GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASEDocument Transcript
2002 ANNUAL REPORT
THE J. DAVID GLADSTONE INSTITUTES
University of California, San Francisco
San Francisco General Hospital Medical Center
OF NEUROLOGICAL DISEASE
2002 ANNUAL REPORT
by The J. David Gladstone Institutes
All rights reserved
Editors: Gary Howard and Stephen Ordway
Designers: John C. W. Carroll and John Hull
Photographers: Stephen Gonzales and Christopher Goodfellow
Project Coordinator: Sylvia A. Richmond
THE J. DAVID GLADSTONE INSTITUTES
P.O. Box 419100, San Francisco, CA 94141-9100
Telephone (415) 826-7500 • Facsimile (415) 826-6541
TABLE OF CONTENTS
DESCRIPTION OF THE INSTITUTES..........7
MEMBERS OF THE INSTITUTE..........13
REPORTS FROM THE LABORATORIES..........15
Behavioral Core Laboratory..........40
Gladstone Genomics Core..........43
EDUCATION AND COMMUNITY OUTREACH..........46
LENNART MUCKE, M.D.
Neurodegenerative disorders rob people ating a protein fragment that readily adopts a disease-associated con-
of their ability to remember, speak, write, formation. Longitudinal monitoring of neurons in culture with the
ambulate, and control their lives. These robotic microscope allowed the investigators to demonstrate that a
conditions are on the rise because people protein kinase, Akt, promotes neuronal survival much more potent-
are living longer, and aging strongly ly than had been appreciated from initial reports that measured sur-
increases the risk of being afflicted by vival at single time points. The potency of Akt makes it an especial-
these conditions. The enormous cost of ly attractive therapeutic target to block neurodegeneration.
caring for individuals with these condi- The laboratory of Dr. Fen-Biao Gao focuses on the genes and molec-
tions threatens our health care system. A ular pathways that regulate the development and maintenance of
medical breakthrough is clearly needed, and the surest way to such neuronal dendrites. Dendrites are tree-like extensions of neurons that
a breakthrough is to determine exactly how these diseases result in receive signals and participate in information processing and stor-
the dysfunction and degeneration of nerve cells. In addition, neuro- age. These structures account for more than 90% of the surface of
logical diseases raise a range of fascinating questions that are of fun- some neurons. In many neurological disorders, including
damental scientific interest. While the investigation of neurological Alzheimer’s disease (AD) and fragile X syndrome, the number of
diseases has promoted basic neuroscientific discoveries for over a dendritic branches is altered. However, little is known about the
century, there has never been a more promising and exciting con- mechanisms that control dendritic branching in vivo. Last year, Dr.
vergence of basic and disease-related neuroscience than now. Gao’s laboratory selectively mutated genes in individual neurons
Investigators at the Gladstone Institute of Neurological Disease and studied the consequences of this manipulation on dendritic
(GIND) have continued to examine the possibility that many, if not branching in living fruit flies. This year, the investigators used this
all, neurodegenerative disorders are caused by the accumulation of approach to study the pathogenesis of fragile X syndrome, the most
proteins that have assumed pathogenic conformations. Although dif- common form of inherited mental retardation in humans. The syn-
ferent proteins accumulate in different neurodegenerative disorders, drome is caused by mutations in the fragile X mental retardation 1
the ways in which their neurotoxic assemblies damage nerve cells (fmr1) gene. Their studies revealed that a close homologue of the
may overlap. This possibility raises hope that it will be feasible to fmr1 gene is highly expressed in the fruit fly nervous system, where
develop treatments that can prevent, stall, or even reverse more than the protein is localized to both dendrites and axons of specific neu-
one of these conditions. Other studies have focused on the molecu- rons. Studies are under way to determine whether and how muta-
lar mechanisms of neural plasticity, which is critical for the devel- tions in the fly gene alter the function and integrity of these neuronal
opment of the nervous system as well as for its adaptations to envi- branches. Dr. Gao and his associates also set up automated behav-
ronmental stimuli and for the remodeling of its circuitry after injury. ioral tests to quantify the functional consequences of disrupting gene
expression in the fruit fly nervous system. Using such tests, they dis-
covered that mutations in the presenilin gene alter the linear loco-
The laboratory of Dr. Steven Finkbeiner studies how a genetic muta- motion of fly larvae. Presenilin is a major drug target in AD research
tion leads to Huntington’s disease and how the nervous system because it is necessary for the production of the neurotoxic amyloid-
adapts to brief experiences by making long-lasting changes in its β peptides (Aβ), which are presumed to cause AD. Dr. Gao’s further
structure and function. Huntington’s disease is a fatal inherited neu- analysis of presenilin in the fruit fly may identify ways to inhibit its
rodegenerative disorder that is associated with increasingly disrup- AD-promoting activities while preserving its beneficial functions.
tive involuntary movements and a progressive loss of motor control.
It is caused by abnormal polyglutamine expansions in the protein The laboratory of Dr. Yadong Huang investigates the lipid carrier
huntingtin, which affect protein folding. Last year, Dr. Finkbeiner apolipoprotein (apo) E and its relationship to AD. The three major
developed a robotic microscope for the large-scale analysis of neu- human isoforms of apoE differentially affect the risk of developing
ronal cell cultures expressing different forms of huntingtin or relat- AD (E4 > E3 > E2). Roughly 10 years after the discovery of this
ed proteins. This year, he used this powerful new tool to reveal that link, apoE4 remains the main known inherited risk factor for the
mutant huntingtin causes a defect in the formation and maintenance most frequent form of AD. Yet, it is still uncertain how exactly it
of the branches (neurites) through which brain cells communicate increases the risk and accelerates the onset of this illness. Last year,
with each other. These new findings further validate the cell culture Dr. Huang and his collaborators demonstrated that apoE4 has a
model Dr. Finkbeiner uses in his studies, since abnormalities in neu- greater proclivity to be broken down into fragments than apoE3,
rite extension and regeneration are also early pathological features both in people with AD and in genetically modified (transgenic)
of Huntington’s disease. Using an antibody they developed, Dr. mice expressing human apoE in the brain. The accumulation of
Finkbeiner and his associates also found that cleavage within a spe- apoE4 fragments was associated with abnormal phosphorylation of
cific domain of mutant huntingtin promotes pathogenesis by gener- tau and cytoskeletal derangements. Abnormally phosphorylated tau
is the major constituent of neurofibrillary tangles, a pathological
4 Director’s Report
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE
hallmark of AD. This year, Dr. Huang demonstrated that the deficits in our mouse models, they constitute reliable molecular indi-
increased fragmentation of apoE is dependent, at least in part, on an cators of clinically relevant deficits and, hence, could improve the
intramolecular domain interaction that occurs in apoE4 but not in assessment of novel AD treatments. We also found marked reduc-
apoE3. Furthermore, when expressed by themselves in the brains of tions in calcium-dependent proteins in granule cells of humans with
transgenic mice, the apoE fragments elicited neurodegenerative AD, although this neuronal population is relatively resistant to AD-
alterations in various brain regions, including regions involved in related cell death. This result demonstrates that neuronal populations
learning and memory. Inhibitors of the protease presumed to cleave resisting cell death in AD can still be drastically altered at the molec-
apoE within neurons might block the adverse effects of apoE4 and ular level. They also suggest that at least some of the neurological
thereby benefit the many apoE4 carriers suffering from or at risk for deficits seen in AD may not reflect loss of brain cells. This conclu-
developing AD. Therefore, the isolation and further characterization sion has important implications because lost neurons are still hard or
of this enzyme is an important goal of ongoing studies. impossible to replace, whereas molecular alterations in surviving
The laboratory of Dr. Robert W. Mahley maintains its long-term brain cells may be more readily reversible by pharmacological inter-
focus on the mechanisms underlying the pathogenic activities of ventions. We look forward to exploring this possibility further in the
apoE4 in AD and other conditions. ApoE3 promotes neurite out- next year.
growth, protects the nervous system against diverse injuries, and The laboratory of Dr. Robert E. Pitas has followed up on their dis-
facilitates regeneration of the nervous system after trauma. In con- covery of apoE-binding protein (EBP), a novel brain protein that
trast, apoE4 is not protective and, in many instances, even worsens belongs to a previously uncharacterized family of proteins with
the outcome of neural injuries. This year, Dr. Mahley and his asso- unknown function. The proteins are encoded by four homologous
ciates extended their investigation of the most common apoE iso- genes in humans and mice and by one related gene in the fruit fly.
forms to the production of Aβ peptides. They examined the effects The association of EBP with apoE3, initially identified by the yeast
of apoE3 and apoE4 on the processing of the amyloid precursor two-hybrid approach, has now been confirmed in other biochemical
protein (APP) and on Aβ production in cultures of neuronal cells. assays. Dr. Pitas and his associates have also produced evidence that
Both apoE isoforms stimulated Aβ production, but apoE4 did so to EBP interacts with the cytoskeleton and have developed polyclonal
a significantly greater extent than apoE3. Interestingly, this differ- antibodies that detect both human and mouse EBP. Staining of
ential effect of the apoE isoforms was mediated by stimulating cell- mouse brain sections with these antibodies revealed neuron-specific
surface APP recycling rather than by altering cellular cholesterol expression of EBP in many regions, including the hippocampus and
content or activities of enzymes involved in Aβ production. When dentate gyrus. Ablating the expression of apoE or expressing human
the investigators disrupted the intramolecular domain interaction APP and Aβ in neurons altered the expression pattern of EBP in
that occurs in apoE4, but not in apoE3, the differential effect of mouse brains, suggesting that the expression of EBP is affected by
apoE3 and apoE4 on Aβ production was attenuated, suggesting that factors critically involved in the pathogenesis of AD. Interestingly,
this effect involves conformational differences among the apoE in cell cultures, EBP protected neuronal cells against oxidative
variants. Since Aβ plays a central role in the pathogenesis of AD, stress. This neuroprotective function of EBP may relate to its inter-
the stronger stimulatory effect of apoE4 on Aβ production could action with apoE, which also appears to fulfill neuroprotective func-
contribute to the increased AD risk associated with inheritance of tions. Studies are under way to ablate EBP expression in fruit flies
this apoE isoform. In addition, apoE4 may increase AD risk through and mice to determine the physiological functions of EBP and the
Aβ-independent mechanisms, as demonstrated in collaborative roles it might play in neurological disease.
studies with Dr. Huang (see above). The laboratory of Dr. Karl H. Weisgraber investigates the relation-
My own laboratory has continued to investigate the molecular path- ship between the three-dimensional structure of different apoE mol-
ways that link genetic determinants or risk factors of AD to neu- ecules and their normal and pathological activities. While much is
rodegeneration and cognitive decline. We have demonstrated in known about the structure of lipid-free apoE, very little is known
transgenic mouse models that human Aβ causes reductions in calci- about apoE’s structure when it is bound to lipids, although apoE
um-dependent proteins in granule cells of the dentate gyrus, a neu- probably exists in the body primarily in a lipid-bound (lipidated)
ronal population critically involved in learning and memory. Our state. This year, Dr. Weisgraber and his coworkers have made great
data suggest that the reductions in calcium-dependent proteins are strides in this area by producing crystals of lipidated apoE whose
caused by small soluble neurotoxic Aβ assemblies rather than by the structure can now be investigated by crystallography and other bio-
large deposits of Aβ that make up amyloid plaques. Notably, plaque physical approaches. In other studies, the investigators discovered
burden remains the most widely used pathological endpoint measure that apoE4 is more likely than apoE3 to assume a particular folding
in the preclinical assessment of AD treatments even though it often state (molten globule) that could promote its binding to and translo-
does not correlate well with cognitive deficits. Since the molecular cation across membranes. By assuming this folding state, apoE4
alterations we identified correlated extremely well with learning might escape from vesicular compartments within the cell and gain
Director’s Report 5
2002 ANNUAL REPORT
access to the cytoskeleton and to enzymes and other molecules in the Klingenstein Fellowship Award in Neuroscience from the
cytoplasm. Collaborative studies are under way to determine Klingenstein Foundation, which supports new approaches to neuro-
whether this process contributes to the cytoskeletal destabilization scientific research. My own research efforts on AD were recognized
and susceptibility to proteolytic cleavage that Drs. Huang and by a Metropolitan Life Foundation Award for Medical Research and
Mahley have found to be associated with apoE4. a MERIT award from the National Institute on Aging. Any award
reflects the participation of many, and I consider all members of my
Education, Special Initiatives, and Recognition
laboratory and my collaborators important contributors. I thank them
The Gladstone Institutes and UCSF provide state-of-the-art research for their tremendous loyalty and support. We all share these awards
facilities and a highly interactive academic environment that is ideal together.
for training in neuroscience and biomedical research. GIND investi-
gators participate actively in the training of students and residents Dr. Tony Wyss-Coray, a member of the GIND since its inauguration,
from various UCSF departments and interdepartmental programs, has been recruited to Stanford University, where he now holds an
including the Departments of Neurology and Physiology, the appointment as assistant professor in the Department of Neurology
Neuroscience Program, the Biomedical Sciences Program, the and Neurological Sciences. The staff research investigator position
Pharmaceutical Sciences and Pharmacogenomics Program, and the he held at the GIND aims to give exceptional young scientists the
Medical Scientist Training Program, as well as from graduate and opportunity to focus solely on developing a successful and creative
undergraduate programs at UC Berkeley and other institutions. research program before accepting a standard faculty position. Dr.
Wyss-Coray made the most of this opportunity and left the institute
Several members of our institute have collaborated to make our well published and supported by a substantial amount of independ-
training environment even more inspiring and rewarding for stu- ent NIH funding. We wish him and his terrific group the very best in
dents of all biomedical disciplines. For example, Dr. Finkbeiner has all their future endeavors and look forward to welcoming the next
developed a curriculum for the neurology house staff called the outstanding candidate into the position he vacated. We are equally
Neuroscience of Disease Seminar series. He has also served as the excited about the recruitment of a new faculty member to the GIND.
neuroscience liaison for UCSF’s Brain Interest Program, which The search for suitable candidates was launched in the fall and will
aims to foster interest in the brain and provides an opportunity for be conducted by an eight-member joint UCSF/Gladstone committee.
students to learn about different faculty, their careers, and their This faculty recruitment represents the first step toward expanding
interests. Also noteworthy in this context is the continued success the GIND as we prepare for the relocation of our research program
of our weekly GIND seminar series, which is organized by Dr. Gao. to UCSF’s new Mission Bay campus.
These well-attended seminars provide a stimulating forum for edu-
cation in disease-related neuroscience and for scientific exchange Gladstone investigators have continued to participate actively in
among members of the institutes and colleagues from the greater finalizing the design of the new research facility that will accommo-
UCSF community. date all three Gladstone Institutes at Mission Bay. The GIND will
occupy the third floor of the new Gladstone building, which is
GIND investigators have extended their efforts to promote education scheduled for completion in 2004. The consolidation of our labora-
and scientific exchange in disease-related neuroscience far beyond tories, cores, and animal care facilities into one building at the new
the boundaries of our institute. They have organized and participat- campus will further enhance our ability to contribute to research and
ed in a number of national and international conferences that have training in neurology and neuroscience.
advanced our field of research in various ways. Despite their busy
schedules and expanding research efforts, GIND members have con- In closing, I would like to thank the participants of this year’s
tinued to devote time to community outreach. As described in the Scientific Advisory Board meeting, Drs. Dale E. Bredesen (Buck
Education and Community Outreach section of this report, these Institute for Age Research), Dennis Selkoe (Harvard Medical
efforts included participation in activities aimed at educating the School), and Eric Shooter (Stanford University) for their outstand-
public about AD and neuroscientific research in general. Our syner- ing input. It contributed greatly to our success. The progress we
gism with the UCSF Memory and Aging Center, the local chapter of made in 2002 reflects the work of all the researchers at the GIND
the Alzheimer’s Association, and the Hereditary Disease Foundation and of the administrative staff at the Gladstone Institutes. As out-
has allowed us to maintain and expand fruitful links between our lined in this report, we have shed new light on molecular processes
research and the patients afflicted by the diseases we study. causing devastating neurological diseases as well as on mechanisms
that might be utilized to prevent and cure them.
I am delighted that our accomplishments have not gone unnoticed,
as reflected by the honors and awards institute members received
this year. Dr. Gao received a McKnight Neuroscience of Brain
Disorders Award from the McKnight Endowment Fund for
Neurosciences, one of six awards to scientists whose research is
directed toward finding new ways to diagnose, treat, and cure disor- Lennart Mucke, M.D.
ders of the brain and central nervous system. He also received a Director
6 Director’s Report
Description of Trustees Executive Director
Richard S. Brawerman Richard Hille
THE J. DAVID GLADSTONE Albert A. Dorman Chief Financial Officer
Richard D. Jones
INSTITUTES Hal Orr, C.M.A.
Robert W. Mahley, M.D., Ph.D.
The J. David Gladstone Institutes is the product of the wisdom and
rimary research efforts at the J. David Gladstone Institutes
focus on three of the most important clinical problems of mod- hard work of many individuals. The first was J. David Gladstone him-
ern times: cardiovascular disease, AIDS, and neurodegenera- self, a Los Angeles real-estate entrepreneur. Others are the trustees.
tive disorders. Cardiovascular disease, the nation’s leading killer, The original trustees, all of whom had known or worked closely with
claims the lives of over one million Americans each year. Despite Mr. Gladstone, were Richard S. Brawerman, his attorney and execu-
more effective treatments, AIDS remains a leading cause of death in tor of his estate; Richard D. Jones, his real-estate attorney; and David
the United States. Worldwide, more than 42 million people are living Orgell, his cousin and confidant. When Mr. Orgell died in 1987, he
with HIV/AIDS, and more than 21 million have died as a direct result was succeeded on the board by Albert A. Dorman, a southern
of HIV infection. Alzheimer’s disease, the most recent focus of inves- California executive with experience in managing large organizations.
tigation by Gladstone scientists, robs people of their ability to think, At the time of Mr. Gladstone’s death in 1971, the southern California
remember, and control their lives. It currently affects over 4 million real-estate market was just beginning to flourish. His estate, left almost
people in the United States alone and is predicted to affect 14 million entirely for medical education and research, was relatively modest by
Americans by 2050. The impact of these diseases on world health later standards. However, the trustees recognized the estate’s potential
infuses Gladstone scientists with a sense of purpose and urgency. for growth and, through their inspired management, increased its worth
Gladstone is composed of three institutes, each of which issues its own severalfold within the first decade. From the beginning, the decisions
annual report. The Gladstone Institute of Cardiovascular Disease of the trustees had profound and positive effects on the research organ-
(GICD), which opened in 1979, focuses on atherosclerosis and its ization that evolved. They continue to manage and enlarge the assets of
complications. In 1992, the Gladstone Institute of Virology and the J. David Gladstone Institutes and to oversee their use.
Immunology (GIVI) was established to study HIV, the causative agent Gladstone Institute of Cardiovascular Disease
of AIDS. The 1993 discovery that apolipoprotein (apo) E—long stud-
Close ties already existed between the UCSF School of Medicine and
ied at GICD for its role in heart disease—plays a role in Alzheimer’s
SFGH, the hospital of the City and County of San Francisco, when the
disease as well led to the establishment of the Gladstone Institute of
trustees leased vacant space from the City in 1977 in which to create
Neurological Disease (GIND) in 1998. The three institutes are located
laboratories and offices. The partnership has flourished. Gladstone
at the San Francisco General Hospital (SFGH) campus of the
scientists collaborate with their colleagues at UCSF and SFGH and
University of California, San Francisco (UCSF), and will soon move
provide service to those organizations as professors and staff physi-
into a new facility at UCSF’s Mission Bay campus. While independ-
cians. The mutually beneficial association between the Gladstone,
ent, Gladstone is formally affiliated with UCSF, and Gladstone inves-
UCSF, and SFGH has created a productive and supportive environ-
tigators hold university appointments and participate in many univer-
ment in which scientists conduct basic research while availing them-
sity activities, including the teaching and training of graduate students.
selves of clinical and academic opportunities.
Although autonomous in their areas of specialization, the institutes
To choose a director for the developing research facility, the trustees
share a common approach. Each institute is organized into research
sought guidance from the scientific community. The choice was
units consisting of scientists, postdoctoral researchers, research asso-
Robert W. Mahley, M.D., Ph.D. At the time of Mr. Gladstone’s death,
ciates, and students. This structure is designed to accommodate small
he was just completing his internship. However, by 1979, when he
groups of scientists who work together closely but who also benefit
was appointed director, Dr. Mahley had established himself as a lead-
from collegial interactions with other research groups. Collaborations
ing researcher in the field of lipoprotein metabolism and atherosclero-
among staff members with various areas of expertise create a stimu-
sis. He came to the Gladstone from the National Institutes of Health,
lating environment that fortifies the scientific lifeblood of the organ-
where he headed the Laboratory of Experimental Atherosclerosis.
Less than a year after his appointment, Dr. Mahley had assembled a
Each institute receives expert input on the progress of its science from staff of 25, and the new organization, then called the Gladstone
an advisory board of distinguished scientists. The scientific advisory Foundation Laboratories for Cardiovascular Disease, officially
boards provide a twofold service in reviewing the quality of the opened on September 1, 1979. Dr. Mahley is professor of pathology
research and in advising the president, directors, and trustees. and medicine at UCSF and is a member of the National Academy of
The work of the scientific staff at all three institutes also extends Sciences and the Institute of Medicine.
beyond the laboratory to the wider community. The mission of the By the end of 2002, the research staff of the GICD had grown to more
institutes includes the education of graduate and medical students, than 100 scientists, postdoctoral fellows, students, and research asso-
postdoctoral fellows, and visiting scientists; specialized training for ciates, occupying about 48,000 square feet of laboratory and office
practicing physicians; and educational outreach to the local and space in buildings 9 and 40 on the SFGH campus. In 22 years of oper-
extended community. ation, the institute has attained an international reputation for excel-
2002 ANNUAL REPORT
are John M. Taylor, Ph.D., Stephen G. Young, M.D., and Robert V.
