars) and Aβ1–42 (gray bars) levels in neocortex (left) and hip-pocampus (right) of hAPP and hAPP/sCrry mice. Values are mean± SEM. **p = 0.028; * p = 0.039 by Mann-Whitney U test. Scale bar:250 µm (A, B).

the increase in Aβ accumulation and plaque forma-tion, 10–12-month-old hAPP/sCrry, but not hAPP,mice showed a severe loss of hippocampal CA3pyramidal NeuN-positive neurons (Figure 3A). Thenumber of NeuN-positive neurons correlatedinversely with the extent of Aβ deposition (Figure3B) and with the number of Aβ-immunopositiveplaques. Likewise, the number of NeuN-positivec e l l s w a s d e c r e a s e d i n t h e c o r t e x o f a g e dhAPP/sCrry mice and correlated inversely with thenumber of plaques and with Aβ1–42 levels. NeuN is amarker of differentiated neurons and stains mostneurons in the adult neocortex, but degeneratingneurons seem to loose this marker. Indeed, the CA3carea that showed the most dramatic loss of NeuNimmunostaining in hAPP/sCrry mice (Figure 3A)also exhibited atypically intense cresyl violet stain-ing, suggesting a shrinkage or degeneration of thesecells. The extent and specificity of neuronal cell lossand degeneration observed in hAPP/sCrry mice hadnot been reported in hAPP transgenic mice, and thelack of neuronal cell loss was regarded by some as amajor weakness of using such mice to model AD.Although the exact mechanisms leading to the cellloss and neurodegeneration in hAPP/sCrry mice arenot yet known, we demonstrate that abnormal Aβ

accumulation and reduced complement activationare both necessary to cause severe pathology.

In line with our previous observations in hAPP/TGF-β1 mice and with the increase in microglial C3immunostaining they exhibit (Figure 1A), microglialactivation in hAPP and hAPP/sCrry mice correlatedinversely with Aβ accumulation. Microglia in theneocortex were significantly less activated inhAPP/sCrry mice than in hAPP mice, supporting apossible function for these cells in Aβ clearance.

Together the data indicate that inhibiting complementactivation in the brain can accelerate amyloid accu-mulation and neurodegeneration in a transgenicmouse model of AD-like pathology. Increased com-plement C3 production in TGF-β1 transgenic micewas associated with reduced Aβ deposition. Theseresults are consistent with the role of complementfactors as recognition molecules for phagocytosisand with TGF-β1 as a possible modulator of thisprocess. Complement deficiency results in the accu-mulation of apoptotic and degenerating cells in vivoand causes autoimmune inflammation. It is conceiv-able that complement inhibition in our model pro-motes neurodegeneration in part by decreasing the

2001 ANNUAL REPORT


Figure 3. C3 inhibition in hAPP mice results in neurodegenera-tion. Brain sections from 10–12-month-old mice from a cross ofhAPP with sCrry mice were analyzed for neurodegeneration andmicroglial activation. (A) NeuN immunostaining of 40-µm brainsections showed markedly less staining in the hippocampal CA3region in hAPP/sCrry mice than in singly transgenic or nontrans-genic (Non-tg) mice. The small panels (bottom right) show at high-

er magnification the CA3c subregion in the large panels. A =hAPP; C = sCrry; N = Non-tg; A/C = hAPP/sCrry (corresponds topanel in upper right corner). Scale bars: 100 µm (large panels), 50µm (small panels). (B) Relative number of NeuN-positive neuronsand Aβ immunoreactive (IR) deposits (3D6 antibody) in theCA2/CA3 region of the hippocampus of hAPP mice (open circles)and hAPP/sCrry mice (filled circles).

clearance of cellular debris and protein aggregatesthat are generated through normal cell turnover.Furthermore, TGF-β1 release during phagocytosis isthought to inhibit local inflammation and enhancephagocytosis. While clearance of Aβ from brains ofhAPP mice can also be stimulated artificially byinducing an immune response and antibody produc-tion against Aβ, complement-mediated clearance ofAβ may represent a natural mechanism by which thebrain prevents Aβ accumulation and neurodegenera-

tion. Alternatively, complement factors such as C3aor C5a may have novel neuroprotective effects, andinhibition of complement activation in our modelmay result in reduced protection. In summary, wedemonstrate a novel function of complement in AD-type pathology in mice that is consistent withincreased clearance of Aβ and reduced neurodegen-eration. Our findings suggest that enhancement ofspecific complement functions could be of therapeu-tic benefit for AD.



Selected References

Wyss-Coray T, Masliah E, Mallory M, McConlogueL, Johnson-Wood K, Lin C, Mucke L (1997)Amyloidogenic role of cytokine TGF-β1 in transgenicmice and Alzheimer’s disease. Nature 389:603–606.

Akiyama H, Barger S, Barnum S, Bradt B, Bauer J,Cole GM, Cooper NR, Eikelenboom P, Emmerling M,Fiebich BL, Finch CE, Frautschy S, Griffin WST,Hampel H, Hull M, Landreth G, Lue LF, Mrak R.,MacKenzie IR, O’Banion K, McGeer PL, Pachter J,Pasinetti G, Plata-Salaman C, Rogers J, Rydel R,Shen Y, Streit W, Strohmeyer R, Tooyoma I, VanMuiswinkel FL, Veerhuis R, Walker D, Webster S,Wegrzyniak B, Wenk G, Wyss-Coray T (2000)Inflammation and Alzheimer’s disease. Neurobiol.Aging 21:383–421.

Wyss-Coray T, Lin C, Sanan D, Mucke L, Masliah E(2000) Chronic overproduction of TGF-β1 in astro-cytes promotes Alzheimer’s disease–like microvascu-lar degeneration in transgenic mice. Am. J. Pathol.156:139–150.

Wyss-Coray T, Mucke L (2000) Ibuprofen, inflamma-tion and Alzheimer disease. Nat. Med. 6:973–974.

Masliah E, Ho G, Wyss-Coray T (2001) Functionalrole of TGF-β in Alzheimer’s disease: Lessons fromtransgenic mice. Neurochem. Int. 39:393–400.

Wyss-Coray T, Lin C, Yu GC, McConlogue L,Masliah E, Mucke L (2001) TGF-β1 promotesmicroglial amyloid-β clearance and reduces plaqueburden in transgenic mice. Nat. Med. 7:612–618.

BEHAVIORAL CORE LABORATORY

Staff Research Scientist

Jacob Raber, Ph.D.

Research Associates

Lisa N. Kekonius

Anthony D. LeFevour

Student

Gerold Bongers




Interpretation of Behavioral Data

In assessing complex behaviors, it is crucial to deter-mine if there are specific deficits in more basic func-tions. For example, it is important to distinguish learn-ing impairments from performance deficits. Vestibulardeficits are often associated with increased horizontallocomotor activity, including circling, reduced rearing(raising of both forefeet off the ground and extension ofthe body), abnormal posture, and poor swimming abil-ity. In assessing olfactory memory, reduced ability todetect a particular odorant may be a confounding fac-tor. In the water maze test, which assesses spatial learn-ing and memory, vision and motivation are required tolocate a hidden platform by using visual cues outsidethe maze. To assess visual and motivational problems,we test the ability of mice to locate a visible platform.The swim speed of the mice is also measured as anindicator of motivation, motor function, and coordina-tion, all of which can influence the time required tolocate the platform (latency). Metabolic alterations areconfounding factors in certain tests, such as the hole-board 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, anddifferent strains of mice vary in their ability to masterdifferent tests. Therefore, the protocols for the behav-ioral testing of disease models established on differentgenetic backgrounds often must be adapted to makethe task neither so easy that all mice can perform itequally well nor so difficult that none of the mice canperform it successfully. The following sections high-light behavioral paradigms that are sensitive to ADrisk factors and exemplify some of the contributionsour laboratory has made to major research programsat the Gladstone Institutes.


Behavioral Core Laboratory

Jacob Raber, Ph.D.

The Behavioral Core Laboratory was estab-lished in 1997 to analyze central nervoussystem function in experimental mouse mod-

els of human neurological diseases. Through collab-orative interactions and consultations, it serves sci-entists at all three Gladstone Institutes and otherinvestigators at the San Francisco General Hospitalcampus, as well as colleagues at the Gallo Center,the Mount Zion and Parnassus campuses of UCSF,the University of California, Berkeley, and otherinstitutions.

From Mice to Humans

We focus on evaluating the behavior of mouse mod-els of human dementing illnesses. Understandingwhat impairs learning and memory in these modelsis providing important insights into both central ner-vous system functions and clinically relevant diseaseprocesses. For example, together with Dr. BruceMiller and his colleagues at the UCSF Memory andAging Center, we are investigating specific linksbetween cognitive impairments in patients withdementia and behavioral deficits in mouse models ofthese diseases. A major long-term goal of this inter-action is the development of suitable tests and noveltreatment strategies to improve cognition in patientssuffering from Alzheimer’s disease (AD) and relatedconditions.

Behavioral Tests

We routinely use a comprehensive battery of tests(Table 1) to fully characterize the neurological condi-tion of mouse models and to validate complex learn-ing paradigms.

2001 ANNUAL REPORT


Table 1. Behavioral tests

Apolipoprotein E4 Impairs Not OnlySpatial but Also Nonspatial Learningand Memory in Female Mice

The three major human apolipoprotein (apo) E iso-forms (E2, E3, and E4) differ in their effect on AD.Compared with apoE2 and apoE3, apoE4 increasesthe risk of AD and, possibly, also of age-related cog-nitive decline in general. ApoE4 appears to interactwith female gender, further increasing the risk of ADand diminishing the effectiveness of treatments inwomen. To assess how interactions between genderand apoE isoforms affect cognition, we studiedfemale and male mice lacking mouse apoE (Apoe–/–)and expressing human apoE3 or apoE4 in the brain atcomparable levels. As they aged, female, but notmale, apoE4 (Apoe–/–) mice developed progressiveimpairments in spatial learning and memory in thewater maze test, compared with age- and sex-matched mice expressing apoE3, endogenous mouseapoE, or no apoE at all. We previously observed thatandrogens and androgen receptor (AR)–dependentpathways protect against the detrimental effects ofapoE4 on cognition. Even brief periods of androgentreatment reduced memory deficits in female apoE4mice. In addition, apoE4 male mice, which per-formed normally in a water maze test at baseline,developed prominent deficits in spatial learning andmemory after blockade of ARs, whereas apoE3 malemice did not.

Several lines of evidence suggest that females may bemore susceptible to spatial memory impairments thanmales. For example, others studying rodents withmedial frontal cortical lesions have found that maleswere less impaired than females in mazes requiringthe use of multiple visuospatial cues for a successfulsolution. We investigated whether apoE4 also exertsgender-dependent detrimental effects on nonspatiallearning and memory. Six-month-old male and femaletransgenic mice expressing apoE3 or apoE4 in neu-rons were tested for novel object recognition. Duringthe training session, mice were allowed to explore anopen field containing two objects for 15 minutes. Forthe retention session (24 hours later), they wereplaced back into the same open field for 15 minutes,after one of the familiar objects was replaced with a

novel object and the other familiar object with anexact replica. The amount of time the mice spentexploring the novel versus the familiar object relativeto the total amount of time they spent exploring eitherobject in the retention session was used as a measureof object recognition memory.



Figure 1. ApoE4 impairs object recognition memory in femalemice. Novel object recognition was tested in untreated 6-month-old male (M) and female (F) mice. Results shown are from thememory retention session. Expression of human apoE was direct-ed by the neuron-specific enolase (NSE) promoter (A) or the glialfibrillary acidic protein (GFAP) promoter (C). (A) NSE-apoE4 mice(n = 11 males, 7 females) and NSE-apoE3 mice (n = 4 males, 7females). (B) Wildtype (Wt) mice (n = 11 males, 6 females) andApoe–/– mice (n = 7 males, 13 females). (C) Apoe–/– mice (n = 6females) and GFAP-apoE4 mice (n = 6 females). Most miceshowed normal object recognition memory. Only female miceexpressing apoE4 in neurons (A) or astrocytes (C) failed to spendsignificantly more time with the novel than with the familiar object.˚p < 0.05, *p < 0.01 versus time exploring the familiar object(Tukey-Kramer test). n.s., not significant.

2001 ANNUAL REPORT


In the training session, all groups of mice spent a com-parable amount of time exploring each object. In theretention session, only female apoE4 mice showed sig-nificant deficits, whereas male apoE4 mice and maleor female apoE3, wildtype, and Apoe–/– mice had intactobject recognition memory (Figure 1A and B). FemaleapoE4 mice spent a significantly smaller proportion oftime exploring the novel object than did female apoE3mice or wildtype controls (p < 0.05, Tukey-Kramertest). These results demonstrate that apoE4 inducesgender-dependent deficits not only in spatial but alsoin nonspatial learning and memory. The resistance ofmale apoE4 mice to deficits in object recognitionmemory is consistent with our previous finding thatAR-dependent pathways protect against apoE4-induced deficits in spatial learning and memory.

Interestingly, deficits in object recognition memorywere also identified in female mice of an indepen-dent transgenic line in which expression of apoE4was targeted to astrocytes with the glial fibrillary

acidic protein promoter (Figure 1C). The indepen-dence of the adverse effects from the cell type pro-ducing apoE4 suggests that they are mediated bysecreted forms of apoE4.

Cytosolic AR Levels

To assess how apoE4 might affect AR-dependent path-ways, we used an AR binding assay to determinecytosolic AR levels in the neocortex and hippocampusof apoE transgenic mice. Female and male apoE4 micehad lower cytosolic AR levels in the neocortex thanapoE3, wildtype, or Apoe–/– mice (Figure 2A). Why doonly female apoE4 mice show deficits in learning andmemory even though both female and male apoE4mice have decreased cortical AR levels? Cognitiveperformance likely depends on a critical balancebetween plasma androgen levels and cytosolic AR lev-els in the brain, and the higher endogenous plasmatestosterone levels in male apoE4 mice may providerelative protection. Consistent with this notion, plasma

Figure 2. Cytosolic AR levels in the neocortex and response toandrogen treatment. Total cytosolic AR levels were determinedfrom AR saturation curves using a single curve fit analysis. Resultsare expressed as fmole [3H]R1881 bound per mg protein. Therewere no differences in Kd among the groups. (A) Untreated maleand female NSE-apoE4 mice had lower cytosolic AR levels thanuntreated NSE-apoE3, Apoe–/–, and wildtype (Wt) mice (n = 3–7

mice per gender and genotype). (B–D) Effect of placebo (B),testosterone (C), and dihydrotestosterone (D) on AR saturationcurves in female NSE-apoE4 mice (n = 3–6 mice/treatment). Thisresponse is in line with the positive regulation of ARs by androgensin wildtype brains observed by others. It suggests that the apoE4-dependent reduction in AR levels does not preclude responsivityof ARs to androgen treatment.

testosterone levels in testosterone-treated femaleapoE4 mice were roughly twice as high as those inuntreated male apoE4 mice. Testosterone and dihy-drotestosterone treatments increased AR levels in theneocortex of female apoE4 mice (Figure 2B–D).

The fact that AR levels and cognitive performancewere significantly lower in apoE4 mice than in miceexpressing no apoE at all suggests an adverse gain-of-function of apoE4. Together with the age-related

decline in plasma testosterone levels in men andwomen, the AR-lowering effect of apoE4 could con-tribute to cognitive decline also in human APOE ε4carriers and promote the development of AD. Thefinding that even brief treatment with testosterone sig-nificantly improved learning and memory in adultfemale apoE4 mice is encouraging. Increased effortsare warranted to test the efficacy of androgens orandrogen derivatives in humans and to investigatetheir modes of action in related animal models.