Gladstone Institute of Cardiovascular Disease Farese, Jr., M.D.
Vascular Biology. This research aims to elucidate how mono-
Scientific Advisory Board cytes/macrophages are attracted to sites of atherosclerotic lesion
Göran K. Hansson, M.D., Ph.D. formation and to delineate the role of platelets in forming the occlu-
Professor of Cardiovascular Research sive thrombus that leads to myocardial infarction. Another research
Center for Molecular Medicine goal is to elucidate cell-signaling pathways that can be used to confer
Karolinska Institute, Karolinska Hospital proliferative advantages to genetically modified cells. The investiga-
tors in this unit are Israel F. Charo, M.D., Ph.D., and Bruce R.
Joachim J. A. Herz, M.D.
Professor of Molecular Genetics Conklin, M.D.
University of Texas Southwestern Medical Center Clinical Molecular Genetics. Patient studies and national and inter-
national population screening projects conducted by Gladstone
Aldons J. Lusis, Ph.D.
Professor of Medicine researchers aim to identify unique genetic abnormalities that cause
and of Microbiology and Molecular Genetics hypercholesterolemia and premature myocardial infarction.
University of California, Los Angeles Researchers in this unit operate the Lipid Disorders Training Center,
which trains medical personnel to manage dyslipidemic patients,
Karen Reue, Ph.D. and the Lipid Clinic, which provides consultation on disease man-
West Los Angeles Veterans Administration Medical Center agement to SFGH patients and to private, referring physicians. This
Associate Professor of Medicine unit also conducts the Turkish Heart Study, which investigates car-
University of California, Los Angeles diovascular risk factors in a developing nation with a high incidence
of heart disease. The investigators in this unit are Dr. Mahley and
Donald M. Small, M.D. Thomas P. Bersot, M.D., Ph.D.
Chairman, Department of Biophysics
Professor of Biophysics, Medicine and Biochemistry Gladstone Genomics Core. The Genomics Core assists scientists
Boston University School of Medicine with the unprecedented research opportunities presented by the
decoding of the mouse and human genomes. Directed by
Daniel Steinberg, M.D., Ph.D. Christopher S. Barker, Ph.D., this laboratory provides state-of-the-
Professor Emeritus, Department of Medicine
University of California at San Diego art technologies in the area of functional genomics for Gladstone
scientists and other investigators at SFGH. The core focuses on
Alan R. Tall, M.D. DNA microarray technology, including the preparation of custom
Professor of Medicine oligonucleotide microarrays and customized microarray hybridiza-
Columbia University College of Physicians and Surgeons tion, array scanning, and data analysis.
Gladstone Transgenic Core. The GICD also maintains a sophisti-
cated core facility for the generation of transgenic mice that is
lence. Its productivity is documented in the more than 900 scientific heavily used by investigators of all three institutes. The core’s
papers published by GICD scientists. activities are coordinated by Dr. Taylor.
Research at the GICD is conducted in five areas and is supported by Gladstone Microscopy Core. The Microscopy Core, under the
three core laboratories. direction of Dr. Young, provides expertise, instrumentation, service,
Lipoprotein Biochemistry and Metabolism. A major focus of and training for the generation and capture of research data in the
research in this area is to correlate the structure and function of the form of microscopic images and for the quantitation, analysis, and
apolipoproteins involved in cholesterol transport, with particular interpretation of those images to all three institutes.
emphasis on apoE. One of the structural tools that scientists in this
Gladstone Institute of Virology and Immunology
unit use is x-ray crystallography to determine the three-dimensional
structures of proteins. The investigators in this unit are Dr. Mahley The GIVI resulted from the convergence of several factors. On the
and Karl H. Weisgraber, Ph.D. forefront of the battle against AIDS since the beginning of the pan-
demic, SFGH is widely recognized as one of the world’s leading
Cell Biology. Studies in this unit examine how the body’s various clinical research centers for the study of HIV disease. The State of
cells regulate the storage and use of cholesterol as it relates to the California provided funding to build an AIDS research center at
development of atherosclerosis. The focus is on the roles of apoB, SFGH under the auspices of the UCSF School of Medicine.
apoE, and class A scavenger receptors in cellular cholesterol metab- Additional funds were needed to finish and equip the center and to
olism and atherogenesis. The investigators in this unit are Robert E. undertake the research. The success of the established relationship
Pitas, Ph.D., and Yadong Huang, M.D., Ph.D. of UCSF and the City with the Gladstone formed the foundation for
Molecular Biology. Scientists in this unit apply the latest DNA tech- a unique agreement by which the Gladstone would lease the center,
niques to understand the regulation of genes important in controlling establish the research program, and manage the ongoing studies.
cholesterol, triglycerides, and apolipoprotein production. Studies Gladstone and UCSF were able to attract an outstanding physician-
focus on apoE and apoB, which mediate the interaction of lipopro- scientist to direct the new institute. Warner C. Greene, M.D., Ph.D.,
teins with cell-surface receptors. Enzymes controlling cholesteryl an internationally recognized immunologist and virologist, official-
ester and triglyceride production represent a new area of research. ly took the helm in September 1991. Before coming to Gladstone,
This has led to studies of adipose tissue metabolism and obesity. In Dr. Greene was professor of medicine and investigator in the
addition, transgenes and homologous recombination are used to cre- Howard Hughes Medical Institute at Duke University Medical
ate animal models of human diseases. The investigators in this unit Center. Currently, Dr. Greene is also a professor of medicine and of
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE
microbiology and immunology at UCSF, co-director of the UCSF
Center for AIDS Research, and a member of the executive commit- Gladstone Institute of Virology and Immunology
tees of the UCSF AIDS Research Institute and UCSF Biomedical
Sciences Graduate Program. Scientific Advisory Board
Formally dedicated on April 19, 1993, the GIVI occupies 27,000 Elizabeth H. Blackburn, Ph.D.
square feet of space on the top two floors of SFGH’s building 3. Professor of Biochemistry and Biophysics
Studies at the GIVI are conducted under the direction of an out- University of California, San Francisco
standing group of physician-scientists in five state-of-the-art labora-
tories and three supporting laboratories. Robert C. Gallo, M.D.
Director, Institute of Human Virology
Laboratory of Molecular Immunology. The Laboratory of Professor of Medicine, Microbiology and Immunology
Molecular Immunology studies the mechanisms by which proteins University of Maryland at Baltimore
within the immune cells harboring HIV may act to trigger the growth
of the virus and how the virus’s own proteins subsequently amplify its Beatrice H. Hahn, M.D.
Professor of Medicine and Microbiology
replication and pathogenic effects in primary T cells and macrophages. University of Alabama at Birmingham
This work, headed by the director of the institute, Dr. Greene, specifi-
cally focuses on the HIV proteins Vpr and Nef and select host factors, Edward W. Holmes, M.D.
including the NF-κB/Rel family of transcription factors. Vice Chancellor for Health Sciences
Dean, School of Medicine
Laboratory of Molecular Evolution. The Laboratory of Molecular University of California, San Diego
Evolution focuses on evolution and its implications for medicine and
epidemiology. Genetic variations in host susceptibility and in micro- Stanley J. Korsmeyer, M.D.
bial replication capacity, virulence, and drug susceptibility typically Sidney Farber Professor of Pathology
and Professor of Medicine
determine who develops disease and who remains healthy. The lab- Harvard Medical School
oratory examines several consequences of molecular evolution, Director, Program in Molecular Oncology
including HIV-1 drug resistance, selection pressures bearing on Department of Cancer Immunology and AIDS
HIV-1 populations during transmission and in tissues, and nonpath- Dana-Farber Cancer Institute
ogenic simian immunodeficiency virus infection in natural host
Joseph R. Nevins, Ph.D.
species. This laboratory is directed by Robert M. Grant, M.D.,
James B. Duke Professor and Chairman
M.P.H., an assistant investigator in GIVI and assistant professor of Department of Genetics
medicine at UCSF. Duke University Medical Center
Laboratory of Viral Pathogenesis. The Laboratory of Viral
Thomas A. Waldmann, M.D.
Pathogenesis focuses on the pathogenic mechanisms of HIV in vivo, Chief, Metabolism Branch
with the specific intent of finding better ways to prevent or suppress National Cancer Institute
HIV-induced disease. The work falls into two areas: effects of HIV National Institutes of Health
on the central hematopoietic system and transmission of HIV across
mucosal and placental barriers. Research in this laboratory is direct- Robin A. Weiss, Ph.D.
Professor of Viral Oncology
ed by Joseph M. McCune, M.D., Ph.D., a senior investigator in GIVI Wohl Virion Centre
and professor of medicine at UCSF. Windeyer Institute of Medical Sciences
Laboratory of Molecular Virology. The Laboratory of Molecular University College London
Virology studies how HIV transcription is controlled by host chro-
matin structure and by viral proteins such as HIV Tat. More recent-
ly, this laboratory has also investigated the molecular basis for HIV- Gladstone-UCSF Laboratory of Clinical Virology. The
induced T-cell death, focusing on the role of apoptosis. This labora- Gladstone-UCSF Laboratory of Clinical Virology, directed by Dr.
tory is directed by Eric M. Verdin, M.D., a senior investigator in Grant, provides key virological testing in support of HIV-related
GIVI and professor of medicine at UCSF. clinical research projects at UCSF. Established in collaboration with
Laboratory of Cellular Immunology. The Laboratory of Cellular the UCSF AIDS Research Institute, this laboratory evaluates
Immunology investigates innate and adaptive cellular immune patients who are failing combination antiviral therapy, studies HIV
responses against HIV and simian immunodeficiency virus at replication in the central nervous system, and investigates mecha-
mucosal and systemic sites. The work focuses on understanding host nisms of primary HIV infection and sexual transmission. This labo-
immune/pathogen interactions that might be manipulated by vacci- ratory is also developing state-of-the-art assays for genotypic and
nation or therapeutic drugs. The laboratory is headed by Douglas F. phenotypic drug resistance and assessment of viral loads using ultra-
Nixon, M.D., Ph.D., an associate investigator in GIVI and associate sensitive techniques to further enhance clinical AIDS research at
professor of medicine at UCSF. SFGH and UCSF.
Flow Cytometry Core Laboratory. The Flow Cytometry Core Antiviral Drug Research Division. The Antiviral Drug Research
Laboratory is dedicated to providing cutting-edge techniques in Division evaluates potential new antiviral drugs. Using an animal
fluorescence-based cell sorting and analysis to Gladstone and model system, the SCID-hu mouse, the group is developing new
UCSF scientists. This laboratory operates both a Becton-Dickinson methods for drug evaluation and extending its work to the field of
FACS Vantage and FACS DiVa for sorting and a FACScan for viral pathogenesis. The laboratory is directed by Cheryl A. Stoddart,
analysis. The laboratory is directed by Martin Bigos, M.S., a staff Ph.D., a staff research scientist in GIVI.
research scientist in GIVI.
2002 ANNUAL REPORT
Gladstone Institute of Neurological Disease
The GIND resulted from the natural expansion of highly successful Gladstone Institute of Neurological Disease
research programs. Its predecessor, the Gladstone Molecular
Scientific Advisory Board
Neurobiology Program, was created in 1996 as a joint venture of the
Gladstone Institutes and the UCSF Department of Neurology. Lennart Dale E. Bredesen, M.D.
Mucke, M.D., recruited to head the new program, brought with him a President and CEO
group of researchers with expertise in diverse areas of disease-related Buck Institute for Age Research
neuroscience. With its establishment, neuroscientists in the new pro- Professor of Neurology
University of California, San Francisco
gram could leverage Gladstone’s wealth of experience with apoE by
applying it to the field of neurodegenerative disease research. These Gerald D. Fischbach, M.D.
efforts were complemented with research on amyloid proteins, which Dean of Faculty of Medicine and for Health Sciences
play a seminal role in Alzheimer’s disease. Harold and Margaret Hatch Professor
Significant findings were rapidly made in a broad range of research
areas, including molecular biology, cell biology, physical structure, Dennis J. Selkoe, M.D.
signal transduction, experimental pathology, and behavioral neurobi- Co-Director, Center for Neurologic Diseases
ology. In 1998, the trustees expanded the program to create the GIND. Brigham and Women’s Hospital
Its goal is to advance the understanding of the nervous system to the Professor of Neurology and Neuroscience
point where rational strategies can be developed for the treatment and
prevention of Alzheimer’s disease, cerebrovascular disease, and other Eric M. Shooter, Ph.D.
major neurological conditions. Productive synergies exist with scien- Professor of Neurobiology
tists studying cardiovascular disease and AIDS in the other institutes. Stanford University
As was the case with the two other institutes, Gladstone and UCSF Sidney Strickland, Ph.D.
were able to attract an outstanding physician-scientist to direct the Dean of Educational Affairs
new institute. Dr. Mucke was educated at the Max-Planck-Institute for Professor of Neurobiology and Genetics
Biophysical Chemistry in Germany, the Massachusetts General Rockefeller University
Hospital, and Harvard Medical School. He came to Gladstone from
Marc Tessier-Lavigne, Ph.D.
The Scripps Research Institute to first expand Gladstone’s research Susan B. Ford Professor of Humanities and Sciences
efforts in disease-oriented neuroscience in the context of the Professor of Biological Sciences
Molecular Neurobiology Program and then to direct the new institute. Stanford University
Dr. Mucke teaches neurology and neuroscience at UCSF, where he is
the first holder of the Joseph B. Martin Distinguished Professorship in
Neuroscience. treatments for Alzheimer’s disease and other neurological conditions.
The GIND was formally dedicated on September 11, 1998. Its labora- Investigators involved in research on this topic are Drs. Mahley,
tories are housed in buildings 1, 9, and 40 of the SFGH campus. Weisgraber, Huang, Mucke, and Robert E. Pitas, Ph.D.
Studies at the GIND are conducted in eight state-of-the-art laborato- Huntingtin and Other Polyglutamine-Repeat Proteins.
ries and a behavioral core laboratory. The research focuses on six Huntington’s disease, the most common inherited neurodegenerative
major areas relating to neurodegenerative disorders, cognitive func- disorder, is caused by an abnormally long stretch of the amino acid
tion, and brain inflammation as outlined below. glutamine within the protein called huntingtin. Abnormal polygluta-
Physiological and Pathophysiological Roles of Amyloidogenic mine stretches within other proteins are responsible for several other
Proteins in the Brain. While amyloidogenic molecules, such as the midlife neurodegenerative disorders. Determining how abnormal
amyloid β protein precursor and α-synuclein, may normally facilitate polyglutamine stretches cause neurons to die may make it possible
learning and memory, they can be broken down into peptides or to develop specific therapies for these disorders. It may also reveal
altered in their conformation to form neurotoxic aggregates in cells general mechanisms of neurodegeneration that are relevant to other
and tissues. Understanding how these toxic proteins form and act neurological diseases. This topic is a major focus of Steven M.
could facilitate the design of better treatments for Alzheimer’s disease Finkbeiner, Ph.D.
and other neurodegenerative disorders. Defining the normal function
Neural Plasticity. Plasticity is a property of the nervous system that
of the amyloidogenic precursor molecules is of fundamental neurosci-
enables it to undergo long-lasting, sometimes permanent adaptive
entific interest. Investigators involved in research on this topic are Dr.
responses to brief stimuli. Plasticity is believed to be important for
Mucke, Robert W. Mahley, M.D., Ph.D., Yadong Huang, M.D., Ph.D.,
establishing precise patterns of synaptic connections during early neu-
and Karl H. Weisgraber, Ph.D.
ronal development and for learning and memory in adults.
Role of ApoE in Neurodegeneration and Cognitive Impairment. Disturbances in plasticity and synaptic function could contribute sig-
The apoE4 allele is the main known genetic risk factor for the most nificantly to memory disorders characteristic of many neurodegenera-
common form of Alzheimer’s disease and for poor neurological out- tive diseases, such as Alzheimer’s disease and Huntington’s disease.
come after head injury. Defining the effects of the three main human An understanding of the molecular mechanisms that regulate the for-
apoE isoforms (E2, E3, and E4) on the structure and function of the mation, activity, degeneration, and regeneration of synapses and neu-
brain should provide crucial insights into the contribution of the ronal dendrites could form the basis for therapeutic strategies to pre-
apoE4 variant to neurological disease. Characterizing how changes in vent memory loss and cognitive decline in diverse diseases.
the x-ray crystallographic three-dimensional structure of apoE affect Investigators involved in research on this topic include Drs.
its activity may result in the development of novel apoE-targeted drug Finkbeiner, Mucke, and Fen-Biao Gao, Ph.D.
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE
Neurobiological Function of Glial Cells and Their Role in Behavioral Core Laboratory. Because many of our mouse models
Neurological Disease. Glial cells are specialized brain cells that sup- are designed to simulate aspects of human diseases resulting in mem-
port the health and function of neurons. In response to brain injuries, ory deficits, behavioral disturbances, or movement disorders, the
these cells produce a large number of molecules that participate in detailed behavioral characterization of these models plays an important
inflammatory and immune responses. While acute glial responses may role in the assessment of their clinical relevance. Behavioral alterations
help prevent neuronal damage and facilitate the removal of toxic amy- in transgenic models can shed light on the central nervous system
loid proteins, abnormal activation of these cells could contribute to effects of diverse molecules and are used to assess novel therapeutic
neurological disease. Genetic and pharmacological strategies are used strategies at the preclinical level. This core is engaged in collaborative
to characterize the beneficial and detrimental roles of glial cells in studies with investigators at all three Gladstone Institutes, as well as
cerebral amyloidosis, neurodegeneration, and HIV-associated demen- with scientists in various UCSF departments and other institutions.
tia. Investigators involved in this research include Drs. Mucke and Pitas.
New Gladstone Laboratories at the UCSF Mission Bay Campus
onstruction of Gladstone’s new facility at Mission Bay is on
schedule. In late 2004, all three institutes will move to a new
six-story building, containing about 190,000 square feet of
space, adjacent to UCSF’s new basic science campus. The move is a
milestone for Gladstone. “This decision was an investment in the
future,” said Gladstone president Robert W. Mahley. “The move will
enhance our ability to fulfill our mission of contributing to the health
and well-being of all people.”
The programming and schematic design phases for the facility have
been completed, and the design development phase is proceeding on
schedule. The San Francisco Redevelopment Agency has approved
the schematic design. Utilities, lighting, trees, and sidewalks at the
Model showing Gladstone’s location at Mission Bay.
building site have been installed. Preparation of the site has begun,
and the steel for the building has been purchased. of downtown San Francisco and the bay. One main entrance will face
In conjunction with NBBJ Architects of San Francisco, Todd Sklar, the entrance to UCSF’s campus, and the other will open onto a land-
Gladstone’s project development and management consultant, has scaped plaza.
directed a team of more than 20 firms through a rigorous planning The new building will greatly enhance the research environment for
and review process. The review process has included significant Gladstone scientists. Having all three institutes under one roof will
input from the institute directors, investigators, and others. foster a sense of unity. Larger and more open laboratories will facil-
The first floor will hold the administrative offices and building oper- itate communication and collaborations among the researchers. The
ations, a 150-person lecture hall, and four seminar rooms. The sec- additional space will allow Gladstone to increase its staff of scientists
ond floor will be left as shell space for future expansion or may be and administrators from about 300 currently to more than 500 with-
developed and leased out temporarily. The third, fourth, and fifth in the next 7 years.
floors will house the offices and laboratories of the three institutes. The move will also strengthen the interactions between Gladstone
The plans provide for a pleasant work environment. The building’s and UCSF. The Gladstone building will be directly across the street
long and relatively narrow footprint will maximize the amount of from UCSF’s new basic science campus. “We foresee a constant
natural light in the offices and laboratories. Equipment areas will be exchange of ideas that will benefit researchers at both institutions,”
located in the center of each floor. Stairwells at the ends of the build- said Dr. Mahley. “There will be a synergy that will promote the
ing will have large windows to take advantage of the beautiful views progress of research and the advancement of our basic science.”
2002 ANNUAL REPORT
Michael Sechman Associates
Top: artist’s rendering of the new Gladstone laboratories at Mission Bay. Bottom: aerial view showing the location of UCSF’s new campus within the larger Mission Bay
redevelopment area. Asterisk indicates approximate location of Gladstone.
12 Mission Bay
Members of the Gladstone Institute of Neurological Disease
Director Jukka Puoliväli, Ph.D. Laboratory Associate
Lennart Mucke, M.D. Juan Santiago-Garcia, Ph.D. John A. Gray
Joseph B. Martin Distinguished Professor Miki Tamura, Ph.D.
of Neuroscience Kanyan Xu, Ph.D. Administrative Staff
Qin Xu, Ph.D. of the J. David Gladstone Institutes
Investigators Shi-Ming Ye, Ph.D.
Steven Finkbeiner, M.D., Ph.D. President
Assistant Professor of Neurology Robert W. Mahley, M.D., Ph.D.
and Physiology Brigitte Bogert
Sarah Carter Vice President
Fen-Biao Gao, Ph.D. Patrick Chang for Administrative Affairs
Assistant Professor of Neurology Ruomeng Dong
Jennifer Hsu Daniel M. Oshiro, M.S.
Peter Li Administrative Assistants
Robert W. Mahley, M.D., Ph.D.
Professor of Pathology and Medicine Catherine Massaro Catharine H. Evans
Siddhartha Mitra Karina G. Fantillo
Robert E. Pitas, Ph.D. Sean Pintchovski Mariena D. Gardner
Professor of Pathology Vikram Rao Leslie T. Manuntag
Kimmy Su Marlette A. Marasigan
Karl H. Weisgraber, Ph.D. Neal T. Sweeney Kelley S. Nelson
Professor of Pathology Amy Tang Jennifer L. Polizzotto
Brigitte Watkins Emily K. O’Keeffe
Staff Research Investigator
Senior Research Associates Executive Assistants
Yadong Huang, M.D., Ph.D.
Assistant Professor of Pathology Kay S. Arnold Brian Auerbach
Maureen E. Balestra Denise Murray McPherson
Staff Research Scientist Walter J. Brecht Sylvia A. Richmond
Zhong-Sheng Ji, Ph.D.
Christopher S. Barker, Ph.D. Yvonne M. Newhouse Communications
Gui-Qiu Yu, M.S. Daniel M. Oshiro, M.S., Officer
Susan H. Dan
Robert L. Raffaï, Ph.D. Research Associates Sylvia A. Richmond
Kimberly A. Scearce-Levie, Ph.D. Elizabeth S. Brooks, M.S. Teresa R. Roberts
Visiting Scientists Kenneth Cheung Editorial
Andrea J. Barczak Nhue L. Do Gary C. Howard, Ph.D.
Liming Dong, Ph.D. Kristina Hanspers, M.S. Stephen B. Ordway
Chandi Griffin, M.S. Yanxia Hao, M.S.
Graphics and Photography
Dionysos Slaga Gregor Hare
John C.W. Carroll, Manager
Tony Wyss-Coray, Ph.D. Faith M. Harris
Christopher A. Goodfellow
Postdoctoral Fellows Shyamal G. Kapadia
John R. Hull, M.F.A.
Lorenzo Arnaboldi, Ph.D. Lisa N. Kekonius
Montserrat Arrasate, Ph.D. Alan Lee Public Affairs
John Bradley, Ph.D. Sam Loeb Laura Lane, M.S., Manager
Shengjun Chang, Ph.D. Maya Mathew
Irene Cheng, Ph.D. Rene D. Miranda Finance and Accounting
Jeannie Chin, Ph.D. Hilda C. Ordanza
Marc E. Minardi, M.A., Officer
Luke A. Esposito, Ph.D. David S. Peterson
Richard S. Melenchuk, M.S., Manager
Christian Essrich, Ph.D. Belma Sadikovic
Emilia M. Franco
Danny M. Hatters, Ph.D. Kristina P. Shockley
Marga R. Guillén, M.P.A.
Paul C.R. Hopkins, Ph.D. Richard M. Stewart
Kai Yun W. Sun
Wenjun Li, Ph.D. Maryam A. Tabar
Kenneth J. Weiner
Jorge J. Palop, Ph.D. Chunyao Xia
Clare A. Peters-Libeu, Ph.D. Fengrong Yan
Members of the Institutet 13
2002 ANNUAL REPORT
Grants and Contracts Intellectual Property/ Purchasing
Rex F. Jones, Ph.D., Officer Technology Transfer P.J. Spangenburg, Manager
Frank T. Chargualaf, M.B.A. Joan V. Bruland, J.D., Officer Tyler G. Campos
Lynne M. Coulson Erin Madden P. Sidney Oduah
Martin C. Rios Anne Scott, M.S. Alberto L. Reynoso
Yvonne L. Young Benjamin V. Young
Office of the President
Human Resources Receptionist
Susan H. Dan
Migdalia Martinez, M.S., Officer Teresa R. Roberts Hope S. Williams
John R. LeViathan, M.A., Manager
Wendy M. Foster Operations Student Assistants
Chad E. Popham Deborah S. Addad, Officer Shannon P. Chi
Alyssa S. Uchimura Christina N. Luna
Information Services Vincent J. McGovern, M.S., Manager
Reginald L. Drakeford, Sr., Officer David R. Bourassa
Jon W. Kilcrease, Manager Randy A. Damron
Matthew L. Lyon Roger A. Shore
Sarah K. Mays
Joseph R. Solanoy
14 Members of the Institute
Behavioral Core Laboratory.........40
Gladstone Genomics Core.........43
Reports from the Laboratories 15
Assistant Investigator Students Research Associates
Steven Finkbeiner, M.D., Ph.D. Sarah Carter Elizabeth Brooks
Patrick Chang Kenneth Cheung
Postdoctoral Fellows Jennifer Hsu Shyamal Kapadia
Montserrat Arrasate, Ph.D. Peter Li
John Bradley, Ph.D. Siddhartha Mitra Administrative Assistant
Sean Pintchovski Kelley Nelson
Molecular Mechanisms of Plasticity and Neurodegeneration
Steven Finkbeiner, M.D., Ph.D.
eration in HD by manually examining immunocytochemically
ur laboratory is interested in two biological questions. First,
how does the nervous system adapt to brief experiences by stained neurons transfected with wildtype or disease-associated
making long-lasting changes in its structure and function? forms of huntingtin. This approach was painstaking and relatively
The molecular mechanisms that underlie this process, collectively insensitive. With the microscope, we can observe neurons as they
known as plasticity, are important for the proper development of the begin to express transfected huntingtin and then monitor them over
nervous system and for forming memories. We are especially inter- time, observing their survival, degeneration, and eventual death.
ested in the role of new gene expression in coupling transient neu- The new approach is 100-fold faster than our old assay, more sen-
ronal activity to long-term changes in synaptic function. The second sitive, and less susceptible to user bias.
question is how a genetic mutation leads to an adult-onset progres- We also designed the microscope and the controlling computer
sive neurodegenerative disease. We focus on Huntington’s disease programs to be able to return to the same field of neurons or the
(HD), the most common inherited neurodegenerative disorder in the same neuron after the tissue-culture plate has been removed from
United States. Unlike Alzheimer’s disease and Parkinson’s disease, the microscope stage. In fact, although the high-throughput capa-
the cause of HD is known. HD is caused by a mutation that leads to bilities of the microscope are very useful, the ability to follow cells
an abnormal polyglutamine expansion in the huntingtin protein. longitudinally may be the key capability that helps us unravel
Since HD shares some common pathological features with cause and effect mechanisms. By observing a neuron and the adap-
Parkinson’s disease and Alzheimer’s disease, we hope that, by tive or maladaptive changes that it undergoes over time, we can
understanding HD, we will understand the molecular mechanisms of reconstruct dynamic relationships that we lose by examining a sin-
neurodegeneration more broadly. gle snapshot. For example, longitudinal analysis of huntingtin-
Applications of Robotic Microscopy transfected neurons revealed a polyglutamine-expansion-depend-
Is it a cause or an effect? That question is often difficult to address ent defect in dendrite and axon formation (Figure 1A). Initially,
experimentally but it must be answered to establish biological or neurons transfected with either wildtype or mutant huntingtin
pathological mechanisms and to identify appropriate therapeutic extended neurites similarly; however, neurons transfected with
targets. The difficulty lies in understanding the true relationship mutant huntingtin eventually failed to extend neurites and then
between the outcome of a long-term dynamic process and a partic- retracted them before the neuron died. Dendritic dystrophy and
ular subsidiary change. The conundrum is especially common in axon regeneration are early pathological features of HD and so our
studies of pathogenesis in which multiple changes occur in paral- ability to recapitulate another feature of HD with our simple neu-
lel. Some changes are a direct consequence of an inciting event and ronal model adds to the validity of the model. We expect that lon-
mediate morbidity, others are beneficial coping responses, and still gitudinal analysis will be useful to trace pathogenic pathways
others are neither helpful nor harmful. The interrelatedness of backwards through time to elucidate critical inciting events.
many molecular and cellular processes can also make it challeng- In the past year, we discovered that the ability to monitor the same
ing to isolate one process and to manipulate it to evaluate its role. neuron or population of neurons over time permits us to use pow-
Last year, we described our invention of a computer-controlled erful statistical tools that are commonly used in clinical studies,
robotic microscope that performs automated imaging and analysis such as Kaplan-Meier analysis and Cox proportional hazards
of living or fixed cells. The capabilities of the microscope have regression analysis. In collaboration with UCSF biostatistician Dr.
fundamentally altered the way we perform many of our experi- Marc Segal, we have begun to adapt these tools to analyze data sets
ments. For example, we used to study mechanisms of neurodegen- generated by the robotic microscope. These tools are powerful for
16 Reports from the Laboratories
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE
GFP fluorescence within individual neurons, serving as a surrogate
for Akt expression within that neuron, could predict which neurons
survived. We found that the levels of GFP in neurons cotransfect-
ed with empty vector did not predict survival, whereas the levels
of GFP in neurons cotransfected with Akt were highly predictive
(Figure 1C and D). The observation is important because it demon-
strates our ability to measure a variable in a living neuron that can
predict its fate.
The approach is especially well suited to studying neurodegenera-
tive diseases. In HD, a mutation increases the risk to all neurons of
initiating the biochemical processes that lead to death. However,
stochastic processes appear to play a major role in determining
when a particular neuron is affected and eventually dies. Because
these processes may take time to unfold, specific changes may be
visible in only a small fraction of neurons at one point in time.
Thus, the significance and even the detection of some changes may
not be recognized with conventional methods. On the other hand,
the ability to track the fate of individual neurons with the robotic
microscope and apply these statistical tools makes it possible to
Figure 1. Applications of robotic microscopy. (A) Mutant huntingtin (Htt) induces identify highly significant and predictive relationships even if the
neurite retraction. Striatal neurons were transfected with amino-terminal hunt- changes are transient or affect a small fraction of neurons at a time.
ingtin fragments fused to GFP and containing either a normal (Htt-25Q-GFP) or
Another reason the approach could be useful to study pathogenesis
disease-associated (Htt-103Q-GFP) stretch of polyglutamines. After automated
imaging approximately daily, corresponding microscope fields were examined to is its potential for identifying therapeutic targets. The approach
reconstruct the pattern of neurite extension of individual neurons over the should help us distinguish factors that predict survival or dysfunc-
course of the experiment. (B) Akt potently protects neurons. Cultured cortical tion and thereby identify therapeutic targets before we fully under-
neurons were transfected with GFP together with either a control vector or an stand the molecular pathways that link them.
active form of the Akt kinase. Transfected neurons were quantified by automat-
ed imaging and analysis for 10 minutes each day, resulting in over 31,000 obser- Molecular Mechanisms of Synaptic Plasticity
vations. Neurons transfected with Akt survived significantly better than those
transfected with control vector. The extraordinary statistical significance
It is widely held that new gene transcription must occur in neurons
achieved here reflects the large number of observations and the power of for long-term memories to form. Our laboratory is interested in a
Kaplan-Meier analysis. (C, D) The level of Akt expression predicts neuronal number of questions concerning the role of new gene expression in
longevity. The expression of the GFP marker directly varies with the expression plasticity. What synaptic signals trigger new gene transcription?
of cotransfected Akt. Therefore, we used the level of GFP fluorescence as a sur-
What are the gene targets of these signals and how do the encoded
rogate for Akt expression in living neurons. Expression of Akt (GFP) in a partic-
ular neuron predicted how long it would survive (C). However, the expression of proteins promote synaptic plasticity? How do genes transcribed in
GFP did not predict longevity in neurons transfected with empty vector (D). the nucleus strengthen only a subset of synapses made by a neuron
Censored data were removed from both Akt and control graphs for clarity. that are directly involved in forming a particular memory?
Previous work from our laboratory and others has identified Ca2+
influx and neurotrophins as two synaptic signals that influence
two reasons. First, Kaplan-Meier analysis is extraordinarily sensi- synaptic plasticity and potently regulate neuronal gene transcription.
tive, in part because it can detect important differences that emerge Ca2+ influx through either of two ion channels, the N-methyl-D-
or accrue over the course of an experiment. We used this approach aspartate (NMDA) receptor or the L-type voltage-sensitive Ca2+
to evaluate the ability of a protein kinase, Akt, to promote neuronal channel (L-VSCC), appears to be particularly effective in regulating
survival (Figure 1B). Longitudinal monitoring and Kaplan-Meier neuronal gene transcription. Neurotrophins are a family of peptides
analysis showed that Akt promotes neuronal survival much more that are essential for the normal growth, development, and differen-
potently than had been appreciated from initial reports that meas- tiation of neuronal subpopulations in the central and peripheral nerv-
ured survival at single time points. The newly found potency of ous systems. One neurotrophin, brain-derived neurotrophic factor
Akt makes it an especially attractive therapeutic target. (BDNF), has also been shown to regulate synaptic plasticity and
Perhaps the most important reason that statistical tools such as Cox learning and memory during adulthood.
proportional hazards regression analysis are so powerful is that Earlier, we had shown that Ca2+ influx through the L-VSCC direct-
they enable us to identify and quantify variables that have predic- ly induces BDNF gene transcription. We hypothesized that BDNF
tive value. This method provides a way to use longitudinal data and Ca2+ influx signals might cooperate more broadly to regulate
(the appearance of identified changes within a particular neuron neuronal gene transcription and synaptic plasticity. The hypothesis
and the knowledge of that neuron’s fate) to identify whether and to attracted us because it offered a potential biochemical mechanism to
what extent a change that appears on one day predicts a particular transduce rapidly varying electrical activity (through the production
fate for that neuron on another day. We have applied this method of transient Ca2+ influx signals) into longer-lasting biochemical sig-
of analysis to neurons cotransfected with the gene for green fluo- nals (BDNF signaling). BDNF-induced signaling cascades generate
rescent protein (GFP) and either an active form of Akt or a control very long-lasting adaptive responses, such as those envisioned for
plasmid. Previously, we showed that the expression level of GFP learning and memory.
correlated with the expression level of Akt in individual neurons
To test this hypothesis, we introduced reporter genes into cortical
even though the overall expression of these two proteins varied
neurons and examined the responses to Ca2+ influx and BDNF. To
from neuron to neuron. Therefore, we asked whether the amount of
Reports from the Laboratories 17
2002 ANNUAL REPORT
our surprise, we discovered that the efficacy with which BDNF
elicits neuronal gene expression is highly dependent on the level of
L-VSCC activity. For example, modest plasma membrane depolar-
ization significantly potentiated the ability of BDNF to induce
expression of the immediate-early gene c-fos (Figure 2A).
Conversely, application of any one of three structurally distinct and
specific L-VSCC antagonists significantly reduced BDNF-induced
c-fos expression. In contrast, application of both NMDA and
BDNF led to additive gene expression responses, and coapplication
of an NMDA receptor antagonist had no effect on responses to
BDNF. These results suggest that Ca2+ influx signals synergistically
regulate BDNF responses in a channel-specific fashion.
Ca2+ influx through L-VSCCs could potentiate BDNF signals at any
site from the plasma membrane (where BDNF initiates a signal) to
the nucleus (where the c-fos promoter is located). The c-fos pro-
moter contains two DNA elements that can each mediate responses
to either Ca2+ influx or growth factor signals. One element is the
Ca2+ and cyclic AMP response element (CaRE) and the other is the
serum response element (SRE). We tested whether luciferase
reporter genes containing only CaREs or SREs could mediate syn-
ergistic responses to BDNF and L-VSCC activation. We found that
only the SRE or Elk-1, one of the transcription factors that binds the
SRE, showed clear evidence of synergistic activation. These results
suggest that Ca2+ influx through L-VSCCs potentiates BDNF-
induced gene expression in a response element–selective fashion.
Since BDNF induces the phosphorylation and activation of Elk-1
through the Ras pathway, Ca2+ influx might potentiate BDNF
responses by upregulating this pathway. We examined the effects of
L-VSCC activation and BDNF on extracellular signal–regulated
kinase (ERK), a downstream component of the Ras pathway and
the major Elk-1 kinase. We found that L-VSCC activation and
BDNF had additive effects on ERK activation (Figure 2B). These
results suggest that the site of synergy is in the nucleus, downstream
of ERK but upstream of transcription initiation.
Recently, a family of factors called histone deacetylases (HDACs)
has been identified. These factors regulate gene expression through
effects on specific transcription factors and on chromatin structure.
Deacetylation of specific histones suppresses gene expression by
making the associated DNA less available to the transcription
machinery. Importantly, Ca2+ influx can promote the translocation
of two HDAC family members from the nucleus to the cytoplasm,
thereby removing their suppressive effects. We found that pharma-
Figure 2. Synaptic signals can cooperate to synergistically regulate neuronal gene cological agents known to inhibit a subset of HDACs (e.g., tricho-
transcription. (A) Cortical neurons were left unstimulated or were stimulated with
statin A or tripoxin) do not by themselves induce expression of a c-
either modest membrane depolarization (KCl, 10 mM), BDNF (10 ng/ml), or both.
Transcription of the immediate-early gene c-fos was evaluated by reverse-tran- fos-luciferase reporter gene in neurons. However, when added
scriptase polymerase chain reaction. Costimulation with KCl and BDNF had a syn- together with BDNF, HDAC inhibitors significantly potentiate neu-
ergistic effect on c-fos induction (the shaded portion of the bar corresponds to the ronal gene expression, similar to L-VSCC activation (Figure 2C).
amount of induction that exceeds an additive response). (B) Costimulation with KCl
(10 mM) and BDNF (10 ng/ml, 15 minutes) did not produce synergistic phospho- In summary, we have elucidated a new signaling pathway in neu-
rylation and activation of ERK or CREB, two factors that critically determine the rons by which Ca2+ influx through L-VSCCs cooperates with
activity of the c-fos promoter. Thus, the site of synergy is downstream of ERK and BDNF to regulate gene expression. We hypothesize that synergy
is probably in the nucleus. The additional lane of BDNF (10 ng/ml, 45 minutes)
arises from the integration of two pathways: BDNF signals that
stimulation is a later time point meant to show that the failure to observe synergis-
tic ERK and CREB phosphorylation earlier was not due to signal saturation. (C) activate Elk-1 bound to the SRE and L-VSCCs signals to HDACs
Synergy may depend on Ca2+ influx–mediated inhibition of HDACs. Cortical neu- that derepress the chromatin structure of the SRE-containing pro-
rons were transfected with a luciferase reporter gene under the control of the c-fos moter.
promoter (fos-luc). The addition of the HDAC inhibitor trichostatin A (TSA, 400 nM)
had almost no effect alone compared with untreated neurons. BDNF modestly Molecular Mechanisms of Huntington’s Disease
induced c-fos-luciferase; however, application of TSA and BDNF synergistically Abnormal deposits of huntingtin known as intranuclear inclusions
induced neuronal gene expression. Ca2+ influx induces the nuclear export of at
least two HDACs and could indicate that synergy arises from the spatial coinci-
are a hallmark of HD. Although our previous work showed that
dence of two signals at the promoter: Ca2+-mediated HDAC inhibition and BDNF- inclusion formation was not required for huntingtin-induced degen-
induced activation of critical nuclear enhancer transcription factors (e.g., Elk-1). eration, nuclear localization was nonetheless important. Reduction
18 Reports from the Laboratories
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE
in the nuclear accumulation of huntingtin eliminated degeneration polyglutamine expansions, we discovered that, in the absence of the
in our HD neuronal model, and since we reported this finding, sev- adjacent domain, the polyglutamine expansion appeared to be more
eral laboratories have reproduced the observation in other cell mod- likely to adopt a conformation recognized by our antibody and less
els and in transgenic and knock-in mice. likely to aggregate. This result is interesting because cleavage of hunt-
The factors that govern the subcellular localization of huntingtin are ingtin at a site within the domain that we have defined has been pro-
unknown. In fact, although huntingtin was discovered almost a posed to play a pathogenic role. Our findings suggest that cleavage
decade ago, its normal function remains a mystery. Therefore, we promotes pathogenesis by generating a fragment of huntingtin that can
performed a large-scale screen of the huntingtin protein (3144 more readily adopt a disease-associated conformation. In the coming
amino acids) for sequences that govern its subcellular localization. year, we plan to use the robotic microscope to prospectively examine
To facilitate the study, the full-length protein was subdivided into whether this disease-associated conformation of huntingtin is a better
smaller overlapping pieces. Each huntingtin fragment was tagged predictor of neurodegeneration than inclusion body formation.
with GFP at the amino terminus and with β-galactosidase at the car- Selected References
boxyl terminus. We used the GFP tag to image the location of the Saudou F, Finkbeiner S, Devys D, Greenberg ME (1998) Huntingtin
attached huntingtin fragment in living neurons, and the β-galactosi- acts in the nucleus to induce apoptosis but death does not correlate
dase tag was used to develop a novel and sensitive luminescence with the formation of intranuclear inclusions. Cell 95:55–66.
assay for its presence in different subcellular fractions.
Finkbeiner S (2000) Calcium regulation of the brain-derived neu-
We found that a domain in the amino terminus of huntingtin, adjacent rotrophic factor gene. Cell. Mol. Life Sci. 57:394–401.
to the polyglutamine stretch, plays a role in governing the localization
of huntingtin and regulating the conformation of the polyglutamine Arrasate Iraqui M, Brooks E, Chang P, Mitra S, Finkbeiner S (2002)
stretch. The presence of this domain of huntingtin promoted its move- Prospective analysis of huntingtin conformation and degeneration in
ment from a subcellular fraction enriched in cytosolic proteins to one neurons. Soc. Neurosci. 29:293.12 (abstract).
enriched with components of the nucleus and the endosomal system. Bradley J, Finkbeiner S (2002) An evaluation of specificity in activ-
However, this region of huntingtin does not appear to mediate the ity-dependent gene expression in neurons. Prog. Neurobiol.
translocation of huntingtin into the nucleus. Our imaging studies 67:469–477.
showed that these fragments of huntingtin remained primarily in the Chang P, Arrasate M, Brooks L, Xia J, Truant R, Finkbeiner S (2002)
cytoplasm of neurons. This domain of huntingtin contains four pro- A domain within huntingtin adjacent to the polyglutamine stretch
tein-interaction domains known as HEAT repeats; however, the bind- that regulates its aggregation and subcellular localization. Soc.
ing partners for these HEAT repeats have not been identified. When Neurosci. 29:293.11 (abstract).
we further subdivided the domain of huntingtin, its localizing ability
was lost, leading us to speculate that this domain may function as a Humbert S, Bryson EA, Cordelières FP, Connors NC, Datta SR,
unit within huntingtin. Finkbeiner S, Greenberg ME, Saudou F (2002) The IGF-1/Akt path-
way is neuroprotective in Huntington’s disease and involves hunt-
Since this new domain of huntingtin is adjacent to the polyglutamine ingtin phosphorylation by Akt. Dev. Cell 2:831– 837.
stretch, we suspected that it might affect the conformation of the
polyglutamine stretch or the ability of huntingtin to aggregate into Kapadia S, Bradley J, Finkbeiner S (2002) Cooperative regulation of
intranuclear inclusions. Using an antibody we developed that recog- neuronal gene expression by neurotrophic factors and calcium chan-
nizes a conformation preferentially formed by disease-associated nels. Soc. Neurosci. 29:752.16 (abstract).