Selected References

Raber J, Wong D, Buttini M, Orth M, Bellosta S, PitasRE, Mahley RW, Mucke L (1998) Isoform-specificeffects of human apolipoprotein E on brain functionrevealed in ApoE knockout mice: Increased suscepti-bility of females. Proc. Natl. Acad. Sci. USA95:10914–10919.

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


Smith SJ, Cases S, Jensen DR, Chen HC, Sande E,Tow B, Sanan DA, Raber J, Eckel RH, Farese RV Jr(2000) Obesity resistance and multiple mechanismsof triglyceride synthesis in mice lacking DGAT. Nat.Genet. 25:87–90.

Raber J, LeFevour A, Mucke L (2001) Androgen treat-ment reduces cognitive deficits in female apoE4 trans-genic mice. In: Alzheimer’s Disease: Advances inEtiology, Pathogenesis and Therapeutics (Iqbal K,Sisodia SS, Winblad B, eds) John Wiley & Sons,Chichester, West Sussex, England, pp 747–757.

GLADSTONE GENOMICS CORE

Staff Research Scientist


Visiting Scientists

Shirley Zhu, Ph.D.

Andrea J. Barczak

Research Associates

Anita Chow

Kristina Hanspers, M.S.

Yanxia Hao, M.S.


Emily K. O’Keeffe



Gladstone Genomics Core


The Genomics Core provides technology, suchas DNA microarrays, to assist Gladstone sci-entists in their studies of the unprecedented

volume of information resulting from the HumanGenome Project and others. This past year has beenone of tremendous growth and change within theGenomics Core as it expands to meet the needs of theGladstone research community. Services are providedto the Gladstone Institutes of Cardiovascular Disease(GICD), Virology and Immunology (GIVI), andNeurological Disease (GIND) and, as resources allow,to the greater UCSF community.

The Genomics Core offers two DNA microarray plat-forms, which use similar, but distinct, methods. Inboth systems, the researcher uses the arrays to deter-mine if there are differences between two biologicalsamples in the expression levels of large numbers ofgenes. When established in 2000, the Genomics Corefocused on implementing the Affymetrix GeneChipsystem. In this system, investigators use arrays pur-chased from the manufacturer, and samples areprocessed according to protocols provided byAffymetrix. Two hands-on training workshops areoffered. The first, which provides one-on-one trainingin the methods needed to prepare materials for arrays,is extremely popular and has been fully enrolled everytime it has been offered. Fifteen researchers from theCharo, Conklin, Dichek, Goldsmith, Grant, Greene,Mahley, McCune, Nixon, and Verdin laboratories atGladstone have completed this course. In addition, wehave provided training to six researchers from theUCSF Departments of Pathology, Pediatrics, andPhysiology and the Diabetes Center. A second, moreadvanced, 6-hour short course teaches researchershow to use the software tools needed to analyze the

data generated with DNA microarrays. Sevenresearchers from the Charo, Conklin, Goldsmith,Greene, and McCune laboratories and four from theUCSF Departments of Pathology, Physiology, andStomatology have completed this course. TheGenomics Core also provides advanced one-on-onetraining in sample preparation and data analysis uponrequest. This year, advanced training was provided tothe Mucke laboratory and the Bluestone laboratoryfrom the Diabetes Center at UCSF.

A major initiative of the Genomics Core this past yearhas been to develop the resources needed to prepareand use custom-printed DNA microarrays. Because ofthe high cost of the Affymetrix GeneChips, we intro-duced the capability to use printed arrays. The diffi-culties of this approach are the need for expensive,specialized equipment and the need to develop reli-able protocols. An early step in this process was toestablish a collaboration with the Sandler Center forBasic Research in Asthma at UCSF. The GenomicsCore and the Sandler Center each agreed to purchasesome of the equipment and to share access to theirequipment. The Genomics Core purchased equipmentto prepare samples for use on arrays, while theSandler Center purchased equipment to print and scanthe arrays. The Genomics Core also purchased alibrary of 70-base oligonucleotides representing 6868mouse genes. We have performed two successfulprinting runs with this library and have on hand over200 arrays available for use. We recently purchased asecond, supplemental library representing an addi-tional 6575 mouse genes. Printing from this largercombined library commenced in mid-December,2001. As our library grows, we will need to designadditional oligonucleotides. To do this, we have

obtained the software program PIK70, which designsoligonucleotides from large numbers of DNAsequences. In our tests of this program, we havedesigned unique, 70-base oligonucleotides against15,275 cDNA sequences from the RIKEN mousecDNA database. This suggests that with proper anno-tation, we will be able to meet the oligonucleotidedesign needs as we increase the size of our arrays.

The second phase of this initiative was to developrobust, consistent working protocols for RNA isola-tion, cDNA synthesis, cDNA labeling, and arrayhybridization. We have completed the development ofour protocols and have begun to characterize the sys-tems underlying the arrays. In a large-scale experi-ment, we prepared eight arrays. Two large cDNApreparations were made from normal adult mouseliver and total brain RNA, and the arrays werehybridized to them in parallel under identical condi-tions. Under these conditions, we detected 3830 genesthat were present on all arrays. Using very stringentselection criteria, we found that 3253 of the detectedgenes were present in both tissues, 30 genes were pre-sent in adult liver alone, and 70 genes were presentonly in adult total brain (p ≤ 0.05 for all differences).We have also begun to test the transfer of our proto-cols to the laboratories of Gladstone investigators. Inan intensive one-on-one test with the Verdin laborato-ry (GIVI), we are using their samples to assess therole of histone deacetylases in activation-inducedgene expression in T cells.

While we have made significant strides on the datageneration side of DNA microarrays, we also need tomove forward in data analysis. The first step in thisprocess has been to train core personnel in the use ofGeneSpring, Microsoft Access, and Microsoft Excel.We now provide training in these programs, alongwith Eisen Cluster and GenMAPP, to researchers. Wehave also begun training of core personnel on a newsoftware program, Significance Analysis ofMicroarrays (SAM), designed to help eliminate falsepositives in our expression databases. Finally, wehave arranged for investigators needing moreadvanced assistance to consult with the UCSF Centerfor Bioinformatics and Molecular Biostatistics.

Investigators UsingGenomics Core Resources

Israel Charo, M.D., Ph.D. (GICD). The Charo labo-ratory has initiated a study to examine the regulation ofchemokines and chemokine receptors. They are com-paring gene expression in the lungs of CCR2 knockoutmice with or without Mycobacterium tuberculosisinfection. Additional comparisons are being made withcorresponding samples from wildtype mice.

Bruce Conklin, M.D. (GICD). The Conklin labora-tory has begun two studies. In the first, gene expres-sion is being analyzed in mouse hearts that have car-diomyopathy induced by transgenes related to G pro-tein signaling. The second is a time course study ofchanges in the patterns of gene expression inmyometrium during normal pregnancy that arethought to be related to G protein signals.

Steven Finkbeiner, M.D., Ph.D. (GIND). TheFinkbeiner laboratory has initiated a study to betterunderstand how the common second messenger Ca2+

can regulate the transcription of different neuronalgenes. Laboratory staff members are focusing theirefforts on genes modulated by either the N-methyl-D-aspartate receptor or the L-type voltage-sensitive cal-cium channel. Results of these studies could lead tothe identification of gene targets that are important forsynaptic plasticity and reveal the molecular mecha-nisms by which Ca2+ can induce distinct but stimulus-specific adaptive responses.

Mark Goldsmith, M.D., Ph.D. (GIVI). TheGoldsmith laboratory began to characterize thehematopoietic system in in vivo models by examiningthe effects of 5-fluorouracil on mouse bone marrowand fetal liver and kidney and comparing the geneexpression levels in these tissues to those in untreatedtissues.