Reports from the Laboratories 19
Assistant Investigator Students Research Associates
Fen-Biao Gao, Ph.D. Brigitte Bogert Nhue L. Do
Emily Lesch Alan Lee
Postdoctoral Fellows Neal T. Sweeney Belma Sadikovic
Wenjun Li, Ph.D. Kimmy Su
Miki Tamura, Ph.D. Amy Tang Administrative Assistant
Kanyan Xu, Ph.D. Tyson Jue
Molecular Mechanisms Underlying Dendritic Morphogenesis
and Their Involvement in Neurological Diseases
Fen-Biao Gao, Ph.D.
little from embryo to embryo, suggesting that a genetic program
ignaling between neurons requires specialized subcellular struc-
tures, including axons and dendrites. Dendrites can be highly controls dendritic morphogenesis. Indeed, a genetic screen has iden-
branched and may account for more than 90% of the postsy- tified a number of important proteins that control different aspects of
naptic surface of some neurons. Only recently have dendrites been dendrite development, including Flamingo, a G protein–coupled
appreciated as having much more active roles in neuronal function. In receptor–like protein with seven transmembrane segments and a
addition, the number of dendritic branches and dendritic spines is large amino-terminal domain containing nine cadherin repeats, and
altered in many neurological disorders, such as Alzheimer’s disease Sequoia, a tramtrack-related novel zinc finger protein.
and fragile X syndrome. Despite the importance of dendrites in neu-
As the building block of the nervous system, individual neurons
ronal function and dysfunction, the molecular mechanisms underlying
need to maintain their structural and functional integrity throughout
dendritic morphogenesis in vivo remain unknown. We continue to
adult life. In a number of age-related neurodegenerative diseases,
investigate how dendritic outgrowth is regulated and how alterations
neuronal morphology is altered long before cell death occurs from
in neuronal morphology contribute to disease processes.
accumulated insults. It is highly likely that the progressive decline in
Using Drosophila as a Model System the function of individual neurons plays a major role in the progres-
to Study Disease Mechanisms sion of these neurological diseases. Thus, we believe that under-
As a genetic model system, the fruit fly Drosophila melanogaster has standing alterations in neuronal morphology and function at the sin-
greatly contributed to our understanding of normal animal develop- gle neuron level throughout life will contribute to the understanding
ment. More recently, Drosophila has been used to dissect the molecu- of disease mechanisms.
lar mechanisms underlying a number of human neurological disor- To study how neuronal morphology is regulated by various factors,
ders. Studies of diseases in model organisms will offer insights into our laboratory uses the mosaic analysis with a repressible marker
normal developmental processes and will likely lead to new therapeu- (MARCM) technique to visualize single wildtype or mutant PNS neu-
tic interventions for these seemingly intractable diseases. rons in living Drosophila larvae. This single-neuron assay system pro-
One of the major reasons we use the peripheral nervous system vides several advantages. First, each dorsal cluster contains only six
(PNS) of Drosophila as our primary model system is its simplicity. MD neurons, which elaborate their dendrites in a two-dimensional
In each abdominal hemisegment of Drosophila embryos or larvae, plane. We can study dendritic growth and branching of the same iden-
there are only 44 PNS sensory neurons, which can be grouped into tifiable single MD neuron in vivo and compare wildtype and mutant
dorsal, lateral, and ventral clusters. In the dorsal cluster, there are six neurons with ease and precision. Second, the dendritic field of a par-
multiple dendritic (MD) neurons, four external sensory neurons, one ticular MD neuron in the dorsal cluster is stereotyped, with limited
bipolar dendritic neuron, and one internal sensory neuron. These variation between abdominal segments of the same larva or between
neurons elaborate their dendrites in a two-dimensional plane just different larvae at the same stage. Therefore, we can analyze quantita-
beneath the epidermal cell layer and can be labeled by green fluo- tively the effects of loss-of-function mutations and overexpression of
rescent protein using the UAS-GAL4 system. Therefore, we can genes of interest on the dendritic morphology of each MD neuron in
directly visualize the dendrites and axons of dorsal MD neurons in vivo. Third, we can continuously image the dendrites and spine-like
living Drosophila embryos and larvae and follow their growth, processes of a single wildtype or mutant MD neuron in a living ani-
branching, and remodeling in real time. Our previous studies have mal over several days and study age-dependent alterations in vivo.
shown that the dendritic branching pattern of the MD neurons varies Using this single-neuron assay system, we found that Flamingo has a
20 Reports from the Laboratories
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE
Figure 1. Schematic representation of the domain structures and homology of about 70% identical between dFMR1 and hFMR1. The RGG box, another
Drosophila and human FMR1 proteins. The Drosophila Genome Project has RNA-binding motif found in hFMR1, is also present in dFMR1. In addition, a
revealed only one fly homolog of the human fmr1 gene (hfmr1), referred to as region that mediates protein–protein interactions between hFMR1 and
dfmr1. Dfmr1 is located on the third chromosome and encodes a protein with hFXR1/hFXR2 shows about 50% identity with dFMR1. The high degree of
681 amino acids (aa) that shares a high degree of conservation with hFMR1. sequence conservation suggests that dFMR1 is the functional homolog of
Most notably, the two KH domains and the ribosome interaction domain are hFMR1 in flies.
cell-autonomous function in controlling dendritic extension and axon- recent studies have identified a number of mRNAs that contain
al elongation in vivo. Similar approaches are being used to study the G-quartet structures and are present in FMR1 messenger-ribonu-
functions of disease genes in neural development (see below). cleoprotein (mRNP) complexes. However, the in vivo binding
specificity and the in vivo RNA targets of FMR1, as well as its
Genetic Dissection of Disease Gene Function in Drosophila
physiological function, remain largely unknown.
We are interested in understanding the functions of several genes that
have been implicated in neurological disorders. During the past year, The Drosophila Genome Project has identified only one fmr1 gene
we have continued to focus on the fragile X syndrome. This is the homolog (dfmr1) in flies, which encodes a protein that shares a high
most common form of inherited mental retardation in humans, with an degree of amino acid identity with human FMR1 in several key
estimated incidence of 1 in 4000 males and 1 in 8000 females. The domains (Figure 1). For instance, the KH domains and the ribosome
syndrome is characterized by learning disabilities and mild to severe interaction domains of the two proteins are more than 70% identical,
mental retardation that is often associated with autistic behavior, suggesting functional conservation among species. Our in situ and
hyperactivity, attention deficit disorder, facial dysmorphism, and immunostaining analyses indicate that dFMR1 is highly expressed in
enlarged testes in postpubertal males. The genetic defect of fragile X the Drosophila nervous system and localized to both dendrites and
syndrome was discovered in 1991, when a single gene, known as the axons of MD neurons. Through a large-scale genetic screen, we iden-
fragile X mental retardation 1 (fmr1) gene, was identified. Fmr1 is tified loss-of-function mutations in the dfmr1 gene. Using a western
located at a fragile site near the end of the long arm on the X chromo- blot with an anti-dFMR1 antibody, we have analyzed larvae that were
some. A CGG trinucleotide repeat is present in the 5′ untranslated homozygous for the mutated chromosome (Figure 2). None of the
region of fmr1 mRNA. This CGG repeat is normally highly polymor- three dfmr1 mutant lines studied, dfmr11, dfmr12, and dfmr14,
phic in length, ranging from a few to 60 repeats, and is often inter- expressed the dFMR1 protein. We cloned and sequenced the genomic
rupted with AGG repeats. However, most patients with fragile X syn- DNA at the dfmr1 locus and identified an 11-nucleotide deletion in the
drome have over 200 CGG repeats and a hypermethylated CpG island dfmr11 allele and a single nucleotide mutation in the dfmr14 allele that
within the promoter region that results in the transcriptional silencing resulted in a stop codon (Figure 2). We did not find mutations in the
of fmr1. In a few cases of sporadic fragile X syndrome without CGG dfmr1 coding region of the dfmr12 allele, which suggests that the
repeat expansion, deletion or missense point mutations were found in mutations that result in the loss of dFMR1 expression may be in the
the coding region of fmr1, further supporting the notion that the loss promoter or introns. Dfmr1 mutant flies are viable and develop to
of fmr1 activity is the cause of fragile X syndrome. adulthood at the predicted Mendelian ratio.
Fmr1 encodes a putative RNA-binding protein that contains two Establishment of Behavioral Assays in Drosophila to Study
ribonucleoprotein K homology (KH) domains and an arginine- and the Roles of Disease Genes in Neuronal Function
glycine-rich domain (RGG box). FMR1 is most abundantly Besides using genetic approaches to investigate the function of disease
expressed in the brain and in the testis. In both human and murine genes, we have expanded our efforts into behavioral studies. Before
brains, FMR1 is highly expressed in neuronal perikaryon and den- metamorphosis, Drosophila larvae stop feeding and begin to wander
drites, with little expression in glial cells. FMR1 preferentially binds in search of a pupation site. Larvae crawl by anterior extension and
to poly(G), poly(U), and a subset of brain mRNAs in vitro. More posterior retraction. The wandering behavior of the third-instar larvae
Reports from the Laboratories 21
2002 ANNUAL REPORT
Figure 2. Generation of dfmr1 mutant fly lines. (A) A P-element is inserted in would abolish dfmr1 activity and the overexpression phenotype. (B) Western
the first intron of the dfmr1 gene. The ATG start codon is in the third exon. blot analysis demonstrates dFMR1 in wildtype (wt) flies but not in three dfmr1
Since the upstream activation sequence (UAS) is engineered into the P-ele- mutants. (C) Point mutations or small deletions generated by the chemical
ment, the endogenous dfmr1 gene can be overexpressed when Gal4 mole- mutagen ethyl methanesulfonate were found in dfmr1 in different mutant
cules are present. Mutations could be introduced in the dfmr1 gene, which lines. nt, nucleotide.
is relatively simple and stereotypic and can be separated into two Selected References
phases: linear locomotion and relatively nonlocomotive turning events 1. Gao F-B, Brenman JE, Jan LY, Jan YN (1999) Genes regulating
between two periods of linear locomotion. During turning events, lar- dendritic outgrowth, branching and routing in Drosophila. Genes Dev.
vae search for a new direction and afterward crawl in a different or the 13:2549–2561.
same direction as previous linear locomotion. We have set up the com-
puter-assisted dynamic image-analysis system to analyze quantitative- 2. Gao F-B, Kohwi M, Brenman JE, Jan LY, Jan YN (2000) Control
ly the locomotion behavior of Drosophila larvae at the wandering of dendritic field formation in Drosophila: The roles of Flamingo and
stage. The parameters analyzed include the average speed of linear competition between homologous neurons. Neuron 28:91–101.
locomotion, average directional change, and average duration of lin- 3. Brenman JE, Gao F-B, Jan LY, Jan YN (2001) Sequoia, a tramtrack-
ear locomotion. To simplify our analysis, larvae locomotion behavior related zinc finger protein functions as a pan-neural regulator for den-
is recorded in a defined environment without cues for phototaxis, drite and axonal morphogenesis in Drosophila. Dev. Cell 1:667–677.
chemotaxis, and presumably geotaxis. Because a large number of ani- 4. Gao F-B (2002) Understanding fragile X syndrome: Insights from
mals can be recorded and analyzed fairly quickly with the computer retarded flies. Neuron 43:859–862.
software, subtle differences in specific aspects of locomotion behav-
ior can be detected with statistical significance. Our preliminary stud- 5. Sweeney NT, Li W, Gao F-B (2002) Genetic manipulation of sin-
ies indicate that mutations in the presenilin gene, which has been gle neurons in vivo reveals essential roles of Flamingo in neuronal
implicated in Alzheimer’s disease, reduce the speed of larval linear morphogenesis. Dev. Biol. 247:76–88.
locomotion by 30%, but do not affect their directional change. These
studies will help analyze the functional consequence of mutated genes
and dissect the neuronal circuits that control a particular behavior.
22 Reports from the Laboratories
Staff Research Investigator Senior Research Associate Summer Student
Yadong Huang, M.D., Ph.D. Walter J. Brecht Ruomeng Dong
Postdoctoral Fellows Research Associate Administrative Assistant
Shengjun Chang, Ph.D. Faith M. Harris Jennifer Polizzotto
Qin Xu, Ph.D.
Apolipoprotein E Proteolysis and Alzheimer’s Disease
Yadong Huang, M.D., Ph.D.
polipoprotein (apo) E4 is a major risk factor for Alzheimer’s
disease. Biochemical, cell biological, and transgenic animal
studies have suggested several potential mechanisms to
explain apoE4’s contribution to the pathogenesis of Alzheimer’s dis-
ease. These include the modulation of the deposition and clearance
of amyloid β peptide (Aβ) and the formation of plaques, impair-
ment of the antioxidative defense system, dysregulation of the neu-
ronal signaling pathways, disruption of cytoskeletal structure and
function, the alteration of the phosphorylation of tau, and the for-
mation of neurofibrillary tangles (NFTs). However, the mechanisms
Figure 1. Working model of apoE proteolysis and
of these apoE4-mediated effects are still largely unknown. Likewise,
Alzheimer’s disease. ER, endoplasmic reticulum.
it is not known which of these pathophysiological effects of apoE4 is
the primary effect and which are subsequent or downstream effects.
Studies from our laboratory have demonstrated a biological event that Neuronal Production of ApoE and Its Regulation
could play a major role in apoE4-related neuropathology. Specifically, by Astroglia
we found that apoE is subject to cleavage in both human and mouse To investigate how apoE expression is regulated in neurons, we trans-
brains by a neuron-specific protease that generates bioactive carboxyl- fected neuronal cell lines with a large fragment of human apoE
terminal-truncated fragments of apoE. ApoE4 is more susceptible to genomic DNA, which includes a 5-kilobase (kb) 5′ flanking region, a
this cleavage than apoE3. The carboxyl-terminal-truncated fragments 3-kb 3′ flanking region, and a 3-kb tissue-specific control element.
of apoE are neurotoxic, leading to cell death and the formation of Although the baseline expression of apoE in these transfected cells
intracellular NFT-like inclusions in some of the dying cells. Since was very low, conditioned medium from an astrocytic cell line (C6) or
apoE is synthesized by neurons under diverse pathological conditions, mouse primary astrocytes increased apoE mRNA expression by 3–4-
this cleavage could be an initial event in apoE4-related neuropatholo- fold (Figure 2A) and apoE protein expression by 4–10-fold (Figure
gy. Thus, our hypothesis is that apoE4 produced in neurons in 2B). These results suggest that astrocytes secrete a factor or factors
response to aging, oxidative stress, brain injuries, or Aβ deposition is that regulate apoE expression in neuronal cells. This regulation
uniquely susceptible to proteolytic cleavage and that the resulting appears to be controlled by the extracellular signal–regulated kinase
bioactive carboxyl-terminal-truncated fragments, probably together (ERK) pathway (Figure 2C). The ERK pathway inhibitor U0126, at a
with other Alzheimer’s disease–related factors (e.g., Aβ), induce level that causes no cytotoxicity, almost abolished apoE expression in
cytoskeletal alterations and other neuropathology (Figure 1). neuronal cells, whereas other mitogen-activated protein kinase
In the past year, we tested this hypothesis by addressing the following (MAPK) pathway inhibitors—c-jun N-terminal kinase (JNK)-
three questions. What regulates apoE expression in neurons? Why is inhibitor-1 and SB203580 (for the p38 pathway)—had no significant
apoE4 more susceptible than apoE3 to proteolysis? Does expression effect on apoE expression (Figure 2C). Furthermore, the human neu-
of carboxyl-terminal-truncated apoE4 in neurons cause neuronal and ronal precursor NT2/D1 cells expressed apoE constitutively (Figure
behavioral deficits in transgenic mice? 2D). As these cells differentiated into neurons induced by retinoic
Reports from the Laboratories 23
2002 ANNUAL REPORT
acid, their apoE expression increased initially and then diminished Neuronal Deficit in Transgenic Mice Expressing
(Figure 2D). However, treatment of the fully differentiated neurons ApoE4(∆272–299) in Neurons
with astrocyte-conditioned medium rapidly upregulated apoE expres- To determine whether expression of carboxyl-terminal-truncated apoE
sion (Figure 2D). These observations and previous studies showing in transgenic mice induces neuropathological changes, we created
that excitotoxic stress induces neuronal expression of apoE led us to transgenic mouse lines expressing apoE4(∆272–299) at various levels
hypothesize that, in response to brain injury, gliosis, or Aβ neurotoxi- in central nervous system (CNS) neurons, including levels similar to
city, neurons are induced to express apoE for purposes of repair or those observed in human cortex. To avoid the cytotoxic effect of the
remodeling. However, in apoE4 carriers, these events trigger prote- truncated apoE4 (observed in vitro in transfected neuronal cells) on
olytic processing of apoE, which is detrimental to repair and remodel- embryonic development, we used a neuron-specific Thy-1 promoter
ing processes. that induces transgene expression at day 15 after birth. The high
Domain Interaction Is Responsible for ApoE4’s expressers died from severe neurodegeneration at 2–4 months of age,
Susceptibility to Proteolysis while the low expressers were viable and fertile. Anti-apoE immuno-
To determine whether domain interaction, which is mediated by for- staining of brain sections showed carboxyl-terminal-truncated apoE4
mation of a salt bridge between Arg-61 and Glu-255, is responsible for in neurons in the neocortex (Figure 4A), hippocampus (Figure 4B and
the susceptibility of apoE4 to proteolysis, we incubated recombinant C), and cerebellum (data not shown). Inclusion bodies containing
apoE4-Thr-61 or apoE4-Ala-255, both of which lack domain interac- truncated apoE4 were observed in neurons of mice at 2–4 months of
tion, with lysates from apoE knockout mouse brains or Neuro-2a cells age (Figure 4A–C). Hematoxylin-eosin staining revealed degenera-
at 37°C for 3 hours and analyzed the proteolysis of apoE by anti-apoE tion of neurons expressing the truncated apoE4 in CA1 (Figure 4D)
western blotting. ApoE4-Thr-61 and apoE4-Ala-255 were both much and CA3 (Figure 4E) neurons of the hippocampus. Importantly,
more resistant to proteolysis than wildtype apoE4 (Figure 3), suggest- expression of a shorter truncated form of apoE4, apoE4(∆241–299) at
ing that apoE4 domain interaction is responsible for its susceptibility similar levels did not induce neuropathology (Figure 4F), suggesting
to proteolysis. This conclusion is supported by our preliminary study that the lipid-binding domain within the truncated apoE4 fragments is
in transgenic mice expressing apoE4-Thr-61 or apoE4-Ala-255 in responsible for the neurotoxic effect.
neurons, in which apoE4 proteolysis is abolished. Western blotting with anti-phosphorylated tau (p-tau) demonstrated
accumulation of monomeric p-tau and sodium dodecyl sulfate
(SDS)–resistant p-tau aggregates in the brains of transgenic mice
expressing high levels of the truncated apoE4 at 2–4 months of age,
which is 6–11-fold higher than in nontransgenic littermates at the
same age. Notably, the amounts of SDS-resistant p-tau aggregates in
the brains of 2–4-month-old transgenic mice expressing high levels of
the truncated apoE4 were similar to those in 18-month-old neuron-
specific enolase (NSE)–apoE4 transgenic mice, suggesting that the
truncated apoE4 enhances tau phosphorylation in transgenic mice.
Figure 2. Expression of apoE in neuronal cells and its regulation by astroglia.
Mouse neuroblastoma Neuro-2a cells were stably transfected with a human
apoE4 genomic DNA construct. The regulation of apoE4 expression by the con-
ditioned medium from an astrocytic cell line (C6 CM) or mouse primary astro-
cytes (1° astrocyte CM) was determined at the mRNA (A) and protein (B) levels
by real-time polymerase chain reaction and anti-apoE western blotting, respec-
tively. (C) Effects of various MAPK pathway inhibitors on the regulation of apoE4
expression in Neuro-2a cells by C6 CM. (D) Human neuronal precursor NT2/D1
cells were induced to differentiate by incubation with retinoic acid (RA) for 0, 1, Figure 3. Domain interaction is responsible for apoE4’s susceptibility to pro-
or 4 weeks (w). Secretion of apoE into the medium was determined by anti- teolysis. Recombinant human apoE4, apoE3, apoE4-Thr-61, or apoE4-Ala-
apoE western blotting. After induction with RA for 4 weeks, some cells were 255 (1 µg protein) was incubated with apoE knockout mouse brain lysates (20
treated with C6 CM for 24 hours and then apoE secretion was determined (D, µl) at 37°C for 3 hours. Fragmentation of apoE was determined by anti-apoE
three far right lanes). western blotting.
24 Reports from the Laboratories
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE
Figure 4. Neuronal deficits in transgenic mice expressing apoE4(∆ 272–299) but mation of truncated apoE4-containing inclusion bodies in cortical (A), CA1 (B), or
not in mice expressing apoE4(∆ 241– 299) at 2– 4 months of age. Brain sections CA3 (C) neurons and neurodegeneration in the CA1 (D, upper panel) and CA3 (E,
from transgenic mice expressing apoE4(∆ 272–299) (A–E) or apoE4(∆241–299) upper panel) regions. (G) Gallyas silver staining of a brain section from an
(F) were stained with anti-apoE (A–C) or hematoxylin-eosin (D–F). Note the for- apoE4(∆ 272–299) mouse revealed NFT-like structures in cortical neurons.
Gallyas silver staining revealed NFT-like structures in neurons in the Huang Y, Liu XQ, Wyss-Coray T, Brecht WJ, Sanan DA, Mahley RW
neocortex of apoE4(∆272–299) transgenic mice (Figure 4G). These (2001) Apolipoprotein E fragments present in Alzheimer’s disease
data suggest that apoE4 fragments increase tau phosphorylation in brains induce neurofibrillary tangle-like intracellular inclusions in
vivo in transgenic mice. We are expanding the transgenic lines in neurons. Proc. Natl. Acad. Sci. USA 98:8838–8843.
which low levels of the carboxyl-terminal-truncated apoE are Brecht WJ, Harris FM, Tesseur I, Wyss-Coray T, Yu G-Q, Mucke L,
expressed to explore in more detail the effects of the truncated apoE4 Mahley RW, Huang Y (2002) ApoE proteolysis and hyperphosphory-
on neuronal and behavioral deficits. lation of tau in transgenic mice expressing apoE4 in neurons. Soc.
In summary, our findings suggest that apoE synthesized in neurons Neurosci. 29:592.13 (abstract).
undergoes proteolytic processing, probably in the secretory pathway, Buttini M, Yu G-Q, Shockley K, Huang Y, Jones B, Masliah E,
to generate carboxyl-terminal-truncated fragments that cause neu- Mallory M, Yeo T, Longo FM, Mucke L (2002) Modulation of
rodegeneration, with apoE4 being more susceptible than apoE3 to the Alzheimer-like synaptic and cholinergic deficits in transgenic mice by
cleavage. This process may not occur to a significant extent under human apolipoprotein E depends on isoform, aging, and overexpres-
physiological conditions, since little apoE is normally expressed in sion of amyloid β peptides but not on plaque formation. J. Neurosci.
neurons. However, in response to aging, oxidative stress, brain injury, 15:10539–10548.
or Aβ deposition, neurons may turn on or increase their apoE expres-
sion for purposes of repair or remodeling, thereby passively activating Harris FM, Brecht WJ, Tesseur I, Mahley RW, Wyss-Coray T, Huang
this proteolytic process, especially for apoE4 carriers. We are testing Y (2002) Astroglial regulation of apoE expression in neuronal cells.
this hypothesis now. We are also attempting to identify the putative Soc. Neurosci. 29:193.16 (abstract).
protease that cleaves apoE at its carboxyl terminus, as it may serve as Ji Z-S, Miranda RD, Newhouse YM, Weisgraber KH, Huang Y,
a therapeutic target for the prevention and treatment of neurodegener- Mahley RW (2002) Apolipoprotein E4 potentiates amyloid β peptide-
ative diseases associated with apoE4. induced lysosomal leakage and apoptosis in neuronal cells. J. Biol.
Mahley RW, Huang Y (1999) Apolipoprotein E: From atherosclerosis
to Alzheimer’s disease and beyond. Curr. Opin. Lipidol. 10:207–217.
Reports from the Laboratories 25
Senior Investigator Senior Research Associates Executive Assistant
Robert W. Mahley, M.D., Ph.D. Maureen E. Balestra Sylvia A. Richmond
Walter J. Brecht
Postdoctoral Fellow Zhong-Sheng Ji, Ph.D. Administrative Assistant
Shiming Ye, Ph.D. Catharine H. Evans
Rene D. Miranda
David S. Peterson
Apolipoprotein E: Impact on Neurodegeneration
and Alzheimer’s Disease Pathobiology
Robert W. Mahley, M.D., Ph.D.
long-term major focus of this laboratory relates to under-
standing the function of apolipoprotein (apo) E in neurobiol-
ogy. By the mid-1980s, clues indicating that apoE played an
important role in neurological diseases had begun to surface. ApoE
was produced in abundance in the brain and served as the principal
lipid transport vehicle in cerebrospinal fluid. It was induced at high
concentrations in peripheral nerve injury, appeared to play a key role
in repair by redistributing lipids to regenerating axons and to Schwann
cells during remyelinization, and modulated neurite outgrowth in cul-
tured rabbit dorsal root ganglion cells or Neuro-2a cells. The stage was
set for the discovery by Roses and associates (Duke University) that
apoE4 is a major susceptibility gene associated with approximately
40–65% of cases of sporadic and familial Alzheimer’s disease and
increases the occurrence and lowers the age of onset of the disease.
Furthermore, the apoE4 allele is associated with poor clinical outcome
in patients with acute head trauma, whereas apoE2 may be protective
Figure 1. Hypothetical role of apoE in the pathogenesis of neurodegeneration and
against neurodegenerative diseases.
A key to understanding the role of apoE in neurological diseases
resides in determining how apoE modulates neuronal repair, remodel-
ing, or protection (Figure 1). Injurious agents can cause neuronal dam- cell death and more than twofold greater DNA fragmentation in
age, requiring neuronal repair of synaptodendritic connections. We apoE4-secreting than in apoE3-secreting or control cells. H2O2 or
would suggest that apoE3 and apoE2 may be effective in mediating staurosporine enhanced cell death and apoptosis in apoE4-transfected
the repair process and in protecting neurons from excessive damage, cells but not in apoE3-transfected cells. A caspase-9 inhibitor abol-
whereas apoE4 may be relatively ineffective. In addition, as reviewed ished the potentiation of Aβ1–42-induced apoptosis by apoE4. Similar
below, apoE4 may have detrimental effects on central nervous system results were obtained with conditioned medium from cells secreting
neurons. The mechanisms responsible for the isoform-specific effects apoE3 or apoE4. Cells preincubated for 4 hours with a source of
of apoE are the focus of the laboratory. apoE3 or apoE4, followed by removal of apoE from the medium and
from the cell surface, still exhibited the isoform-specific response to
ApoE Potentiation of Aβ-Induced Lysosomal Leakage and Aβ1–42, indicating that the potentiation of apoptosis required intra-
Apoptosis in Neuronal Cells cellular apoE, presumably in the endosomes or lysosomes. Studies of
We assessed the isoform-specific effects of apoE on the response of phospholipid [dimyristoylphosphatidylcholine (DMPC)] bilayer vesi-
Neuro-2a cells to the amyloid β peptide (Aβ1–42). As determined by cles encapsulating 5-(and-6)-carboxyfluorescein dye showed that
the intracellular staining pattern and the release of β-hexosaminidase apoE4 remodeled and disrupted the phospholipid vesicles to a greater
into the cytosol, apoE4-transfected cells treated with aggregated extent than apoE3 or apoE2 (Figure 2). In response to Aβ1–42, vesi-
Aβ1–42 showed a greater tendency toward lysosomal leakage than cles containing apoE4 were disrupted to a greater extent than those
neo- or apoE3-transfected cells. Aβ1–42 caused significantly greater containing apoE3. These findings are consistent with the idea that
26 Reports from the Laboratories
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE
activities, but by stimulating cell-surface APP recycling, as deter-
mined by monitoring the internalization of radiolabeled 1G7 anti-
body against the amino terminus of APP (Figure 3B). Furthermore,
preincubation of the B103-APP cells with a low concentration (25
nM) of the receptor-associated protein, which blocks the low density
lipoprotein receptor–related protein pathway, abolished the differen-
tial effects of apoE3 and apoE4 on Aβ production. This result sug-
gests that this pathway may be involved in the more pronounced
stimulatory effect of apoE4 on Aβ production. Finally, replacement
of Arg-61 with threonine in apoE4, which abolishes the intramolecu-
lar domain interaction of apoE4, also attenuated the differential
effects of apoE3 and apoE4 on Aβ production, suggesting that apoE4
domain interaction is involved in this process. Thus, apoE4 not only
affects Aβ deposition or clearance but also modulates APP process-
ing and Aβ production, which may provide an alternative explanation
Figure 2. Release of fluorescent dye from DMPC phospholipid vesicles by apoE3 as to why apoE4 is associated with increased risk for Alzheimer’s dis-
and apoE4. After a 10-second baseline measurement, apoE was added to the
DMPC and the fluorescence released over 50 seconds was measured. ApoE4
ease. These studies are conducted in collaboration with Shiming Ye,
markedly destabilizes the phospholipid bilayer vesicles. Ph.D., and Dr. Huang.
Bioactive Carboxyl-Terminal-Truncated Fragments of ApoE4
Alter the Cytoskeleton and Cause Neurodegeneration
apoE4 forms a reactive molecular intermediate that avidly binds phos- Several lines of evidence indicate that apoE4 has a major effect on the
pholipid and may insert into the lysosomal membrane, destabilizing it cytoskeleton of neurons and disrupts cytoskeletal structure and func-
and causing lysosomal leakage and apoptosis in response to Aβ1–42. tion. Strittmatter and associates demonstrated differential effects of
These studies are conducted in association with Zhong-Sheng Ji, apoE isoforms on tau. Others, including our laboratory, have shown
Ph.D., and Yadong Huang, M.D., Ph.D. that apoE4 enhances tau phosphorylation. We have shown that, in the
ApoE Enhances Aβ Peptide Production presence of a lipid source, apoE3 stimulates neurite outgrowth in cul-
in Cultured Neurons tured neurons, whereas apoE4 inhibits neurite outgrowth and causes
Many studies have suggested that apoE has isoform-specific effects on microtubular instability. ApoE4 also has detrimental effects in apoE-
the deposition or clearance of Aβ peptides. Few studies, however, null transgenic mice overexpressing apoE4, including behavioral
focus on whether apoE isoforms also influence Aβ production. We abnormalities and significant decreases in immunostained synapto-
examined the effects of lipid-poor apoE isoforms on the processing of physin and microtubule-associated protein 2 in the hippocampus and
amyloid precursor protein (APP) and on Aβ production in rat neuro- the cortex. These findings suggested that apoE4 affects the cytoskele-
blastoma B103 cells stably transfected with human wildtype APP695 tal system and neural plasticity.
(B103-APP). ApoE3 and apoE4 both stimulated Aβ production in Our most recent studies have examined the cellular and molecular
B103-APP cells, but apoE4 did so to a significantly greater extent mechanisms by which apoE3 and apoE4 differentially modulate the
(Figure 3A). The enhanced Aβ production by apoE4 was abolished at cytoskeleton of neurons. Carboxyl-terminal-truncated fragments of
low temperatures (22°C), which block endosome recycling. The dif- apoE occur in cultured neurons and in the brains of patients with
ferential effects of apoE3 and apoE4 on Aβ production were mediat- Alzheimer’s disease and induce the formation of neurofibrillary tan-
ed not by altering cellular cholesterol content or α- and β-secretase gle–like inclusions in neurons and neurodegeneration. These inclu-
Figure 3. ApoE3 and apoE4 exert isoform-specific effects on Aβ production times for each condition. * p < 0.05. (B) ApoE4 increased the internalization
through their differential effects on intracellular APP recycling. (A) Blockage of cell-surface APP to a greater extent than apoE3. Internalization of cell-sur-
of APP recycling by culturing cells at low temperature abolished the apoE4- face APP after apoE treatment was determined by measuring the uptake of
enhanced Aβ production. Recombinant human apoE3 or apoE4 (7.5 µg/ml) radioiodinated 1G7 antibody. The results are expressed as a ratio of the
was incubated with B103-APP cells at either 22°C or 37°C for 24 hours. The radioactivity associated with the internalized versus cell-surface pools of APP.
conditioned media were assayed for Aβ by enzyme-linked immunosorbent Values are the mean ± SD of two experiments, each repeated three times for
assay. Values are the mean ± SD of two experiments, each repeated 4–6 each condition. * p < 0.05.
Reports from the Laboratories 27
2002 ANNUAL REPORT
Figure 4. Bioactive carboxyl-terminal-truncated apoE(∆ 272–299) accumulates intracellularly in neurons and forms a cytosolic
inclusion complexed with p-tau and other cytoskeletal proteins. Carboxyl-terminal-truncated apoE fragments occur in neuronal
inclusions in brains of Alzheimer’s disease patients. GFP, green fluorescent protein.
sions were composed of phosphorylated tau (p-tau), phosphorylated Mahley RW, Huang Y (1999) Apolipoprotein E: From atherosclerosis
neurofilaments of high molecular weight, and truncated apoE. to Alzheimer’s disease and beyond. Curr. Opin. Lipidol. 10:207–217.
Truncated apoE4, especially apoE4(∆272–299), induced neurofibril- Buttini M, Akeefe H, Lin C, Mahley RW, Pitas RE, Wyss-Coray T,
lary tangle–like inclusions in up to 75% of transfected neuronal cells Mucke L (2000) Dominant negative effects of apolipoprotein E4
(Figure 4), but not in transfected nonneuronal cells. ApoE4 was more revealed in transgenic models of neurodegenerative disease.
susceptible to truncation than apoE3 and resulted in much greater Neuroscience 97:207–210.
intracellular inclusion formation. These results suggest that apoE4
preferentially undergoes intracellular processing, creating a bioactive Mahley RW, Rall SC Jr (2000) Apolipoprotein E: Far more than a lipid
fragment that interacts with cytoskeletal components and induces neu- transport protein. Annu. Rev. Genomics Hum. Genet. 1:507–537.
rofibrillary tangle–like structures and cell death. The preferential pro- Raber J, Wong D, Yu G-Q, Buttini M, Mahley RW, Pitas RE, Mucke
teolytic cleavage of apoE4 may represent an important mechanism for L (2000) Apolipoprotein E and cognitive performance. Nature
apoE4 in neurodegenerative disorders. These studies are being con- 404:352–354.
ducted in collaboration with Dr. Huang (see his report for more Huang Y, Liu XQ, Wyss-Coray T, Brecht WJ, Sanan DA, Mahley RW
details). (2001) Apolipoprotein E fragments present in Alzheimer’s disease
brains induce neurofibrillary tangle-like intracellular inclusions in
neurons. Proc. Natl. Acad. Sci. USA 98:8838–8843.
Buttini M, Orth M, Bellosta S, Akeefe H, Pitas RE, Wyss-Coray T, Ji Z-S, Miranda RD, Newhouse YM, Weisgraber KH, Huang Y,
Mucke L, Mahley RW (1999) Expression of human apolipoprotein E3 Mahley RW (2002) Apolipoprotein E4 potentiates amyloid β peptide-
or E4 in the brains of Apoe–/– mice: Isoform-specific effects on neu- induced lysosomal leakage and apoptosis in neuronal cells. J. Biol.
rodegeneration. J. Neurosci. 19:4867–4880. Chem. 277:21821–21828.
28 Reports from the Laboratories
Director and Christian Essrich, Ph.D. Research Associates Administrative Assistant
Senior Investigator Jorge J. Palop, Ph.D. Gregor Hare Leslie Manuntag
Lennart Mucke, M.D. Jukka Puoliväli, Ph.D. Brian Jones
Hilda C. Ordanza
Research Scientist Visiting Scientist Kristina P. Shockley
Kimberly A. Scearce-Levie, Ph.D. Tony Wyss-Coray, Ph.D. Fengrong Yan
Postdoctoral Fellows Student Laboratory Associate
Irene Cheng, Ph.D Catherine Massaro John A. Gray
Jeannie Chin, Ph.D.
Luke A. Esposito, Ph.D. Senior Research Associate Executive Assistant
Gui-Qiu Yu, M.S. Denise Murray McPherson
Neurobiology of Dementia
Lennart Mucke, M.D.
n this laboratory, we study processes that result in memory loss Aβ-Dependent Deficits in Learning and Memory Are Closely
and other major neurological deficits, with an emphasis on Related to Neuronal Depletion of a Calcium-Binding Protein
Alzheimer’s disease (AD) and related neurodegenerative disor- The bright prospects of increasing life expectancy in many popula-
ders. Our long-term goal is to advance the understanding of the tions around the world are tempered by an alarming increase in aging-
healthy and the diseased central nervous systems to a point where related neurodegenerative disorders. AD, the most frequent of these
rational strategies can be developed to prevent and cure these con- conditions, causes inexorable loss of memory and other cognitive
ditions. AD and Parkinson’s disease are the most frequent neu- functions. Although the etiology of most AD cases remains elusive,
rodegenerative disorders. They erode people’s ability to think and the analysis of AD-related transgenic mouse models is beginning to
control their movements, two of the most critical and fascinating unravel the pathogenic importance of specific AD-associated mole-
functions of the central nervous system. Both conditions are on the cules. These models are also used increasingly to assess novel AD
rise and can be neither prevented nor cured. These facts underline treatments. Amyloid plaques are the primary pathological outcome
the significance and urgency of our research efforts. measure in these studies, although their contribution to AD-related
Molecules similar to those involved in neurodegenerative diseases cognitive deficits is controversial. In fact, it remains to be determined
are highly expressed in the nervous system of diverse species and which of the many pathological and biochemical alterations in AD and
appear to function in learning, synaptic plasticity, and regeneration. in transgenic models of the disease contribute most critically to the
We are particularly curious about the functions and pathogenic roles decline in neuronal functions.
of amyloid precursor proteins (APP), apolipoprotein (apo) E, and α- We therefore investigated the relationship between morphological,
synuclein, which play key roles in AD and Lewy body diseases such biochemical, and behavioral alterations in transgenic mice in which
as Parkinson’s disease. neuronal expression of hAPP is directed by the platelet-derived
Mutations in human APP (hAPP) that increase the production of growth factor β chain promoter. Mice expressing FAD-mutant
amyloid-β peptides ending at residue 42 (Aβ42) cause autosomal hAPP (hAPPFAD ) have high levels of human Aβ in the hippocampal
dominant forms of early-onset familial AD (FAD). Aβ42 and other formation. This brain region includes the dentate gyrus, which is
Aβ peptides also accumulate in the brains of sporadic cases of AD, critically involved in spatial learning and memory. We analyzed the
suggesting a central role of these APP metabolites in the pathogene- expression of calcium-dependent proteins in these brain regions
sis of AD in general. In AD-related transgenic models, we previous- because pathological and cell culture studies suggest that alterations
ly discovered that Aβ peptides can damage synapses and disrupt in neuronal calcium homeostasis play an important role in the
neural memory circuits independent of their deposition into the amy- pathogenesis of AD.
loid plaques that form in AD brains. The plaque-independent toxic- Calbindin, a 28-kDa calcium-binding protein, is particularly abundant
ity of Aβ was inhibited by apoE3, but not apoE4, which may relate in neurons of the dentate gyrus and highly responsive to alterations in
to the differential effects of apoE isoforms on AD risk (E4 > E3) and calcium influx. Most hAPPFAD mice had significantly lower neuronal
age of onset (E4 < E3). calbindin levels in the dentate gyrus than nontransgenic controls
During the past year, we focused on the identification of molecular (Figure 1A). Interestingly, granule cells are relatively resistant to AD-
indicators and mediators of hAPP/Aβ-induced neuronal dysfunction associated cell death. Yet, we found marked reductions in neuronal cal-
and degeneration. bindin levels in the dentate gyrus of AD cases, with the most striking
Reports from the Laboratories 29
2002 ANNUAL REPORT
Figure 1. Calbindin depletion in the granular layer of the dentate gyrus in from a different subject. Comparable calbindin reductions were identified in
hAPPFAD mice and humans with AD. Hippocampal sections from hAPPFAD hAPPFAD mice and AD cases. Numbers in parentheses indicate Blessed
mice and a nontransgenic (NTG) mouse (A) and from humans with or with- score, which increases with the severity of the dementia.
out AD (B) were immunolabeled for calbindin. Each panel reflects findings
depletions seen in the most severely demented subjects (Figure 1B). tions in calbindin were caused by Aβ, we analyzed their relationship
Double-labeling of brain sections from hAPPFAD mice for calbindin with Aβ deposits (plaques), levels of soluble Aβ1–42 and Aβ1–x
and the neuronal marker Neu-N (Figure 2) indicated that the calbindin (approximates total Aβ), and Aβ1–42/Aβ1–x ratios. Calbindin reduc-
reduction in the dentate gyrus primarily reflects a decrease in neuronal tions in hAPPFAD mice did not correlate with the extent of Aβ deposi-
calbindin levels rather than a loss of calbindin-producing neurons. tion but correlated with the Aβ1–42/Aβ1–x ratio, which reflects the
These results demonstrate that neuronal populations resisting cell death abundance of Aβ ending at residue 42 relative to other, mostly short-
in AD can still be drastically altered at the molecular level. It is likely er, Aβ peptides.
that such molecular alterations have functional consequences. These results are consistent with mounting evidence that AD-relat-
hAPPFAD mice showed significant interindividual variations in cal- ed neuronal deficits are caused by nonfibrous Aβ assemblies rather
bindin reductions that were detectable at the protein and mRNA lev- than by plaques. They are also consistent with studies suggesting
els (Figure 3A). However, calbindin levels did not correlate with that, above an absolute threshold concentration, the formation of
hAPPFAD levels, suggesting that the calbindin reductions are not neurotoxic Aβ assemblies depends more on relative than absolute
caused by the expression of hAPPFAD per se. To assess whether reduc- levels of Aβ1–42. Although Aβ production is dependent on hAPP
Figure 2. Calbindin depletion in the dentate gyrus of hAPPFAD mice is not tion of calbindin in granule cells of hAPPFAD mice but did not reveal obvious
due to loss of granule cells. Double-immunolabeling of sagittal vibratome changes in the density of their nuclei compared to nontransgenic controls.
sections for calbindin and the neuronal marker Neu-N confirmed the reduc- NTG, nontransgenic.
30 Reports from the Laboratories
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE
levels, the formation of neurotoxic Aβ assemblies may be strongly more consistent with cell-culture models in which expression of
affected by proteins that bind or degrade Aβ. This may explain why FAD-mutant proteins does not induce apoptosis by itself but rather
reductions in calbindin correlated with the relative abundance of increases the susceptibility of cells to apoptosis induced by other
Aβ1–42 but not with hAPPFAD levels. The exact mechanisms by insults. We previously developed such a model by stably expressing
which Aβ assemblies reduce calbindin remain to be determined. different hAPP constructs in neuronal cell lines (B103 cells) at near-
They could involve destabilization of the neuronal calcium home- physiological levels. Cells expressing hAPPFAD were significantly
ostasis by chronic inflammation, formation of pores in cell mem- more susceptible to cell death induced by secondary insults than
branes, and alterations in the function of calcium channels and other cells expressing hAPPWT. As outlined below, this difference is like-
membrane proteins. ly due to increased production of Aβ42 in hAPPFAD cells rather than
To further assess the pathophysiological significance of Aβ-induced to a FAD mutation–related loss of neuroprotective APP functions.
calbindin reductions, we analyzed hAPPFAD mice and nontrans-
genic controls in a Morris water maze test, which provides putative
measures of learning and memory. In contrast to nontransgenic
mice, hAPPFAD mice showed a tight correlation between calbindin
levels and deficits in learning and memory in the spatial component
of this test (Figure 3B).