Lennart Mucke, M.D. (GIND). The Mucke labora-tory has compared transgenic mice expressing in thebrain wildtype versus Alzheimer-mutant forms ofthe human amyloid protein precursor (APP) andAPP mice that were sham-vaccinated or vaccinated

2001 ANNUAL REPORT


with amyloid β (Aβ) peptides to prevent the devel-opment of Alzheimer-like pathologies. Expressionof human APP and APP-derived Aβ peptides elicitedchanges in the expression of multiple mRNAs in thehippocampus of transgenic mice. Differences ingene expression patterns in wildtype and mutantAPP mice are beginning to shed light on the patho-genicity of Alzheimer mutations and Aβ peptides.Differences in gene expression in sham- and Aβ-vaccinated APP mice are being used to identifymediators of vaccination effects and evaluated asend point measures for vaccination trials.

Stephen Young, M.D. (GICD). The Young labora-tory has continued to study gene expression in thebrains of protein carboxylmethyltransferase knock-out and wildtype mice in order to identify the genesresponsible for the seizures to which the knockoutmice are prone.

David Bredt, M.D., Ph.D. The Bredt laboratory(Department of Physiology, UCSF) began a study ofthe molecular basis of postsynaptic organization. Inthis study, they are using polyribosomal RNA as thestarting material for all comparisons to assay onlythose genes being actively transcribed.



Selected Reference

Essrich C, Wang Z, Allen D, Yu G-Q, Ordanza H,Mucke L (2001) Hippocampal mRNA profiles oftransgenic mice expressing comparable levels of

wildtype or Alzheimer-mutant human amyloid proteinprecursors. Soc. Neurosci. 127 (Part 1):332 (abstract).

OUTREACH

Outreach 109

Outreach 111

Special Events, Initiatives, and Contributions

Education in basic and disease-oriented neuro-sciences is an important mission of theGladstone Institute of Neurological Disease

(GIND). GIND investigators promoted this missionthrough several educational and scientific initiatives.They also participated actively in fund-raising effortsfor the Alzheimer’s Association. Through such contri-butions, we demonstrate our commitment to the com-munity as well as to the pursuit of our scientific goals.

Society for Neuroscience Meeting:Neurodegenerative Disorders, MisfoldedProteins, and Apolipoprotein E

The 31st annual meeting of the Society forNeuroscience, held in San Diego, brought togethermore than 28,000 neuroscientists from around theworld. While past meetings focused primarily onbasic neuroscience, the Decade of the Brain and themany fundamental scientific questions raised by neu-rological diseases have resulted in a steady increase indisease-related topics addressed at this conference.For this year’s meeting, Dr. Lennart Mucke organizeda symposium entitled “Protein Aggregation andNeurodegenerative Disease—An Unfolding Story.”This well-attended symposium focused on emergingevidence that many, if not all, neurodegenerative dis-orders are caused by the abnormal folding and aggre-gation of proteins inside or around nerve cells.Although different proteins accumulate in differentneurodegenerative disorders, the mechanisms bywhich the misfolded proteins damage nerve cellsappear to have common features. This possibility rais-es hope for developing treatments to prevent orreverse more than one of these conditions.Mechanisms by which misfolded proteins can damagecells were discussed by five speakers: Drs. Mucke and

Steven Finkbeiner from the GIND, Dr. Bruce Yanknerfrom Harvard Medical School, Dr. David Ron fromthe Skirball Institute and New York University, andDr. Harry Orr from the University of Minnesota.

For the first time, a large group of neuroscientistsgathered at the same meeting for a symposium enti-tled “Apolipoproteins/Lipoprotein Receptors: Role inNeurobiology and Disease.” The all-day symposiumwas organized by Drs. Lennart Mucke and DavidHoltzman (Washington University). Twenty-sevenresearchers from the United States, Europe, and Israelpresented new findings on the roles of apoE and relat-ed molecules in health and disease. Presentationsfocusing on research at the GIND were given by Drs.Robert Mahley, Karl Weisgraber, Robert Pitas,Yadong Huang, Jacob Raber, and Manuel Buttini.

Alzheimer Meetings in the Bay Areaand Beyond

Promising discoveries in Alzheimer research arebeing made with increasing frequency. Several of themost exciting developments were reviewed at a sym-posium entitled “Recent Advances in Alzheimer’sDisease Research,” held in San Mateo, California,and sponsored by the American Health AssistanceFoundation. Drs. Sangram Sisodia (University ofChicago), Edward Koo (University of California atSan Diego), and Donald Price (Johns HopkinsUniversity) organized the event, and Dr. Muckeserved as the local host. Among the topics reviewedwere anti-amyloid vaccines, which have advancedrapidly from mouse models to clinical trials in peoplewith the disease, and the pharmacological manipula-tion of enzymes responsible for the production of theβ-amyloid peptides that accumulate to abnormal levels

in Alzheimer brains. Participants also reviewed theprogress made at the GIND and elsewhere in definingthe molecular pathways that lead from the accumula-tion of β-amyloid peptides to the development of neu-rodegeneration and cognitive decline. A KeystoneSymposium entitled “Vascular and Neuronal Stress: ANew Window on Alzheimer’s Disease” at Durango,Colorado, was co-organized by Dr. Mucke and includ-ed presentations by Drs. Mahley and Tony Wyss-Coray highlighting, respectively, the contributions ofapoE4 and brain inflammation to the development ofAlzheimer’s disease. Dr. Mucke also participated in apublic lecture series organized by the Bay AreaChapter of the Alzheimer’s Association in November,National Alzheimer’s Awareness Month. The advancesdiscussed at these conferences are likely to usher in anew era in disease management and have given rise tojustifiable hope among Alzheimer patients and thoseproviding for their treatment and care.

Genomic Neurology—A Peek at the Future

The initial sequencing and analysis of the humangenome were recently published by Drs. FrancisCollins, Eric Lander, and the International HumanGenome Consortium in Nature and by Dr. CraigVenter and colleagues of Celera Genomics inScience. This extraordinary compendium of newmolecular knowledge offers unprecedented opportu-nities to advance our understanding of the develop-ment, function, and aging of the human nervous sys-tem. The new genetic information will also promotediscoveries in relation to complex neurological dis-eases and expedite the development of rational treat-ments for the most recalcitrant conditions neurolo-gists face in clinical practice. At the 126th annualmeeting of the American Neurological Association inChicago, Drs. Mucke and Roger Rosenberg(University of Texas, Southwestern) organized aworkshop entitled “Genomic Neurology.” Landmarkfindings from the Human Genome Project and themany ways in which the new genetic informationwill affect neurological research and clinical practicewere described by Drs. Francis Collins (NationalHuman Genome Research Institute), KennethFischbeck (National Institute of Neurological

Disorders and Stroke), Allen Roses (Glaxo SmithKline), and Thomas Bird (University of Washington).

Participation in Other Meetings

GIND scientists also gave invited lectures at theElsevier Science 11th Neuropharmacology Conferenceon Molecular Mechanisms of Synaptic Function (SanDiego), Gordon Research Conference on CAG TripletRepeat Diseases (Mount Holyoke College, SouthHadley, Massachusetts), Meeting of Alzheimer’sDisease Center Directors and the National Institute onAging (Chicago), XII Lipid Meeting (Leipzig,Germany), Tenth International Symposium: NewFrontiers of Neurochemistry and Neurophysics onDiagnosis and Treatment of Neurological Diseases(Florence, Italy), and Complement-AssociatedDiseases, Animal Models, and Therapeutics Workshop(Santorini, Greece).