Our findings that hAPPFAD/Aβ is sufficient to reduce neuronal cal-
bindin levels in vivo and that this effect is tightly associated with
behavioral deficits has practical implications, particularly in light of
increasing efforts to assess novel therapies for AD in transgenic
mouse models. The behavioral testing of mice is time consuming,
and test results obtained in different laboratories can vary widely.
Reliable surrogate markers of behavioral deficits could help cir-
cumvent these obstacles and facilitate the preclinical assessment of
AD treatments. Our results suggest that calbindin levels in the den-
tate gyrus are a better diagnostic marker of Aβ-induced neuronal
dysfunction than plaque load (Figure 3C), which remains the most
widely used pathological endpoint measure in the preclinical
assessment of novel AD treatments. Experiments are in progress to
further validate this notion.
Although it is likely that diverse molecular alterations contribute to
AD-associated neuronal dysfunction, the tight association between
molecular and functional alterations we identified makes one won-
der whether reductions in calbindin not only indicate but also medi-
ate hAPPFAD/Aβ-dependent behavioral deficits. Calcium-binding
proteins play key roles in calcium signaling and homeostasis, which
are critical to neuronal function. Calbindin can buffer intracellular
calcium, activate enzymes, modulate ion channels, increase calcium
entry into neurons, facilitate spatial transfer or release of calcium
within the neuronal cytoplasm, and prolong intracellular calcium
signals. Since calbindin can also protect neurons against Aβ-
induced toxicity, the reduction of calbindin by Aβ could be part of
a vicious cycle promoting progressive neuronal dysfunction in
hAPPFAD mice and in AD.
These findings raise the intriguing possibility that reductions in cal-
bindin in hAPPFAD mice and humans with AD not only reflect cog-
nitive deficits but also contribute to them. We will continue to
Figure 3. Calbindin reductions in the dentate gyrus accurately reflect Aβ-
investigate the roles of these and related molecular alterations as induced deficits in learning and memory. (A) Levels of calbindin (CB)
potential indicators and mediators of neurodegenerative disease. immunoreactivity (IR) in the dentate gyrus of hAPPFAD mice correlated tightly
with calbindin protein and mRNA levels in the dentate gyrus of the opposite
Blocking Aβ Production Increases the Resistance hemibrain, as determined by western blot analysis and quantitative fluorogenic
of hAPPFAD -Producing Neurons to Secondary Insults reverse transcriptase polymerase chain reaction, respectively. (B) Reductions
In some cell-culture models, expression of APPFAD or mutant pre- in calbindin correlated tightly with behavioral deficits. hAPPFAD mice (filled
dots) and nontransgenic littermate controls (open dots) were trained in a
senilins is sufficient to kill neurons within a relatively short time.
Morris water maze. The average time it took the mice to locate the platform in
However, even though the mutant proteins are expressed from birth all hidden platform trials (left) and the percentage of time they searched in the
in humans with FAD mutations, it typically takes at least two to target quadrant after removal of the platform (right) were used as putative
three decades for the disease to manifest itself. Aging in humans measures of spatial learning and memory retention, respectively. (C) Aβ likely
and other species is associated with increasing oxidative stress, elicits both plaque-dependent and plaque-independent neuronal alterations.
Cumulatively, our data suggest that plaque-independent neurotoxicity plays a
DNA damage, and decline in mitochondrial function, all of which critical role in the pathogenesis of Aβ-induced cognitive deficits (see text and
might trigger neurodegeneration in neurons primed for excessive references). Reductions of calbindin in the dentate gyrus accurately reflect this
activation of proapoptotic pathways. This scenario would seem form of Aβ toxicity and may even contribute to it.
Reports from the Laboratories 31
2002 ANNUAL REPORT
bition of γ-secretase may indeed increase the survival of neurons,
even in cells expressing hAPPWT at near-physiological levels.
Interestingly, hAPPWT inhibited apoptosis induced by DNA-dam-
aging insults, even when it was retained in the endoplasmic reticu-
lum and intermediate compartment (Figure 4). This finding sug-
gests that its neuroprotective effect does not involve secreted forms
of APP or the transduction of signals by APP that is anchored in the
surface membrane. Studies are under way to determine whether the
antiapoptotic effect of APP in the endoplasmic reticulum and inter-
mediate compartment is mediated by interactions of APP or its
intracellular domain with adapter proteins, inhibitors of the Notch
intracellular domain, p53, or other transcription factors that might
modulate cell death and survival signals in the nucleus.
Hsia A, Masliah E, McConlogue L, Yu G, Tatsuno G, Hu K,
Kholodenko D, Malenka R, Nicoll R, Mucke L (1999) Plaque-inde-
Figure 4. Inhibition of Aβ production increases the resistance of hAPPFAD cells pendent disruption of neural circuits in Alzheimer’s disease mouse
to apoptosis. Stably transfected B103 cells were plated in selection medium, models. Proc. Natl. Acad. Sci. USA 96:3228–3233.
differentiated in N2 medium, exposed to camptothecin (to induce apoptosis),
and analyzed by cell death detection ELISA. The extent of apoptosis is Mucke L, Masliah E, Yu G-Q, Mallory M, Rockenstein EM, Tatsuno
expressed relative to control cultures that express the same construct but G, Hu K, Kholodenko D, Johnson-Wood K, McConlogue L (2000)
were exposed to vehicle instead of camptothecin. * p < 0.001 relative to all High-level neuronal expression of Aβ1-42 in wild-type human amy-
other cell lines (Tukey-Kramer posthoc test). WT, wildtype hAPP; FAD, hAPP
carrying the V717I mutation; WT/ER and FAD/ER, hAPPWT and hAPPFAD con-
loid protein precursor transgenic mice: Synaptotoxicity without
structs carrying a mutation that results in the retention of hAPP in the endo- plaque formation. J. Neurosci. 20:4050–4058.
plasmic reticulum and intermediate compartment; FAD/β–, hAPPFAD construct Masliah E, Rockenstein E, Veinbergs I, Sagara Y, Mallory M,
carrying a mutation that prevents cleavage by β secretase.
Hashimoto M, Mucke L (2001) β-Amyloid peptides enhance α-synu-
clein accumulation and neuronal deficits in a transgenic mouse model
linking Alzheimer’s disease and Parkinson’s disease. Proc. Natl. Acad.
A mutation that prevents hAPP from entering the distal secretory Sci. USA 98:12245–12250.
pathway, where most Aβ peptides are generated, markedly Wyss-Coray T, Lin C, Yan F, Yu G-Q, Rohde M, McConlogue L,
increased the resistance of APPFAD cells to apoptosis (Figure 4). Masliah E, Mucke L (2001) TGF-β1 promotes microglial amyloid-β
Preventing Aβ42 production with a mutation (M596I) that blocks clearance and reduces plaque burden in transgenic mice. Nat. Med.
cleavage of hAPP by β-secretase also drastically increased the 7:612–618.
resistance of hAPPFAD cells, underlining the potential therapeutic Buttini M, Shockley K, Yu G-Q, Masliah E, Mallory M, Yeo T, Longo
value of drugs that specifically block this enzyme activity. FM, Mucke L (2002) Modulation of Alzheimer-like synaptic and
Treatment of hAPPFAD cells with an inhibitor of γ-secretase, the cholinergic deficits in transgenic mice by human apolipoprotein E
other key enzyme required for Aβ production, had a similar effect. depends on isoform, aging and overexpression of Aβ but not on
There is substantial concern that inhibiting cleavage of Notch and plaque formation. J. Neurosci. 22:10539–10548.
other γ-secretase activities might limit the therapeutic usefulness of Raber J, Bongers G, LeFevour A, Buttini M, Mucke L (2002)
γ-secretase inhibitors, but it has been postulated that partial inhibi- Androgens protect against apoE4-induced cognitive deficits. J.
tion of γ-secretase might allow reduction of Aβ production while Neurosci. 22:5204–5209.
leaving intact other γ-secretase activities. Indeed, repeated doses of
active γ-secretase inhibitor within an appropriate dose range partial- Wyss-Coray T, Mucke L (2002) Inflammation in neurodegenerative
ly decreased Aβ production in our experiments and increased the disease—a double-edged sword. Neuron 35:419–432.
resistance of hAPPFAD cells to levels found in hAPPWT cells. This Palop JJ, Jones B, Kekonius L, Chin J, Yu G-Q, Raber J, Masliah E,
treatment also slightly increased the resistance of hAPPWT cells, Mucke L (2003) Neuronal depletion of calcium-dependent proteins in
which produce some Aβ, suggesting that susceptibility to apoptosis the dentate gyrus is tightly linked to Alzheimer’s disease-related cog-
is proportional to the amount of Aβ42 produced and that partial inhi- nitive deficits. Proc. Natl. Acad. Sci. USA. In press.
32 Reports from the Laboratories
Senior Investigator Research Associate
Robert E. Pitas, Ph.D. Richard M. Stewart
Postdoctoral Fellows Administrative Assistant
Lorenzo Arnaboldi, Ph.D. Emily K. O’Keeffe
Paul C. R. Hopkins, Ph.D.
Juan Santiago-García, Ph.D.
Molecular Mechanisms Contributing to Neurological Disorders
Robert E. Pitas, Ph.D.
brain cDNA library. BLAST searches showed that EBP is a member
ur laboratory is interested in identifying previously unchar-
acterized proteins and mechanisms that contribute to the of a previously uncharacterized family of proteins with unknown
development of neurological disorders. These studies may function. The proteins are encoded by four homologous genes in
lead to new strategies for treatment. Mutations in several proteins humans with equivalent genes in mice. There is one related gene in
have been linked to the development of Alzheimer’s disease (AD); Drosophila, which is predicted to produce a protein with 45% amino
however, the mechanism by which many of these mutant proteins acid identity with human EBP. The mRNAs that encode the human
contribute to the disease has not been elucidated. One of the most and mouse forms of this protein are present almost exclusively in the
important risk factors for the development of AD is inheritance of at brain. EBP is predicted to contain a high content of α-helix and to
least one apolipoprotein (apo) E4 allele. ApoE is a lipid-binding pro- form coiled-coil domains. It contains two predicted transmembrane
tein that is produced in the brain and other organs. ApoE has been domains near the carboxyl terminus (Figure 1). The association of
examined for its interaction with proteins known to be important for EBP with apoE3, initially identified by the yeast two-hybrid
the pathogenesis of AD, including the amyloid beta (Aβ) peptide and approach, has been confirmed. Fluorescence quenching experiments
tau components of the defining lesions of AD: neuritic plaques and using recombinant EBP and apoE3 showed that the fluorescence of
neurofibrillary tangles. However, the extent to which these interac- apoE is increased by coincubation with EBP, indicating an interac-
tions increase the risk of developing AD remains uncertain. We are tion between the two proteins. Analysis of coincubated apoE3 and
testing the hypothesis that apoE4 contributes to AD and other neu- EBP by size-exclusion chromatography also demonstrated an inter-
rological diseases through its interactions with intracellular proteins action and suggests that the apoE3–EBP complex has a 1:1 stoi-
that are important for the maintenance of neuronal plasticity or chiometry. Using assays that separate various components of the
through its effects on the metabolism of lipids in the central nervous cytoskeleton by differential centrifugation, we have determined that
system (CNS). We have, therefore, begun to identify and character- EBP interacts with the cytoskeleton. Furthermore, in the presence of
ize brain-derived apoE-binding proteins and to fully characterize EBP but not in control cells, apoE3 also associates with the
lipid abnormalities that occur in the brain during the initiation and cytoskeleton and is found in the cell pellet of cells transfected to
progression of neurological disorders. express EBP.
ApoE-Binding Proteins in the Brain We previously reported the results of in situ hybridization studies of
We have continued to investigate a CNS protein (EBP) that we iden- mouse brain sections that revealed panneuronal expression of EBP.
tified by its binding to apoE in a yeast two-hybrid screen of a human These findings were confirmed in primary cultures derived from
Figure 1. EBP is predicted to contain two coiled-coil domains and, near the carboxyl tity with characterized protein families. Due to the frequent involvement of coiled-coil
terminus, two transmembrane (TM) helices. EBP has no significant sequence iden- domains in multimerization, EBP is shown here as a dimer. aa, amino acids.
Reports from the Laboratories 33
2002 ANNUAL REPORT
Immunostaining of EBP in
mouse brain sections.
Vibratome sections were
anti-EBP. Sections from
hippocampus of wildtype
(WT) (A) or Apoe–/– (B)
mice or from Apoe–/– mice
expressing human apoE3
(C) or apoE4 (D) in
astrocytes are shown.
Black arrowheads indicate
EBP aggregates in apoE4
transgenic mice. These
aggregates are shown at
in the inset.
fetal mouse brain. Reverse transcription–polymerase chain reaction these mice but not in littermate controls. Currently, we are investi-
analysis demonstrated that EBP mRNA is expressed in neurons but gating the nature of these EBP deposits and their relationship to neu-
not in astrocytes. rodegeneration.
To further examine the expression pattern of EBP, we have developed Interestingly, we have also found that stable expression of EBP pro-
polyclonal antibodies to a mixture of three peptides corresponding to tects Neuro-2a cells from H2O2-induced cell death. The results
sequences near the amino terminus of the protein and to the recom- obtained with stably transfected cells were confirmed and extended
binant protein. These antibodies detect both human and mouse EBP. using Neuro-2a cells transiently transfected to express EBP. The
In mouse brain sections, EBP was expressed only in neurons, with transiently transfected cells are a mixed population of nonexpressing
the highest level of expression in Purkinje cells of the cerebellum, cells and cells with a range of EBP expression (Figure 3). These cells
neurons of the hippocampus, cerebral cortex, interpeduncular nucle- were either not treated or were incubated with H2O2, fixed,
us, habenular complex, cochlear nucleus, and olfactory bulb. In the immunostained with anti-EBP and a fluorescein-labeled second
hippocampus, EBP was highly expressed in cell bodies of the antibody, and analyzed with a fluorescence-activated cell sorter. We
pyramidal neurons in the CA1, CA2, and CA3 layers and in the den- observed a decrease in the relative number of nonexpressing cells
tate gyrus. EBP was also observed in cell bodies within the molecu- and a preferential survival of EBP-expressing cells after H2O2 treat-
lar and granular layers of the hippocampus. ment. We are currently determining the mechanism by which EBP
We have begun to analyze EBP expression in brain sections from protects against cell death induced by oxidative stress.
control and Apoe–/– mice (Figure 2A and B) and from Apoe–/– mice Since EBP was previously uncharacterized, is highly conserved
expressing human apoE3 or apoE4 (Figure 2C and D). In the hip- throughout evolution, and is expressed exclusively by neurons, we
pocampus of wildtype mice, predominant immunoreactivity for EBP have initiated studies to assess its function in mice using gene knock-
was seen in the cell bodies of neurons in the hippocampus. out technology and in Drosophila using double-stranded RNA-medi-
Interestingly, in Apoe–/– mice, the EBP staining pattern was much ated gene silencing, also called RNA interference (RNAi). We
more diffuse, suggesting that apoE expression affects the distribu- sequenced ~19 kilobases (kb) of the murine EBP gene and deter-
tion of EBP. These results were confirmed and extended by examin- mined the gene structure. The gene consists of five exons; exon 1
ing the EBP expression in the hippocampus of Apoe–/– mice express- contains a 5′ untranslated region, and exons 2–5 contain the coding
ing apoE3 or apoE4 in astrocytes. Diffuse staining was observed, as region. From online databases and our sequence, we determined that
in the Apoe–/– mice; even more interestingly, however, EBP aggre- the mouse gene spans more than 40 kb. We designed and constructed
gates were seen in the hippocampus in apoE4 mice, but not in a conditional knockout vector to delete exon 2, which contains the
apoE3, Apoe–/–, or wildtype mice (Figure 2). translation start site. LoxP sites were introduced to allow cell- or tis-
In a different line of investigation, we analyzed the expression of sue-specific elimination of expression by breeding with Cre-deleter
EBP in transgenic mice expressing amyloid precursor protein (APP) mice. This vector was introduced into mouse embryonic stem cells.
carrying the Swedish and Indiana mutations, which leads to high Targeted cells were selected, verified, and injected into C57BL/6
levels of Aβ peptide formation, cognitive impairment, and neurode- blastocysts at the Gladstone Blastocyst Core. Chimeric mice have
generation. We also observed EBP aggregates in the hippocampus of been generated. These mice will be bred to obtain mice with germline
transmission and then crossed with Cre-deleter mice.
34 Reports from the Laboratories
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE
terization of CNS lipids. This involves extracting lipids from brain,
separating all lipid classes by thin-layer chromatography, and quan-
titating each phospholipid class (phosphatidylcholine, PE, sphin-
gomyelin, phosphatidylserine, and phosphatidylinositol). The PE is
then separated into the diacyl and plasmalogen forms and quantitat-
ed, and the composition is determined by gas-liquid chromatogra-
phy. In collaboration with Dr. Lennart Mucke, we have performed
initial studies in control mice and transgenic mice from the J20A
line, which express human APP with both the Swedish and Indiana
mutations. These mice develop Aβ deposits and cognitive impair-
ments. We have analyzed the hippocampus, cortex, and cerebellum
separately. Preliminary results showed a decrease in PE plasmalogen
in the hippocampus of 18-month-old transgenic mice (32% versus
41% in controls). We are currently analyzing the lipid content of
J20A mouse brains to determine the temporal pattern of the changes
in PE composition. These studies will show whether the changes
precede or follow the appearance of Aβ deposits and the relationship
of the changes in composition to the onset of cognitive decline. We
Figure 3. Neuro-2a cells were transiently transfected to express EBP and then
incubated in the presence or absence of 150 µM H2O2 for 24 hours. Cells were
will then extend these analyses to examine the effect of apoE3 and
labeled with anti-EBP and analyzed by fluorescence-activated cell sorting. apoE4 expression in the brain on lipid composition with and without
the coexpression of mutant APP.
In experiments performed in collaboration with Dr. Fen-Biao Gao, Raber J, Wong D, Buttini M, Orth M, Bellosta S, Pitas RE, Mahley
we suppressed the expression of mRNA for the Drosophila homo- RW, Mucke L (1998) Isoform-specific effects of human apolipopro-
logue of EBP using the RNAi approach. The consequence of organ- tein E on brain function revealed in ApoE knockout mice: Increased
ism-wide ablation of expression is a delay in the onset of pupation susceptibility of females. Proc. Natl. Acad. Sci. USA 95:10914–
and mortality during the early stages of metamorphosis, a time that 10919.
correlates with increased expression of EBP in the CNS. We are cur-
Buttini M, Orth M, Bellosta S, Akeefe H, Pitas RE, Wyss-Coray T,
rently investigating the effects of cell-specific ablation of expression.
Mucke L, Mahley RW (1999) Expression of human apolipoprotein
Lipid Metabolism and Neurological Disease E3 or E4 in the brains of Apoe–/– mice: Isoform-specific effects on
Several lines of evidence suggest that altered lipid metabolism is neurodegeneration. J. Neurosci. 19:4867–4880.
associated with the development of neurological disease. A relative- Buttini M, Akeefe H, Lin C, Mahley RW, Pitas RE, Wyss-Coray T,
ly consistent finding in humans with AD is a decrease in the phos- Mucke L (2000) Dominant negative effects of apolipoprotein E4
phatidylethanolamine (PE) content of the brain. PE exists in two revealed in transgenic models of neurodegenerative disease.
forms, one having two fatty acids linked to the glycerol backbone of Neuroscience 97:207–210.
the PE by ester bonds (diacyl PE) and the other having an alkyl
Raber J, Wong D, Yu G-Q, Buttini M, Mahley RW, Pitas RE, Mucke
group at position one of the phospholipid in an α, β-unsaturated
L (2000) Apolipoprotein E and cognitive performance. Nature
ether linkage (plasmalogen or alkenylacyl PE). The decrease in the
PE content in AD brains is due primarily to a decrease in the plas-
malogen component of PE. However, it is unclear whether or not the Santiago-García J, Mas-Oliva J, Innerarity TL, Pitas RE (2001)
decrease in plasmalogen is related to the etiology of the disease or is Secreted forms of the amyloid-β precursor protein are ligands for the
simply a result of the changing cellular composition of the brain. class A scavenger receptor. J. Biol. Chem. 276:30655–30661.
As a new initiative in our laboratory, we have begun to examine this Hopkins PCR, Santiago-García J, Hahn SL, Stewart RM, Do NL,
question in transgenic mouse models with certain features of AD. Gao FB, Pitas RE (2002) A neuronal intracellular apoE-binding pro-
We have established the techniques required for a detailed charac- tein. Soc. Neurosci. 29:883.10 (abstract).
Reports from the Laboratories 35
Senior Investigator Postdoctoral Fellows Research Associates
Karl H. Weisgraber, Ph.D. Danny M. Hatters, Ph.D. Sam Loeb
Clare A. Peters-Libeu, Ph.D. Maya Mathew, M.S.
Visiting Scientist Maryam A. Tabar
Liming Dong, Ph.D. Senior Research Associates Chunyao Xia, M.D.
Kay S. Arnold
Research Scientist Yvonne M. Newhouse Executive Assistant
Robert L. Raffaï, Ph.D. Brian Auerbach
Structure and Function of Apolipoprotein E
Karl H. Weisgraber, Ph.D.
ur research focuses on the structural and functional relation-
ships of apolipoprotein (apo) E in lipoprotein metabolism,
heart disease, and neurodegenerative diseases, including
Alzheimer’s disease. ApoE is a 299–amino acid, single-chain protein
with two structural domains that also define functional domains
(Figure 1). The three common human isoforms, apoE2, apoE3, and
apoE4, differ at two positions in the molecule and have very different
metabolic properties and effects on disease. ApoE3 (Cys-112, Arg-
158) binds normally to low density lipoprotein (LDL) receptors and is
associated with normal lipid metabolism, whereas apoE2 (Cys-112,
Cys-158) binds defectively to LDL receptors and, under certain cir-
cumstances, is associated with the genetic disorder type III hyper-
lipoproteinemia. ApoE4 (Arg-112, Arg-158) binds normally to LDL
receptors but is associated with elevated cholesterol levels and
increased risk for cardiovascular disease. In addition, apoE4 is a major
risk factor for Alzheimer’s disease and predictor for poor outcome
from head injury. Our objective is to determine how the structural and
biophysical properties of apoE influence its metabolic properties and
contribute to its isoform-specific effects in disease and injury.
The structures of the amino-terminal domains of apoE2, apoE3, and
apoE4 in the lipid-free state have been determined; all three structures
adopt a four-helix-bundle motif (Figure 1). However, subtle differ-
ences in side-chain conformations and in salt-bridge arrangements of
the isoforms affect their functions and characteristics. In addition,
Figure 1. The two-domain structure of apoE. As determined by x-ray crystal-
since apoE likely performs most, if not all, of its functions in a lipid- lography, the amino-terminal domain assumes a four-helix-bundle folding motif.
associated state, a major focus is to determine the influence of lipid The structure of the carboxyl-terminal domain is not known and is depicted as
binding on apoE structure and function. a series of α-helices, consistent with circular dichroism measurements. The
receptor binding region of apoE is located in the amino-terminal domain on
The successful crystallization of apoE complexed with the phospho- helix 4. The carboxyl-terminal domain contains the elements for binding to
lipid dimyristoylphosphatidylcholine (DMPC) was a breakthrough in spherical lipoprotein particles (boxed area). Amino acid differences at position
studying the interaction of apoE with lipid. This exciting result raises 112 distinguish apoE3 (Cys) and apoE4 (Arg). ApoE3 displays a lipoprotein
for the first time the possibility of obtaining detailed structural infor- preference for HDL, whereas apoE4 displays a preference for VLDL. The con-
cept of domain interaction was introduced to account for the influence of the
mation on protein–lipid complexes. This is important for apoE func- polymorphic site at position 112 in the amino-terminal domain on the lipid-
tion because high-affinity binding to LDL receptors requires lipid binding properties of the carboxyl-terminal domain.
36 Reports from the Laboratories
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE
Effect of apoE4 structure on
function. To assess the impact
of apoE4 structure on function,
our long-term objective is to
introduce each of the known
isoform differences individually
into the mouse apoE gene by
gene targeting. This approach
will provide mouse models to
assess the relative contribu-
tions of these differences to
known isoform-specific effects
in plasma (lipoprotein metabo-
lism and atherosclerosis) and
brain (amyloid-β metabolism
and fibrillogenesis, cognitive
behavior, lipid transport, and
ApoE4•DMPC crystals displayed a fiber-like diffraction pattern ApoE Isoform Differences
with a unit cell spacing of 54 Å along the fiber axis and cell spac- We have identified three structural and biophysical differences
ings of ~150 Å and ~300 Å, respectively, for the two axes approxi- among the three apoE isoforms: (1) apoE4 domain interaction; (2)
mately perpendicular to the fiber axis. These findings are consistent protein stability and folding; and (3) cysteine content (Figure 2). Our
with the model that the apoE•DMPC discs (~150 Å in diameter and working hypothesis is that one or more of these differences are
55 Å thick) stack to form long, fiber-like rod structures. The stack- responsible for the isoform-specific effects of apoE4 on plasma
ing of the discs appears to be well defined along the fiber axis, as lipoprotein metabolism, atherosclerosis, and neurodegeneration. To
indicated by the resolution extending to about 7–9 Å along this axis. test this hypothesis, we will engineer each of these isoform differ-
The connection between the resulting rod-like fibers is less defined ences individually into the mouse Apoe gene by gene targeting. In
and extends to only ~15 Å, indicating a weaker stacking interaction this way, we can determine the contribution of each difference to the
along the long cell axes where the sides of the discs touch each other. isoform-specific effects that are known to occur in plasma and the
Recently, improved crystals were obtained by substituting dipalmi- brain (Figure 2). Sorting out the relative contributions of these dif-
toylphosphatidylcholine for DMPC. These crystals diffract to ferences has important implications in developing effective, apoE4-
approximately 8 Å along all three axes. based therapeutic strategies.
Figure 3. ApoE4 domain interaction. In apoE3, with cysteine at position 112, 112, however, the Arg-61 side chain projects into the aqueous environment,
the Arg-61 side chain is positioned in a cleft between two helices and cannot where it can interact with Glu-255, thereby mediating domain interaction,
interact with the carboxyl-terminal domain. In apoE4, with arginine at position resulting in a different overall tertiary structure for apoE4.
Reports from the Laboratories 37
2002 ANNUAL REPORT
in the Arg-61 mice were similar to those of apoE4 in humans, we
were eager to determine if domain interaction affects the central
nervous system of these mice. In a pilot study, aged Arg-61 mice
(14–18 months old) and wildtype, age-matched controls were inject-
ed with kainic acid to elicit cytotoxic neuronal injury. Mice (10
mice/group) were injected intraperitoneally with either kainic acid
(18 mg/kg) or saline as a control. After 6 days, the mice were sacri-
ficed, and brain sections were stained with a monoclonal anti-mouse
antibody to synaptophysin, a marker for dendrites, followed by a
fluorescein isothiocyanate-conjugated secondary antibody. Images
were captured by laser-scanning confocal microscopy and analyzed
and quantified for synaptic content. Our preliminary results show a
significant loss of synaptophysin immunoreactivity in the kainic
acid–treated Arg-61 mice compared with the controls. These excit-
ing results mirror those obtained by other investigators at the
Gladstone Institute of Neurological Disease using this injury model
Figure 4. Effect of candidate compounds on binding of apoE to emulsion parti- in human apoE4 and apoE3 transgenic mice. More importantly, the
cles. The binding of 125I-labeled apoE3 and apoE4 to triolein and phospholipid
emulsion particles was determined in the absence and presence of candidate
study demonstrates that the effects of apoE4 domain interaction can
compounds and the percentage of protein that bound to the emulsions was be observed in the central nervous system and that domain interac-
determined. The results are the average of two determinations. tion plays a key role in the loss of synaptic connections after kainic
Protein Stability and Folding. In addition to structural differences
among protein isoforms, biophysical properties are important deter-
ApoE4 Domain Interaction. ApoE4 binds preferentially to very minants of their functional properties. An emerging concept in pro-
low density lipoproteins (VLDL), whereas apoE3 binds preferen- tein folding is that the stable folding intermediate, or molten glob-
tially to high density lipoproteins (HDL) (Figure 1). We determined ule, represents a third thermodynamic state that a protein may
that the two domains in apoE4 interact and that this interaction is assume. A molten globule has a semi-rigid structure that is almost
restricted to apoE4. The interaction is mediated by Arg-61 (amino- as compact as the native structure. It retains most of the secondary
terminal domain) and Glu-255 (carboxyl-terminal domain) and is structure and much of the tertiary structure of the native state.
responsible for the VLDL binding preference of apoE4 (Figure 3). Owing to the partial loss of tertiary structure, it usually contains an
We are collaborating with Drs. Fred Cohen and Irwin Kuntz (UCSF) exposed hydrophobic surface. Until recently, it had been assumed
and using their DOCK program to identify small molecules that will that the molten globule was a relatively rare occurrence. However,
bind to apoE4 in the vicinity of Arg-61 but not to apoE3 and there- there is a large body of experimental evidence that the molten glob-
by interfere with domain interaction. We expect that such molecules ule state is a common feature of most proteins and that molten glob-
will represent a therapeutic approach by converting apoE4 into an ules can exist in cells and play key roles in a wide variety of phys-
“apoE3-like” molecule (Figure 3). Sixty-five candidate compounds iological processes, including translocation across membranes,
were identified from the DOCK computer search and tested in an increased affinity for membranes, binding to liposomes and phos-
emulsion binding assay. In the initial screen, 13 of the 65 compounds pholipids, protein trafficking, extracellular secretion, and regulation
were found to reduce the apoE4 emulsion binding levels to the of the cell cycle.
apoE3 range, indicating that they were interfering with apoE4 To compare the physical characteristics of the apoE isoforms, we con-
domain interaction. In a second round of screening, 8 of the 13 pos- ducted guanidine and thermal denaturation studies of apoE2, apoE3,
itive compounds repeated their interference with apoE4 domain and apoE4 and their 22- and 10-kDa fragments. Guanidine denatura-
interaction. Two of these compounds, GIND-25 and -105, reduced tion demonstrated that the two domains unfold independently in the
emulsion binding, with neither compound affecting apoE3 binding three isoforms. However, the amino-terminal fragments of the apoE
(Figure 4). isoforms differed in stability. ApoE4 denatured at the lowest guanidine
In several species, including the mouse, apoE contains arginine and concentration and temperature, while apoE2 denatured at the highest
glutamic acid at positions equivalent to positions 112 and 255, concentration and temperature. Furthermore, the denaturation data
respectively, in human apoE. However, these species lack the criti- suggested that apoE4, unlike apoE2 and apoE3, did not fit a two-state
cal human Arg-61 required for domain interaction. Their apoE con- denaturation equilibrium. The lack of cooperative unfolding suggests
tains threonine and, like human apoE3, displays a preference for that apoE4 forms a stable, partially folded intermediate.
HDL. Based on these observations, we used a “knock-in” gene tar- Since folding intermediates are often more stable at an acidic pH, we
geting approach to introduce an arginine codon into the mouse gene examined urea denaturation of the three 22-kDa fragments at pH 4.0.
to “humanize” mouse apoE at position 61 and introduce domain Fitting the data with a two- or three-stage model demonstrated that
interaction. apoE2 exhibits cooperative two-stage unfolding, whereas both
In heterozygous targeted mice, as in human apoE4 heterozygotes, apoE3 and apoE4 display three-stage unfolding, indicating the pres-
the plasma level of the Arg-61 form is 20–40% lower than that of ence of stable folding intermediates under these conditions. Analysis
wildtype apoE. This characteristic pattern reflects the more rapid of denaturation curves revealed that the stable intermediate of apoE4
clearance of apoE4 from plasma. The “Arg-61” mouse apoE also represents approximately 90% of the mixture at 3.75 M urea, where-
displays the expected preference for VLDL. These results demon- as the intermediate of apoE3 represents approximately 30% at this
strate that domain interaction was successfully introduced in vivo. urea concentration and was increased to approximately 80% at 4.75
Since the effects of Arg-61 apoE on plasma lipoprotein metabolism M urea. The apoE2 fragment did not display a folding intermediate.
38 Reports from the Laboratories
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE
In collaboration with Drs. Anthony Fink and Keith Oberg tively. As a result, apoE2 and apoE3 can form both disulfide-linked
(University of California, Santa Cruz), we used Fourier transmit- homodimers and heterodimers with apoA-II. In apoE3/3 subjects,
tance reflective infrared analysis to examine the characteristics of approximately 50% of the apoE3 exists in one of these disulfide-
the apoE4 folding intermediate in the absence and presence of urea linked forms. In addition, the apoE3 homodimer is present in cere-
at pH 4.0. In the absence of urea, the secondary structure of the brospinal fluid. These disulfide-linked dimers affect lipid-binding
apoE4 22-kDa fragment was estimated to consist of 75% α-helix properties and interaction with LDL receptors, but their effect in
and 3% β-sheet, consistent with previous estimates. In 3.75 M urea, neurobiology has not been systematically assessed. Since mouse
apoE4 consisted of 46% α-helix and 17% β-sheet. Thus, the apoE4 apoE also lacks cysteine, a future objective will be to use gene tar-
intermediate contained 61% of the original helical content and had geting to introduce a cysteine codon to produce the functional equiv-
an increased β-sheet structure. Pepsin proteolysis of apoE4 at pH 4.0 alent of apoE3 and determine its effect on neurodegeneration.
in 0 M and 3.75 M urea showed cleavages between helices 2 and 3,
within helix 3, and between helices 3 and 4 in the presence of urea. Selected References
These results indicate that there is a conformational change in apoE4
at pH 4.0 in the presence of urea. We speculate that the four-helix Dong L-M, Weisgraber KH (1996) Human apolipoprotein E4
bundle is partially unfolded, similar to the unfolded structure when domain interaction. Arginine 61 and glutamic acid 255 interact to
this fragment binds to lipid. direct the preference for very low density lipoproteins. J. Biol.
Characterization of the apoE4 stable folding intermediate indicates
that it is a molten globule. Since molten globules have been impli- Weisgraber KH, Mahley RW (1996) Human apolipoprotein E: The
cated in a variety of physiological processes, including membrane Alzheimer’s disease connection. FASEB J. 10:1485–1494.
binding and translocation, we examined the ability of apoE4 and Raffaï RL, Dong L-M, Farese RV Jr, Weisgraber KH (2001)
apoE3 to bind to and disrupt DMPC vesicles at pH 4.0, with and Introduction of human apolipoprotein E4 “domain interaction” into
without urea. Under both conditions, apoE4 was more effective than mouse apolipoprotein E. Proc. Natl. Acad. Sci. USA 98:11587– 11591.
apoE3, suggesting that the apoE4 molten globule may be involved in Morrow JA, Hatters DM, Lu B, Höchtl P, Oberg KA, Rupp B,
membrane translocation. Weisgraber KH (2002) Apolipoprotein E4 forms a molten globule
Cysteine Content. The three major isoforms differ in cysteine con- state: A potential basis for its association with disease. J. Biol. Chem.
tent: apoE4, apoE3, and apoE2 contain 0, 1, and 2 cysteines, respec- 277:50380–50385.
Reports from the Laboratories 39
BEHAVIORAL CORE LABORATORY
Senior Investigator Postdoctoral Fellow Administrative Assistant
Lennart Mucke, M.D. Jukka Puoliväli, Ph.D. Leslie Manuntag
Research Scientist Research Associate
Kimberly A. Scearce-Levie, Ph.D. Lisa Kekonius
Behavioral Core Laboratory
influence the time required to locate the platform (latency).
he Behavioral Core Laboratory was established to analyze
nervous system functions in experimental mouse models of Metabolic alterations are confounding factors in certain tests, such
human neurological diseases. Through collaborative inter- as the holeboard test, in which mice learn to locate a food or water
actions and consultations, it has served scientists at all three reward by poking their heads into a baited hole.
Gladstone Institutes and other investigators at the SFGH campus, Complex behaviors are regulated by many genes, and different strains
as well as colleagues at the Gallo Center, the Mount Zion and of mice vary in their ability to master different tests. Therefore, the
Parnassus campuses of UCSF, UC Berkeley, and other institutions. protocols for the behavioral testing of disease models established on
Our focus has been the behavioral evaluation of mouse models of different genetic backgrounds often must be adapted to make the task
human dementing illnesses. Understanding what impairs learning neither so easy that all mice can perform it equally well nor so diffi-
and memory in these models is providing important insights into cult that none of the mice can perform it successfully. The following
both central nervous system functions and clinically relevant dis- section and the selected references highlight some of the studies to
ease processes. For example, together with Dr. Bruce Miller and which this core facility has made significant contributions.
his colleagues at the UCSF Memory and Aging Center, we are Effects of Apolipoprotein E and Amyloid Peptides
investigating specific links between cognitive impairments in on Learning and Memory
patients with dementia and behavioral deficits in mouse models of
The three major human apolipoprotein (apo) E isoforms (E2, E3, and
these diseases. A major long-term goal of this interaction is the
E4) differ in their effect on AD. Compared with apoE2 and apoE3,
development of suitable tests and novel treatment strategies to
apoE4 increases the risk of AD and lowers the age of onset. ApoE4
improve cognition in patients suffering from Alzheimer’s disease
appears to interact with female gender, further increasing the risk of
(AD) and related conditions.
AD and diminishing the effectiveness of treatments in women. To
Behavioral Tests assess how interactions between gender and apoE isoforms affect cog-
We routinely use a comprehensive battery of tests (Table 1) to fully nition, we studied female and male mice lacking mouse apoE
characterize the neurological condition of mouse models and to (Apoe–/–) and expressing human apoE3 or apoE4 in the brain at com-
validate complex learning paradigms. In assessing complex behav- parable levels. As they aged, female, but not male, apoE4 (Apoe–/–)
iors, it is crucial to determine if there are specific deficits in more mice developed progressive impairments in spatial learning and mem-
basic functions. For example, it is important to distinguish learn- ory in the water maze test, compared with age- and sex-matched mice
ing impairments from performance deficits. Vestibular deficits are expressing apoE3, endogenous mouse apoE, or no apoE at all.
often associated with increased horizontal locomotor activity, Subsequent studies revealed that androgens and androgen
including circling, reduced rearing (raising of both forefeet off the receptor–dependent pathways protect against the detrimental effects
ground and extension of the body), abnormal posture, and poor of apoE4 on cognition. Even brief periods of androgen treatment
swimming ability. In assessing olfactory memory, reduced ability reduced memory deficits in female apoE4 mice. In addition, apoE4
to detect a particular odorant may be a confounding factor. In the male mice, which performed normally in a water maze test at baseline,
water maze test, which assesses spatial learning and memory, developed prominent deficits in spatial learning and memory after
vision and motivation are required to locate a hidden platform by blockade of androgen receptors, whereas apoE3 male mice did not.
using visual cues outside the maze. To assess visual and motiva- Cognitive performance likely depends on a critical balance between
tional problems, we test the ability of mice to locate a visible plat- plasma androgen levels and cytosolic androgen receptor levels in the
form. The swim speed of the mice is also measured as an indicator brain, and the higher endogenous plasma testosterone levels in male
of motivation, motor function, and coordination, all of which can apoE4 mice may provide a relative protection.
40 Reports from the Laboratories
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE
Table 1. Behavioral tests
Reports from the Laboratories 41
2002 ANNUAL REPORT
While apoE4 is the best established genetic susceptibility factor for Raber J, Akana SF, Bhatnagar S, Dallman MF, Wong D, Mucke L
AD, neurotoxic assemblies of the amyloid β peptide (Aβ) have (2000) Hypothalamic–pituitary–adrenal dysfunction in Apoe –/– mice:
emerged as the likeliest primary cause of the illness. When we com- Possible role in behavioral and metabolic alterations. J. Neurosci.
bined the expression of apoE4 with the expression of human Aβ, 20:2064–2071.
both male and female mice developed deficits in learning and mem- Raber J, Wong D, Yu G-Q, Buttini M, Mahley RW, Pitas RE, Mucke
ory, consistent with the fact that both men and women develop AD. L (2000) Alzheimer’s disease: Apolipoprotein E and cognitive per-
Interestingly, apoE3 prevented or delayed Aβ-induced cognitive formance. Nature 404:352–354.
deficits, whereas apoE4 did not. These results may relate closely to
Smith SJ, Cases S, Jensen DR, Chen HC, Sande E, Tow B, Sanan
the accelerated onset of AD in human apoE4 carriers and provide
DA, Raber J, Eckel RH, Farese RV Jr (2000) Obesity resistance and
useful preclinical outcome measures for therapeutic strategies aimed
multiple mechanisms of triglyceride synthesis in mice lacking
at Aβ, apoE4, or their pathogenic interactions. As described in the
DGAT. Nat. Genet. 25:87–90.
reports by Drs. Lennart Mucke, Robert Mahley, and Yadong Huang,
we have made much progress in identifying the mechanisms by Masliah E, Rockenstein E, Veinbergs I, Sagara Y, Mallory M,
which these molecules erode cognitive functions. Hashimoto M, Mucke L (2001) β-Amyloid peptides enhance α-
synuclein accumulation and neuronal deficits in a transgenic mouse
Selected References model linking Alzheimer’s disease and Parkinson’s disease. Proc.
Raber J, Wong D, Buttini M, Orth M, Bellosta S, Pitas RE, Mahley Natl. Acad. Sci. USA 98:12245–12250.
RW, Mucke L (1998) Isoform-specific effects of human apolipoprotein Raber J, Bongers G, LeFevour A, Buttini M, Mucke L (2002)
E on brain function revealed in Apoe knockout mice–Increased sus- Androgens protect against apoE4-induced cognitive deficits. J.
ceptibility of females. Proc. Natl. Acad. Sci. USA 95:10914– 10919. Neurosci. 22:5204–5209.