Interactions with UCSF Programsand Community Outreach Activities

In their fight against Alzheimer’s disease, GINDresearchers collaborate closely with their colleagues atthe UCSF Memory and Aging Center, directed by Dr.Bruce Miller. On October 6, members of the GIND andthe Memory and Aging Center joined forces in thisyear’s 3-mile Memory Walk, an annual walk-a-thonorganized by the local chapter of the Alzheimer’sAssociation to raise awareness and funds for theAssociation’s efforts to help Alzheimer victims andtheir families. More than 3,500 people walked onTreasure Island in San Francisco Bay this year andhave so far raised $470,000, far surpassing theAssociation’s goal of $375,000. This year, the institutealso participated in the San Francisco Fire Fighters ToyProgram. Established in 1949 and run by off-duty andretired fire fighters and their families, this volunteerorganization distributes toys to underprivileged fami-lies, not just during the holiday season but year round.

The GIND also recognizes the enormous importanceof training the scientists of tomorrow. Drs.Finkbeiner, Fen-Biao Gao, and Mucke taught basicand disease-related neuroscience to UCSF medicalstudents, M.D./Ph.D. students, and Ph.D. students of

2001 ANNUAL REPORT

112 Outreach

the Neuroscience Program, the Biomedical SciencesProgram, and the Pharmaceutical Sciences andPharmacogenomics Program. Dr. Gao also participat-ed in the UCSF Science Education PartnershipProgram. Dr. Mucke served as the attending physi-cian on the neurology service at San FranciscoGeneral Hospital, training medical students, internsand residents in the diagnosis and treatment of neuro-logical disorders. Dr. Finkbeiner collaborated withDr. Miller and the Huntington’s Disease Society ofAmerica to establish a Center of Excellence forHuntington’s Disease at UCSF.

Special Memberships, Honors,and Awards

Several members of the GIND were recognized fortheir contributions to neuroscience and biomedicalresearch by distinguished memberships, honors, andawards. Dr. Mahley was elected to the Institute ofMedicine of the National Academy of Sciences andgave the Dean’s Distinguished Lecture at theUniversity of Arkansas for Medical Sciences. Dr.

Mucke was appointed as chair of the Neuroscience ofAging Review Committee of the National Institute onAging and as a trustee of the recently established ElanNeuroscience Foundation. He was also selected todeliver the Peter W. Lampert Memorial Lecture at theUniversity of California at San Diego and the NamsooLee Memorial Lecture at Duke University. Dr. Wyss-Coray gave the Killam Lecture at the MontrealNeurological Institute and McGill University. Dr. Gaoreceived an Alfred P. Sloan Research FellowshipAward, which recognizes young scholars of outstand-ing promise, and a McKnight Neuroscience of BrainDisorders Award from the McKnight EndowmentFund for Neurosciences, one of six awards to scien-tists whose research is directed toward finding newways to diagnose, treat, and cure disorders of thebrain and central nervous system. Dr. Finkbeinerreceived a Klingenstein Fellowship Award inNeuroscience from the Klingenstein Foundation,which supports new approaches to neuroscientificresearch, and a Distinguished Medical Scholar Awardfrom the W. M. Keck Foundation, which supports pio-neering efforts in medical research.


Outreach 113

PUBLICATIONS

Publications 115

Publications 117

Publications

1. D’Hooge R, Nagels G, Westland CE, Mucke L,De Deyn PP (1996) Spatial learning deficit inmice expressing human 751-amino acid β-amy-loid precursor protein. Neuroreport 7:2807–2811.

2. Fleming LM, Weisgraber KH, Strittmatter WJ,Troncoso JC, Johnson GVW (1996) Differentialbinding of apolipoprotein E isoforms to tau andother cytoskeletal proteins. Exp. Neurol.138:252–260.

3. Mahley RW, Nathan BP, Bellosta S, Pitas RE(1996) Apolipoprotein E: Structure, function, andpossible roles in modulating neurite extension andcytoskeletal activity. In: Apolipoprotein E andAlzheimer’s Disease (Roses AD, Weisgraber KH,Christen Y, eds) Springer-Verlag, Berlin, pp 49–58.

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

5. Masliah E, Sisk A, Mallory M, Mucke L, SchenkD, Games D (1996) Comparison of neurodegen-erative pathology in transgenic mice overexpress-ing V717F β-amyloid precursor protein andAlzheimer’s disease. J. Neurosci. 16:5795–5811.

6. Mohajeri MH, Bartsch U, van der Putten H,Sansig G, Mucke L, Schachner M (1996) Neuriteoutgrowth on non-permissive substrates in vitrois enhanced by ectopic expression of the neuraladhesion molecule L1 by mouse astrocytes. Eur.J. Neurosci. 8:1085–1097.

7. Pitas RE (1996) Microtubule formation and neu-rite extension are blocked by apolipoprotein E4.Semin. Cell Dev. Biol. 7:725–731.

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

9. Roses AD, Einstein G, Gilbert J, Goedert M, HanS-H, Huang D, Hulette C, Masliah E, Pericak-Vance MA, Saunders AM, Schmechel DE,Strittmatter WJ, Weisgraber KH, Xi P-T (1996)Morphological, biochemical, and genetic supportfor an apolipoprotein E effect on microtubularmetabolism. Ann. N.Y. Acad. Sci. 777:146–157.

10. Weisgraber KH, Mahley RW (1996) Humanapolipoprotein E: The Alzheimer’s disease con-nection. FASEB J. 10:1485–1494.

11. Weisgraber KH, Dong LM (1996) Role ofapolipoprotein E in Alzheimer’s disease: Cluesfrom its structure. In: Apolipoprotein E andAlzheimer’s Disease (Roses AD, Weisgraber KH,Christen Y, eds) Springer-Verlag, Berlin, pp11–19.

12. Wyss-Coray T, Masliah E, Toggas SM,Rockenstein EM, Brooker MJ, Lee HS, Mucke L(1996) Dysregulation of signal transduction path-ways as a potential mechanism of nervous systemalterations in HIV-1 gp120 transgenic mice andhumans with HIV-1 encephalitis. J. Clin. Invest.97:789–798.

13. Zhao J, Paganini L, Mucke L, Gordon M, RefoloL, Carman M, Sinha S, Oltersdorf T, LieberburgI, McConlogue L (1996) β-Secretase processingof the β-amyloid precursor protein in transgenicmice is efficient in neurons but inefficient inastrocytes. J. Biol. Chem. 271:31407–31411.

2001 ANNUAL REPORT

118 Publications

14. Gutman CR, Strittmatter WJ, Weisgraber KH,Matthew WD (1997) Apolipoprotein E binds toand potentiates the biological activity of ciliaryneurotrophic factor. J. Neurosci. 17:6114–6121.

15. Mahley RW (1997) Apolipoprotein E: Structureand function in lipid metabolism and neurobiolo-gy. In: The Molecular and Genetic Basis ofNeurological 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) Amyloid precursor proteins pro-tect neurons of transgenic mice against acute andchronic excitotoxic injuries in vivo. Neuroscience78:135–146.

17. Mattson MP, Barger SW, Furukawa K, Bruce AJ,Wyss-Coray T, Mark RJ, Mucke L (1997)Cellular signaling roles of TGFβ, TNFα andβAPP in brain injury responses and Alzheimer’sdisease. 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 apoE2 potentiates β-amyloidprotein activation of complement in vitro. BrainRes. 749:135–138.

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

20. Raber J, Chen S, Mucke L, Feng L (1997)Corticotropin-releasing factor and adrenocorti-cotrophic hormone as potential central mediatorsof OB effects. J. Biol. Chem. 272:15057–15060.

21. Raber J, Koob GF, Bloom FE (1997) Interferon-α and transforming growth factor-β1 regulate cor-ticotropin-releasing factor release from the amyg-dala: Comparison with the hypothalamicresponse. Neurochem. Int. 30:455–463.

22. Wyss-Coray T, Borrow P, Brooker MJ, Mucke L(1997) Astroglial overproduction of TGF-β1enhances inflammatory central nervous systemdisease in transgenic mice. J. Neuroimmunol.77:45–50.

23. Wyss-Coray T, Masliah E, Mallory M,McConlogue L, Johnson-Wood K, Lin C, MuckeL (1997) Amyloidogenic role of 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 regulation of c-Jun N-terminal kinaseactivity in mouse brain by environmental stimuli.Proc. Natl. Acad. Sci. USA 94:12655–12660.

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

26. Buttini M, Westland CE, Masliah E, Yafeh AM,Wyss-Coray T, Mucke L (1998) Novel role ofhuman CD4 molecule identified in neurodegen-eration. Nat. Med. 4:441–446.

27. Coward P, Wada HG, Falk MS, Chan SDH,Meng F, Akil H, Conklin BR (1998) Controllingsignaling with a specifically designed Gi-coupledreceptor. Proc. Natl. Acad. Sci. USA 95:352–357.

28. Ji Z-S, Pitas RE, Mahley RW (1998) Differentialcellular accumulation/retention of apolipoproteinE mediated by cell surface heparan sulfate pro-teoglycans. Apolipoproteins E3 and E2 greaterthan E4. J. Biol. Chem. 273:13452–13460.

29. Krucker T, Toggas SM, Mucke L, Siggins GR(1998) Transgenic mice with cerebral expressionof human immunodeficiency virus type-1 coatprotein gp120 show divergent changes in short-and long-term potentiation in CA1 hippocampus.Neuroscience 83:691–700.

30. Mahley RW, Weisgraber KH, Farese RV Jr(1998) Disorders of lipid metabolism. In:Williams Textbook of Endocrinology, 9th edition(Wilson JD, Foster DW, Kronenberg HM, LarsenPR, eds) W. B. Saunders, Philadelphia, pp1099–1153.

31. Mahley RW (1998) Expanding roles forapolipoprotein E in health and disease. In:Atherosclerosis XI (Jacotot B, Mathé D, FruchartJ-C, eds) Elsevier, Amsterdam, pp 117–124.


Publications 119

32. Marshall DCL, Wyss-Coray T, Abraham CR(1998) Induction of matrix metalloproteinase-2 inhuman immunodeficiency virus-1 glycoprotein120 transgenic mouse brains. Neurosci. Lett.254:97–100.

33. Masliah E, Raber J, Alford M, Mallory M,Mattson MP, Yang D, Wong D, Mucke L (1998)Amyloid protein precursor stimulates excitatoryamino acid transport: Implications for roles inneuroprotection and pathogenesis. J. Biol. Chem.273:12548–12554.

34. Mucke L, Buttini M (1998) Molecular basis ofHIV-associated neurologic disease. In: MolecularNeurology (Martin JB, ed) Scientific American,New York, pp 135–154.

35. Pitas RE, Ji Z-S, Weisgraber KH, Mahley RW(1998) Role of apolipoprotein E in modulatingneurite outgrowth: Potential effect of intracellularapolipoprotein E. Biochem. Soc. Trans.26:257–262.

36. Pitas RE, Ji Z-S, Supekova L, Mahley RW (1998)Divergent metabolism of apolipoproteins E3 andE4 by cells. In: Progress in Alzheimer’s andParkinson’s Diseases (Fisher A, Hanin I, YoshidaM, 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 of human apolipoprotein E onbrain function revealed in ApoE knockout 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 obesity to memory deficits. Mol. Neurobiol.18:1–22.

39. Raber J, Sorg O, Horn TFW, Yu N, Koob GF,Campbell IL, Bloom FE (1998) Inflammatorycytokines: Putative regulators of neuronal andneuro-endocrine function. Brain Res. Rev.26:320–326.

40. Toggas SM, Mucke L (1998) Transgenic modelsto assess the pathogenic potential of viral prod-ucts in HIV-1-associated CNS disease. In: The

Neurology of AIDS (Gendelman HE, Lipton SA,Epstein L, Swindells S, eds) Chapman & Hall,New York, pp 156–167.

41. Bush TG, Puvanachandra N, Horner CH, PolitoA, Ostenfeld T, Svendsen CN, Mucke L, JohnsonMH, Sofroniew MV (1999) Leukocyte infiltra-tion, neuronal degeneration, and neurite out-growth after ablation of scar-forming, reactiveastrocytes in adult transgenic mice. Neuron23:297–308.

42. Buttini M, Orth M, Bellosta S, Akeefe H, PitasRE, Wyss-Coray T, Mucke L, Mahley RW (1999)Expression of human apolipoprotein E3 or E4 inthe brains of Apoe–/– mice: Isoform-specificeffects on neurodegeneration. J. Neurosci.19:4867–4880.

43. D’Hooge R, Franck F, Mucke L, De Deyn PP(1999) Age-related behavioural deficits in trans-genic mice expressing the HIV-1 coat proteingp120. Eur. J. Neurosci. 11:4398–4402.

44. Hsia AY, Masliah E, McConlogue L, Yu G-Q,Tatsuno G, Hu K, Kholodenko D, Malenka RC,Nicoll RA, Mucke L (1999) Plaque-independentdisruption of neural circuits in Alzheimer’s dis-ease mouse models. Proc. Natl. Acad. Sci. USA96:3228–3233.

45. Huang F, Buttini M, Wyss-Coray T, McConlogueL, Kodama T, Pitas RE, Mucke L (1999)Elimination of the class A scavenger receptordoes not affect amyloid plaque formation or neu-rodegeneration in transgenic mice expressinghuman amyloid protein precursors. Am. J. Pathol.155:1741–1747.

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

47. Mahley RW, Huang Y (1999) Apolipoprotein E:From atherosclerosis to Alzheimer’s disease andbeyond. Curr. Opin. Lipidol. 10:207–217.

48. Mahley RW, Ji Z-S (1999) Remnant lipoproteinmetabolism: Key pathways involving cell-surface

2001 ANNUAL REPORT

120 Publications

heparan sulfate proteoglycans and apolipopro-tein E. J. Lipid Res. 40:1–16.

49. Mahley RW, Rall SC Jr (1999) Is ε4 the ancestralhuman apoE allele? Neurobiol. Aging20:429–430.

50. Redfern CH, Coward P, Degtyarev MY, Lee EK,Kwa AT, Hennighausen L, Bujard H, FishmanGI, Conklin BR (1999) Conditional expressionand signaling of a specifically designed Gi-cou-pled receptor in transgenic mice. Nat.Biotechnol. 17:165–169.

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

52. Akiyama H, Barger S, Barnum S, Bradt B, BauerJ, Cole GM, Cooper NR, Eikelenboom P,Emmerling M, Fiebich BL, Finch CE, FrautschyS, Griffin WST, Hampel H, Hull M, Landreth G,Lue L-F, Mrak R, Mackenzie IR, McGeer PL,O’Banion MK, Pachter J, Pasinetti G, Plata-Salaman C, Rogers J, Rydel R, Shen Y, Streit W,Strohmeyer R, Tooyoma I, Van Muiswinkel FL,Veerhuis R, Walker D, Webster S, Wegrzyniak B,Wenk G, Wyss-Coray T (2000) Inflammationand Alzheimer’s disease. Neurobiol. Aging21:383–421.

53. Buttini M, Akeefe H, Lin C, Mahley RW, PitasRE, Wyss-Coray T, Mucke L (2000) Dominantnegative effects of apolipoprotein E4 revealed intransgenic models of neurodegenerative disease.Neuroscience 97:207–210.

54. Finkbeiner S (2000) Calcium regulation of thebrain-derived neurotrophic factor gene. Cell.Mol. Life Sci. 57:394–401.

55. Finkbeiner S (2000) CREB couples neurotrophinsignals to survival messages. Neuron 25:11–14.

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

57. Masliah E, Rockenstein E, Veinbergs I, MalloryM, Hashimoto M, Takeda A, Sagara Y, Sisk A,

Mucke L (2000) Dopaminergic loss and inclu-sion body formation in α-synuclein 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 ofthe glial injury response to the multifactorialpathogenesis of Alzheimer’s disease. In: Neuro-immune Interactions in Neurologic andPsychiatric Disorders (Patterson P, Kordon C,Christen Y, eds) Springer-Verlag, Berlin, pp19–33.

59. Mucke L, Masliah E, Yu G-Q, Mallory M,Rockenstein EM, Tatsuno G, Hu K, KholodenkoD, Johnson-Wood K, McConlogue L (2000)High-level neuronal expression of Aβ1–42 inwild-type human amyloid protein precursortransgenic mice: Synaptotoxicity without plaqueformation. J. Neurosci. 20:4050–4058.

60. Mucke L, Yu G-Q, McConlogue L, RockensteinEM, Abraham CR, Masliah E (2000) Astroglialexpression of human α1-antichymotrypsinenhances Alzheimer-like pathology in amyloidprotein precursor transgenic mice. Am. J. Pathol.157:2003–2010.

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

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

63. Redfern CH, Degtyarev MY, Kwa AT, SalomonisN, Cotte N, Nanevicz T, Fidelman N, Desai K,Vranizan K, Lee EK, Coward P, Shah N,Warrington JA, Fishman GI, Bernstein D, BakerAJ, Conklin BR (2000) Conditional expressionof a Gi-coupled receptor causes ventricular con-duction delay and a lethal cardiomyopathy. Proc.Natl. Acad. Sci. USA 97:4826–4831.

64. Smith SJ, Cases S, Jensen DR, Chen HC, SandeE, Tow B, Sanan DA, Raber J, Eckel RH, Farese


Publications 121

RV Jr (2000) Obesity resistance and multiplemechanisms of triglyceride synthesis in micelacking Dgat. Nat. Genet. 25:87–90.

65. Wyss-Coray T, Lin C, Sanan DA, Mucke L,Masliah E (2000) Chronic overproduction oftransforming growth factor-β1 by astrocytes pro-motes 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 dis-ease–like cerebrovascular pathology in trans-forming growth factor-β1 transgenic mice andfunctional metabolic correlates. Ann. N.Y. Acad.Sci. 903:317–323.

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

68. Finkbeiner S (2001) New roles for introns: Sitesof combinatorial regulation of Ca2+- and cyclicAMP-dependent gene transcription. Science’sSTKE (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 Efragments present in Alzheimer’s disease brainsinduce neurofibrillary tangle-like intracellularinclusions in neurons. Proc. Natl. Acad. Sci. USA98:8838–8843.

70. Masliah E, Ho G, Wyss-Coray T (2001)Functional role of TGFβ in Alzheimer’s diseasemicrovascular injury: Lessons from transgenicmice. 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 accumula-tion and neuronal deficits in a transgenic mouse

model linking Alzheimer’s disease andParkinson’s disease. Proc. Natl. Acad. Sci. USA98:12245–12250.

72. Raber J, LeFevour A, Mucke L (2001) Androgentreatment reduces cognitive deficits in femaleapoE4 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, WeisgraberKH (2001) Introduction of human apolipoproteinE4 “domain interaction” into mouse apolipopro-tein E. Proc. Natl. Acad. Sci. USA 98:11587–11591.

74. Santiago-García J, Mas-Oliva J, Innerarity TL,Pitas RE (2001) Secreted forms of the amyloid-βprecursor protein are ligands for the class A scav-enger receptor. J. Biol. Chem. 276:30655–30661.

75. Scearce-Levie K, Coward P, Redfern CH, ConklinBR (2001) Engineering receptors activated solelyby synthetic ligands (RASSLs). TrendsPharmacol. Sci. 22:414–420.

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

77. Wyss-Coray T, McConlogue L, Kindy M, SchmidtAM, Yan SD, Stern DM (2001) Key signalingpathways regulate the biological activities andaccumulation of amyloid-β. Neurobiol. Aging22:967–973.

78. Scearce-Levie K, Coward P, Redfern CH, ConklinBR (2002) Tools for dissecting signaling path-ways in vivo: Receptors activated solely by syn-thetic ligands. Methods Enzymol. 343:232–248.

Seminars 123

SEMINARS

Gladstone DistinguishedLecture Series

GIND Seminar Series

Seminar Seriesat Gladstone, SFGH,and UCSF

Seminars 125

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

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

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

Joseph 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 Sciences

University of Texas Southwestern Medical SchoolDallas, TXMembrane-bound SREBP: Sterol sensor and tran-scriptional regulator

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

November 21, 1996Günter Blobel, M.D., Ph.D.InvestigatorHoward 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.InvestigatorHoward Hughes Medical InstituteHiggins Professor of Biochemistry and MolecularBiophysicsProfessor of PathologyColumbia UniversityNew York, NYThe molecular biology of smell

Gladstone Distinguished Lecture Series

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

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

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

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

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

2001 ANNUAL REPORT

126 Seminars

Seminars 127

January 25, 2001Linda J. Noble, Ph.D.*Department of Neurological SurgeryUniversity of California, San FranciscoSan Francisco, CATransgenic modeling as a strategyfor understanding regional neuronal vulnerabilityafter traumatic brain injury

February 22, 2001Allan Basbaum, Ph.D.Department of AnatomyUniversity of California, San FranciscoSan Francisco, CAThe neurochemistry of acute and chronic pain

April 5, 2001Grant A. Krafft, Ph.D.*Department of Molecular PharmacologyNorthwestern UniversityEvanston, ILSolving the riddle of amyloidand Alzheimer’s disease

May 17, 2001Ben Barres, M.D., Ph.D.*Department of Neurobiologyand Developmental BiologyStanford UniversityStanford, CANeuron-glial interactions in the developing CNS

May 31, 2001Alex Roher, M.D., Ph.D.*Haldeman Laboratory for Alzheimer’s ResearchSun Health Research InstituteSun City, AZOf mice and men: The amyloidof Alzheimer’s disease

July 5, 2001Julie Andersen, Ph.D.*Buck Institute for Age ResearchNovato, CAWhat role does oxidative stress play in cell deathin Parkinson’s disease?

July 19, 2001William Klein, Ph.D.*Center for Cognitive Neurologyand Alzheimer’s DiseaseNorthwestern University Institute for NeuroscienceEvanston, ILSoluble toxins in Alzheimer’s disease:New vaccine and SOM drug targets

August 6, 2001Thomas van Groen, Ph.D.*Department of Neuroscience and NeurologyUniversity of Kuopio Kuopio, FinlandThe development of pathological changesand behavioral deficits in aging AD mutant mice:Is there a relation?

GIND Seminar Series

2001 ANNUAL REPORT

128 Seminars

August 27, 2001Andrew Holmes, Ph.D.*National Institute of Mental HealthBethesda, MDCognitive and emotional phenotypesin galanin overexpressing transgenic mice

October 4, 2001Michael Shelanski, M.D., Ph.D.*Department of PathologyColumbia UniversityNew York, NYAβ modulation of memory mechanisms

October 18, 2001Scott Small, M.D.*Sergievsky CenterColumbia UniversityNew York, NYImaging metabolism of neuronal populationsin mice and humans

October 25, 2001Matthew Anderson, M.D., Ph.D.*Center for Learning and MemoryMassachusetts Institute of TechnologyCambridge, MAThe study of brain pacemakers using conditionalknockout techniques: An approach to solving brainnetwork mechanisms using mouse genetics

November 16, 2001Denisa Wagner, Ph.D.*The Center for Blood ResearchHarvard Medical SchoolBoston, MANew roles for CD40L and apoE in vascular biology

December 6, 2001Paul Frankland, Ph.D.*Department of NeurobiologyUniversity of California, Los AngelesLos Angeles, CAHippocampal memories:Representations, systems and molecules

*Neurobiology of Alzheimer’s Disease and RelatedDisorders Lecture Series

Bay Area RNA Club

Members of nearly 40 laboratories working on RNAresearch gather to hear three informal talks by partici-pating labs; held the first Thursday of every monthfrom 6:30–9:30 p.m. in Health Sciences West (HSW)302 on the UCSF campus. Information is available athttp://www.ucsf.edu/frankel/RNA_club/RNA_club.html. Contact: Jeff Pleiss (E-mail: [email protected]).

Biochemistry and Biophysics Seminar

Formal presentations by faculty or guest speakersorganized by the UCSF Department of Biochemistryand Biophysics; held every Tuesday from 4:00–5:00p.m. in HSW-301 on the UCSF Parnassus campus andbroadcast to the 5th floor library of San FranciscoGeneral Hospital (SFGH) Building 3 and to the 4thfloor conference room of SFGH Building 40.Information is available at http://biochemistry.ucsf/edu/seminars–b&b.html. Contact: Judy Piccini (Tel:476-1515; E-mail: [email protected]).

BMS Journal Club

Journal club of the UCSF Biomedical Sciences(BMS) Program; held every Wednesday from12:30–1:30 p.m. in the 4th floor conference room ofSFGH Building 40. Articles from any area of biomed-icine are discussed in two half-hour presentations bygraduate students, postdoctoral fellows, and faculty.Contact: Naima Contos (Tel: 695-3729; E-mail: [email protected]).

CVRI Lecture

Formal presentations by faculty or guest speakers orga-nized by the UCSF Cardiovascular Research Institute(CVRI); every Monday from 4:00–5:30 p.m. on theUCSF Parnassus campus (room varies). Contact: JulieTom (Tel: 476-1310; E-mail: [email protected]).

Frontiers in Neurology and Neuroscience

Lectures by faculty or guest speakers focusing on neu-rological diseases and their treatment; organized bythe UCSF Department of Neurology. Lectures areheld every other Wednesday from 5:00–6:00 p.m. inthe N-217 auditorium of the School of Nursing on theUCSF Parnassus campus. Contact: Bill Ramsay (Tel:476-1489; E-mail: [email protected]).

GICD Scientists Meeting

Informal seminars focusing on research progress incardiovascular disease given by graduate students,postdoctoral fellows, or investigators of the GladstoneInstitute of Cadiovascular Disease (GICD); held everyFriday from 9:00–10:00 a.m. in the 4th floor confer-ence room of SFGH Building 40. Contact: BrianAuerbach (Tel: 695-3757; E-mail: [email protected]).

GICD Seminar

Formal presentations focusing on topics related to car-diovascular disease given by guest speakers and can-didates for postdoctoral fellowships at the GICD; heldfrom 12–1:00 p.m. in the 4th floor conference room ofSFGH Building 40 (day varies). Contact: BrianAuerbach (Tel: 695-3757; E-mail: [email protected]).

GIND Seminar

Presentations focusing on research in disease-relatedneuroscience given by graduate students, postdoctoralfellows, or investigators of the Gladstone Institute ofNeurological Disease (GIND) or guest speakers; heldevery Thursday from 9:00–10:00 a.m. in the 5th floorlibrary of SFGH Building 3. Contact: Kelley Nelson(Tel: 695-3885; E-mail: [email protected]).

Seminars 129

Seminar Series at Gladstone, SFGH, and UCSF

GIVI SeminarPresentations focusing on research progress in virolo-gy and immunology, including HIV and AIDS, givenby national and international speakers, alternatingwith presentations by members of the GladstoneInstitute of Virology and Immunology (GIVI); heldevery Thursday from 12:00–1:00 p.m. in the 5th floorlibrary of SFGH Building 3. Contact: Robin Givens(Tel: 695-3801; E-mail: [email protected]).

Microbiology and Immunology SeminarFormal presentations by faculty or guest speakersorganized by the UCSF Department of Microbiologyand Immunology; normally held every Monday from5:00–6:00 p.m. in HSW-301 on the UCSF Parnassuscampus and broadcast to the 5th floor library of SFGHBuilding 3 and to the 4th floor conference room ofSFGH Building 40. Information is available athttp://itsa.ucsf.edu/%7Emicro/immunology/index.htm. Contact: Harriet Romero (Tel: 502-1961; E-mail:[email protected]).

Neurobiology of Alzheimer’s Diseaseand Related Disorders Lecture SeriesMonthly presentations by Bay Area investigators orguest speakers focusing on basic research projects orclinical investigations on Alzheimer’s disease or relat-ed disorders; held in the 5th floor library of SFGHBuilding 3. Contact: Lennart Mucke (Tel: 695-3819;E-mail: [email protected]).

Neuroscience Journal ClubJournal club of the UCSF Neuroscience Program;held every Friday from 4:00–5:00 p.m. in S-214 onthe UCSF Parnassus campus. Articles from any areaof neuroscience are discussed in two half-hour pre-sentations by graduate students and faculty.Refreshments are served after the meeting. Informa-tion is available at http://www.ucsf.edu/neurosc/club.html. Contact: Lisa Magargal (Tel: 476-8370; E-mail:[email protected]).

Neuroscience SeminarFormal presentations by guest speakers focusing onbasic neuroscience; held every Thursday from4:00–5:00 p.m. in HSW-301 on the UCSF Parnassuscampus. Information is available at http://www.ucsf.edu/neurosc/seminars.html. Contact: Lisa Magargal(Tel: 476-8370; E-mail: [email protected]).

PCMM/BMS SeminarLectures of the UCSF Program in Cellular andMolecular Medicine (PCMM) are given by faculty ofthe BMS Program or by guest speakers from any areaof biomedicine; held every Friday from 12:00–1:00p.m. in the 5th floor library of SFGH Building 3.Contact: Eaine Sampior (Tel: 206-6946; E-mail: [email protected]).

PIBS Journal ClubJournal club of the UCSF Program in BiologicalSciences (PIBS); held every Wednesday from2:00–3:30 p.m. in the N-217 auditorium of the Schoolof Nursing on the UCSF Parnassus campus. Articlesfrom any area of biomedicine are discussed in two half-hour presentations by graduate students and faculty.Contact: Maria Realubin (Tel: 476-6178; E-mail: [email protected]).

Seminar in Biomedical SciencesFormal presentations by faculty or guest speakers fromany area of biomedicine; organized by the BMSProgram; held every Wednesday from 4:00–5:00 p.m.in HSW-300 on the UCSF Parnassus campus. Contact:Monique Piazza (Tel: 476-2189; E-mail: [email protected]).

Signaling ClubInformal presentations attended by people from 15–20laboratories at UCSF; held on the first Tuesday of everymonth from 12:00 –1:00 p.m. in room L-1361 of LongHospital on the UCSF Parnassus campus. Research onsignaling is discussed in two half-hour presentations bygraduate students or postdoctoral fellows. Contact:Mark Von Zastrow (Tel: 476-7855; E-mail: [email protected]).

2001 ANNUAL REPORT

130 Seminars


Seminars 131

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(4:00–5:30)UCSF (room varies)

Microbiology andImmunology Seminar HSW-301

Signaling Club(monthly)L-1361

Biochemistry andBiophysics SeminarHSW-301

BMS Journal Club(12:30)SFGH B40

PIBS Journal ClubN-217

Seminar in Biomedical Sciences HSW-300

Frontiers inNeurology and Neuro-science (2 x/month)N-217

GIND Seminar (weekly)and Neurobiology of ADand Related Disorders(monthly), SFGH B3

GIVI SeminarSFGH B3

NeuroscienceSeminar HSW-301

Bay Area RNA Club(6:30; monthly)UCSF (room varies)

GICD Scientists MeetingSFGH B40

PCMM/BMS Seminar SFGH B3

NeuroscienceJournal Club S-214

Calendar of Gladstone, SFGH, and UCSF Seminars

gladstone institute of neurological disease

Documents