42 Reports from the Laboratories
GLADSTONE GENOMICS CORE
Staff Research Scientist Research Associates Visiting Scientists Administrative
Christopher S. Barker, Ph.D. Blanca Cabezas Andrea J. Barczak Assistant
Kristina Hanspers, M.S. Chandi Griffin, M.S. Emily K. O’Keeffe
Yanxia Hao, M.S. Dionysos Slaga
Gladstone Genomics Core
Christopher S. Barker, Ph.D.
design additional custom oligonucleotides for interested investigators
enomics is the study of gene populations. The goal of the
Genomics Core is to assist Gladstone scientists in their stud- to include in arrays produced in this laboratory and others.
ies of the unprecedented volume of information resulting Many investigators want to perform experiments when only very
from the Human Genome Project and related projects describing the small amounts of RNA are available. The current generation of pro-
genomes of other model organisms. Services are provided to tocols for both printed and Affymetrix arrays require 10–20 µg of
Gladstone scientists and, as resources allow, to the greater UCSF com- total RNA as starting material. This amount can be prohibitive for
munity as well as investigators at other institutions. This past year has many projects. This year, in collaboration with the UCSF Sandler
been one of continued growth and change within the Genomics Core Center Functional Genomics Core Facility, we developed methods to
as it expands to meet the needs of the Gladstone research community. amplify RNA from small samples. We modified the T7 RNA poly-
More specifically, the core makes available genomics technology such merase method of Eberwine so that researchers can start with as lit-
as DNA microarrays. These techniques allow scientists to examine the tle as 50 ng of total RNA. This technique has been applied success-
tissues of a transgenic mouse or human with a disease by assessing the fully to both Affymetrix and custom-printed arrays. However, it is
expression levels of all the genes that could be expressed in each tis- laborious and expensive. We have been providing materials and serv-
sue. DNA microarrays contain oligonucleotides corresponding to the ices, on a fee-for-service basis, to NuGEN Technologies, a local
sequences of known or partially characterized genes on distinct biotechnology company that is developing a novel amplification
regions of glass slides. By hybridizing tissue samples to these microar- method that is faster, more robust, and less expensive. We are com-
rays, the relative abundance of tens of thousands of genes can be pleting arrangements to be a beta test site for this company. If this
determined in a single experiment. The ability to monitor many bio- technology lives up to its early promise, we hope to make it available
chemical responses, known and unknown, with a single assay is a for use by our clients in 2003.
great advantage of the technology. Investigators Using Genomics Core Resources
A major event this year was the inauguration of our printed oligonu- Israel F. Charo, M.D., Ph.D. (Gladstone Institute of Cardiovascular
cleotide microarray service. Major roadblocks to printed microarrays Disease). Researchers in the Charo laboratory have initiated a study to
have been the steep learning curve and high failure rate associated examine the regulation of chemokines and chemokine receptors. They
with obtaining quality data. During the past two years, the core has are comparing gene expression in the lymph nodes and lungs of CCR2
devoted a large part of its resources to developing printed oligonu- knockout mice with and without Mycobacterium tuberculosis infec-
cleotide microarrays and the technology to process them quickly, reli- tion. Additional comparisons are being made with corresponding
ably, and consistently. Early this year, we completed a successful test samples from wildtype mice.
of our systems with the assistance of the Verdin laboratory at the
Gladstone Institute of Virology and Immunology (GIVI). Samples Bruce R. Conklin, M.D. (Gladstone Institute of Cardiovascular
were prepared according to our directions and then run on 60 arrays Disease). The Conklin laboratory has isolated heart samples from sev-
over 6 weeks without a single failure. Most laboratories running these eral different transgenic and nontransgenic mouse lines. These lines
arrays expect to have failure rates as high as 50%. This successful final were generated to investigate transcriptional regulation in the context
test allowed us to introduce our printed microarray service in June. of mild to severe cardiac muscle remodeling and myopathy. The
results of microarray analyses will allow the investigators to formulate
Another major event was the introduction of larger printed arrays. New new hypotheses regarding cardiomyopathy and heart remodeling.
mouse and human oligonucleotide libraries based on new design algo-
rithms were obtained from Operon and have replaced the first-genera- Steven Finkbeiner, M.D., Ph.D. (Gladstone Institute of Neurologi-
tion libraries. These new libraries contain 16,443 and 21,329 genes, cal Disease). The Finkbeiner laboratory seeks to understand how Ca2+
respectively. This means that, for the first time, the size of the printed regulates the transcription of neuronal genes. They focus on genes
arrays produced in our laboratory is similar to those produced by modulated by either the N-methyl-D-aspartate receptor or the L-type
industry leader Affymetrix. In addition, for the first time, we designed voltage-sensitive calcium channel. Results of these studies could lead
new oligonucleotides for another laboratory. Using our PIK70 soft- to the identification of gene targets that are important for synaptic
ware, we designed 460 oligonucleotides against novel genes that had plasticity and could reveal the molecular mechanisms by which the
been mapped onto the mouse genome and provided them to the common second messenger Ca2+ induces distinct but stimulus-specif-
Akhurst laboratory at the UCSF Cancer Center for inclusion in arrays ic adaptive responses.
prepared by that facility. In the coming months, we expect to replace Warner C. Greene, M.D., Ph.D. (GIVI). The human T-cell lym-
PIK70 with software based on newer algorithms. We will continue to photropic virus-1 Tax oncoprotein activates viral gene expression and
Reports from the Laboratories 43
2002 ANNUAL REPORT
alters the expression of a wide array of host genes by inducing host terminal repeat. Tat regulates several aspects of cellular function,
transcription factors, such as cyclic AMP response element binding including cellular activation, apoptosis, and general transcription. Tat
protein (CREB), NF-κB, and AP-1. Researchers in the Greene lab can also transduce into cells and exert effects even from the cell sur-
have generated Jurkat T-cell lines inducibly expressing wildtype and face. Given these varied effects on cellular function, an analysis of cel-
two mutant Tax proteins (M22 and M47, which are defective in NF- lular gene expression in response to various forms of the Tat protein
κB and CREB activation, respectively). They are using these cell lines will provide insights into HIV-mediated transcriptional regulation
to identify and differentiate genes that are regulated by Tax-induced within the cell and increase our understanding of HIV pathogenesis.
NF-κB and/or CREB. These experiments will allow identification of genes that are tran-
Joseph M. McCune, M.D., Ph.D. (GIVI). The McCune laboratory scriptionally regulated by Tat and should provide insights into the
has initiated studies to analyze genes upregulated by interleukin 7 in mechanism of Tat-mediated effects of HIV-1.
selected subpopulations of human lymphocytes and to analyze genes Stephen G. Young, M.D. (Gladstone Institute of Cardiovascular
that might be differentially expressed in recent thymic emigrants. Disease). Hepatic steatosis has been clinically associated with
These experiments require amplification of transcripts from relatively increased risk for cirrhosis, but the mechanisms that mediate this
small numbers of phenotypically homogeneous populations of cells injury remain unclear. Dr. Young has developed a strain of mice
(isolated by fluorescence-activated cell sorting in the Flow Cytometry (Reversa mice) in which the hepatic microsomal triglyceride transfer
Core Laboratory) using the new methods described above. protein (MTP) is inactivated. While the mice and their liver function
Lennart Mucke, M.D. (Gladstone Institute of Neurological appear normal, he noticed an accumulation of neutral lipids within
Disease). The amyloid precursor protein (APP) and one of its metabo- hepatocytes when MTP is inactivated. In MTP knockout mice, sus-
lites, the amyloid-β peptide (Aβ), play a central role in Alzheimer’s ceptibility to hepatic injury is increased when challenged by a second
disease, but it remains unknown how Aβ (or other APP fragments) insult. The Young laboratory has begun to use DNA microarrays on
elicits the progressive decline in the function and survival of brain RNA from these livers to determine how the accumulation of intra-
cells associated with this illness. Dr. Christian Essrich in the Mucke cellular lipids enhances this susceptibility to injury.
lab has analyzed transgenic mouse models of Alzheimer’s disease Kenneth Aldape, M.D. (Department of Pathology, M.D. Anderson
with DNA microarrays to determine the effects of APP and Aβ on Cancer Center, Houston, TX). The core provided sample preparation
gene expression in the hippocampus, a brain region critically involved services to Dr. Aldape’s laboratory for use in microarray expression
in learning and memory. This investigation revealed expression studies through the SFGH General Clinical Research Center. The
changes in several gene clusters that might be involved in the neu- goals of this study were to identify candidate genes that may be impor-
rodegenerative and behavioral alterations in these mice. A number of tant for the pathogenesis of glioblastomas, to identify genes important
these gene expression changes have already been confirmed by quan- for prognosis, and to group glioblastomas according to expression pat-
titative fluorogenic reverse transcriptase polymerase chain reaction, terns to identify new targets that might be exploited therapeutically.
western blot analysis, or immunohistochemistry. Notably, changes in Harold Bernstein, M.D., Ph.D. (Department of Pediatrics, UCSF).
the levels of some calcium-dependent gene products in subregions of Muscle hypertrophy occurs in the heart and skeletal muscle as an
the hippocampus correlated tightly with deficits in learning and mem- adaptive process in response to various physiological and pathological
ory, suggesting a mechanistically informative relationship to the cog- stresses. The abnormal hemodynamic states associated with congeni-
nitive decline observed in Alzheimer’s disease. tal heart disease frequently lead to hypertrophy. Although it may be
Douglas F. Nixon, M.D., Ph.D. (GIVI). The Nixon laboratory is com- compensatory in the short term, such pathological hypertrophy even-
paring the differential gene expression in primary human CD4+ T tually outstrips the heart’s metabolic resources, leading to cell death
cells exposed to HIV-1. This is intended as a first step in characterizing and heart failure. Therefore, this project seeks to understand the role
the changes in apoptotic gene expression that occur in macrophages of the cell-cycle machinery in the hypertrophic response.
and CD4+ T cells in response to HIV-1 exposure. Harold Chapman, M.D. (Cardiovascular Research Institute, UCSF).
Eric M. Verdin, M.D. (GIVI). The Verdin laboratory has initiated two The project is focused on the interaction between integrin and the
projects. In the first project, researchers led by postdoctoral fellow urokinase receptor. The goal is to identify the downstream genes reg-
Herbert Kasler are looking for novel targets of the class IIa histone ulated by expression of the urokinase receptor and to shed light on the
deacetylases (HDACs) in T cells. Class IIa HDACs are calcium sig- urokinase receptor signal pathway, especially in the mouse B7 and
nal–dependent corepressors of transcription that play important roles B12 lung cell lines.
in muscle differentiation, neuronal survival, and negative selection of Maryka Quik, Ph.D. (The Parkinson’s Institute, Sunnyvale, CA).
T cells. Recently, they demonstrated a critical role for HDAC7 in the This group has initiated a project to study the mechanism underlying
regulation of Nur77, a gene involved in the negative selection of the occurrence of L-dopa-induced dyskinesia by investigating alter-
developing T cells in the thymus. Since then, they have used a num- ations in signal transduction systems using microarray techniques.
ber of convergent approaches, with DNA microarrays as a readout, to Shaun R. Coughlin, M.D., Ph.D. (Cardiovascular Research
identify other genes in T cells that might be regulated by class IIa Institute, UCSF). This study will examine signaling pathways reg-
HDACs. Thus far, they have identified a functional cassette of at least ulated by the protease thrombin and its cognate receptors, termed
eight class IIa HDAC-regulated genes besides Nur77 that have been protease-activated receptors. RNA will be isolated from primary
implicated in negative selection. Other genes were identified that cultures of human umbilical vein endothelial cells, which express
appear to be relevant to muscle differentiation and neuronal survival. protease-activated receptors 1–3. There are currently four members
A substantial number of additional microarray experiments will be of this family of G protein–coupled receptors, and the signaling
performed in the coming year to confirm and extend these findings. pathways regulated by each receptor exhibit similarities to as well
In the second project, graduate student Prerana Jayakumar is studying as differences from the others. Examination of the genes that are
genes that are transcriptionally regulated by HIV-1 Tat, a protein up- or downregulated by each ligand/receptor pair would help iden-
involved in transcription initiation and elongation from the HIV long tify unique pathways and identify new genes in these pathways.
44 Reports from the Laboratories
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE
Joel D. Ernst, M.D. (Cardiovascular Research Institute, UCSF). differentially expressed by subplate neurons before and after
Infection with the intracellular bacterium Mycobacterium tuberculosis ischemic hypoxia.
leads to a paradoxical situation in which infection persists even though
significant levels of the protective cytokine, interferon γ (IFNγ), are Dean Sheppard, M.D. (Department of Medicine, UCSF). The goal
produced. It has recently been found that M. tuberculosis persists part- of this project is to identify subsets of genes that are involved in anti-
ly because it can disrupt the IFNγ signaling pathway. The expression gen-induced airway hyperresponsiveness and bleomycin-induced
of several genes that regulate macrophage function is controlled by pulmonary fibrosis in mice. The investigators started by analyzing
IFNγ. Some of the better-characterized genes are interferon-inducible baseline gene expression in different strains of mice and have fin-
protein, monokine induced by gamma, and IFNγ-inducible GTPase. ished 45 arrays for baseline samples and expect to start evaluating
As the function of these genes is the activation of macrophages and treated lung samples soon. By evaluating patterns of gene expression
chemoattraction of immune cells, it is important to determine how M. in inbred strains of mice with different degrees of sensitivity to each
tuberculosis affects the IFNγ-dependent induction of these genes. A effect, they hope to increase their ability to identify mechanistically
preliminary study was carried out to identify a pool of genes that is important genes.
regulated chiefly by IFNγ. In this study, wildtype C57BL/6 mice and Ajith Welihinda, Ph.D. (SangStat Corporation, Fremont, CA).
IFNγ–/– mice were infected intravenously with M. tuberculosis SangStat has initiated a proof-of-principle experiment to determine if
H37Rv, and RNA was isolated from lungs of infected mice 14 days they will expand their research efforts to include microarray technol-
later (at which time IFNγ is produced by primed T cells). The RNA ogy. The core primarily assists with preliminary experiments on a fee-
was analyzed with Affymetrix U74A.v2 microarrays. About 150 for-service basis to facilitate their drug discovery efforts.
genes, at least 25 of which were functionally relevant, were more
highly expressed in the wildtype controls than in the IFNγ–/– mice. Selected Publications
Nurith Kurn, Ph.D. (NuGEN Technologies, San Carlos, CA). Lee JH, Kaminski N, Dolganov G, Grunig G, Koth L, Solomon C,
NuGEN Technologies has developed a proprietary technology to Erle D, Sheppard D (2001) Interleukin-13 induces dramatically dif-
amplify RNA and DNA samples. The core has provided microarrays, ferent transcriptional programs in three human airway cell types. Am.
RNA samples, and consultation services to enable the application of J. Respir. Cell Mol. Biol. 25:474–485.
this technology to DNA microarrays.
Dahlquist KD, Salomonis N, Vranizan K, Lawlor SC, Conklin BR
Patrick McQuillen, M.D. (Department of Pediatrics, UCSF). The
(2002) GenMAPP, a new tool for viewing and analyzing microarray
goal of this project is to understand the mechanisms that make the
data on biological pathways. Nat. Genet. 31:19–20.
infant brain unusually vulnerable to injury. The primary hypothesis
of this study was that subplate neurons are vulnerable to early DeFreitas MF, Hamrick SEG, Ferriero DM, McQuillen PS (2002)
hypoxic ischemic brain injury and that the death of those cells Subplate neuron cell death and mRNA expression profiling following
accounts for the unique patterns of injury from ischemic hypoxia in oxygen glucose deprivation. Developmental Cerebral Blood Flow and
the developing brain. A rat brain model was used to identify genes Metabolism Symposium, Hershey, PA (abstract).
Reports from the Laboratories 45
EDUCATION AND COMMUNITY OUTREACH
quality science education for high school students. The program
he Gladstone Institute of Neurological Disease (GIND) pro-
vides a highly interactive academic environment and state- was initiated in 1987 by UCSF professor Bruce Alberts, current
of-the-art research facilities that are ideal for training in president of the National Academy of Sciences.
neuroscience and biomedical research. Our postdoctoral fellows Translation of knowledge gained from basic research into educa-
program offers opportunities for multidisciplinary training in tion, prevention, and treatment programs is indeed an important
diverse areas of basic and disease-related neuroscience. It com- aspect of our mission. The GIND is actively engaged in efforts to
bines rigorous scientific education and hands-on research experi- translate scientific discoveries into better treatments for major dis-
ence with a strong emphasis on mentoring and career development. eases of the nervous system.
Several GIND investigators collaborate closely with colleagues at
For example, GIND has expanded its collaborative interactions
UC Berkeley and Stanford, further broadening the exposure of
with UCSF’s Memory and Aging Center to ensure that advances in
trainees to cutting-edge technologies and scientific concepts.
neurodegenerative disease research are promptly but cautiously
GIND investigators hold joint faculty appointments in different translated into better clinical care of patients suffering from
UCSF departments and graduate programs, and have taught stu- Alzheimer’s disease and related conditions.
dents of the Neuroscience Program, which is a component of
We also take an active role in educating the public about neurode-
the Program in Biological Sciences, the Biomedical Sciences
generative disorders and neuroscientific research in general and
Program, the Pharmaceutical Sciences and Pharmacogenomics
participate regularly in fund-raising efforts of charitable organiza-
Program, and the Medical Scientist Training Program.
tions. GIND investigators have continued to participate in public
Undergraduate students from different programs at UC
lecture series aimed at educating lay people about research
Berkeley have also benefited from training in GIND laborato-
advances in our fields of study. Most of these lectures have focused
ries. Dr. Lennart Mucke, the director of the GIND, also partici-
on the causes of neurodegenerative diseases and on emerging treat-
pates in the training of medical students and neurology resi-
ments that give rise to justifiable hope among patients and those
dents at UCSF’s Department of Neurology, the Memory and
providing for their care.
Aging Center, and SFGH.
Members of the GIND regularly participate in the Memory Walk,
The Gladstone-UCSF community offers a large number of lec-
an annual walk-a-thon organized by the Alzheimer’s Association to
tures, seminars, and journal clubs, featuring local experts as well
raise awareness and funds for the Association’s efforts to help
as outstanding scientists from around the world. The weekly GIND
Alzheimer patients and their families. Since 1989, Memory Walk
seminar series has continued to provide a stimulating and highly
participants have raised more than $100 million for Alzheimer pro-
interactive forum for the presentation and discussion of innovative
grams and services. Over $525,000 was raised with this year’s
research in basic and disease-related neuroscience.
Memory Walk on Treasure Island in San Francisco Bay.
Several GIND laboratories have contributed actively to the
Through contributions such as those highlighted above we demon-
Science & Health Education Partnership, a collaboration between
strate our commitment to the community as well as to the pursuit
UCSF and the San Francisco Unified School District promoting a
of our scientific goals.
partnership between scientists and educators in support of high
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE
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(1996) Spatial learning deficit in mice expressing human 751- transgenic mice is efficient in neurons but inefficient in astro-
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(1996) Comparison of neurodegenerative pathology in trans- T, Mark RJ, Mucke L (1997) Cellular signaling roles of TGFβ,
genic mice overexpressing V717F β-amyloid precursor protein TNFα and βAPP in brain injury responses and Alzheimer’s dis-
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D, Hulette C, Masliah E, Pericak-Vance MA, Saunders AM, tor release from the amygdala: Comparison with the hypothal-
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2002 ANNUAL REPORT
26. Buttini M, Westland CE, Masliah E, Yafeh AM, Wyss-Coray disease. In: The Neurology of AIDS (Gendelman HE, Lipton
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28. Ji Z-S, Pitas RE, Mahley RW (1998) Differential cellular accu- adult transgenic mice. Neuron 23:297–308.
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face heparan sulfate proteoglycans. Apolipoproteins E3 and Coray T, Mucke L, Mahley RW (1999) Expression of human
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63. Redfern CH, Degtyarev MY, Kwa AT, Salomonis N, Cotte N, mice. Nat. Med. 7:612–618.
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GIND Seminar Series
Other Seminar Series
at Gladstone, SFGH,
The Gladstone Distinguished Lectures
November 22, 1993 December 11, 1997
Gerald R. Fink, Ph.D. Richard Axel, M.D.
Director Investigator, Howard Hughes Medical Institute
Whitehead Institute for Biomedical Research Higgins Professor of Biochemistry and Molecular Biophysics
Cambridge, MA Professor of Pathology
Dimorphism in yeast: A model for fungal pathogenesis Columbia University
New York, NY
January 10, 1995 The molecular biology of smell
Eric S. Lander, Ph.D.
Member June 1, 1999
Whitehead Institute for Biomedical Research Richard D. Klausner, M.D.
Professor of Biology Director, National Cancer Institute
Massachusetts Institute of Technology Bethesda, MD
Director, Whitehead/MIT Center for Genome Research The VHL tumor suppressor gene
Mapping genes and genomes October 7, 1999
Joan A. Steitz, Ph.D.
March 7, 1995 Investigator, Howard Hughes Medical Institute
Nobel Laureate Henry Ford II Professor of Molecular
Michael S. Brown, M.D. Biophysics and Biochemistry and Chemistry
Paul J. Thomas Professor of Medicine and Genetics Director, Molecular Genetics Program
Director, Center for Genetic Diseases Yale University
Regental Professor of the University of Texas New Haven, CT
Distinguished Chair in Biomedical Sciences The cell nucleolus: An RNA machine
University of Texas Southwestern Medical School
Dallas, TX April 11, 2000
Judah Folkman, M.D.
Nobel Laureate Julia Dyckman Andrus Professor of Surgery
Joseph L. Goldstein, M.D. Professor of Cell Biology
Professor and Chairman Harvard Medical School
Department of Molecular Genetics Boston, MA
Paul J. Thomas Professor of Medicine and Genetics Angiogenesis research: From laboratory to clinic
Regental Professor of the University of Texas
Louis A. Beecherl, Jr., Chair in Biomedical Sciences December 20, 2000
University of Texas Southwestern Medical School Nobel Laureate
Dallas, TX Eric R. Kandel, M.D.
Membrane-bound SREBP: University Professor
Sterol sensor and transcriptional regulator Senior Investigator, Howard Hughes Medical Institute
Center for Neurobiology and Behavior
January 26, 1996 College of Physicians and Surgeons
Robert J. Lefkowitz, M.D. Columbia University
Investigator, Howard Hughes Medical Institute New York, NY
James B. Duke Professor of Medicine Genes, memory storage, and the search for new types
Professor of Biochemistry of synaptic actions
Duke University Medical Center
Durham, NC January 29, 2002
G protein–coupled receptors and their regulation Elaine Fuchs, Ph.D.
Amgen Professor of Basic Sciences
November 21, 1996 Investigator, Howard Hughes Medical Institute
Nobel Laureate University of Chicago
Günter Blobel, M.D., Ph.D. Chicago, IL
Investigator, Howard Hughes Medical Institute Genetic disorders of the cytoskeleton
John D. Rockefeller, Jr., Professor
Head of the Laboratory of Cell Biology November 13, 2002
The Rockefeller University Huda Y. Zoghbi, M.D.
New York, NY Baylor College of Medicine
Protein traffic into and out of the nucleus Professor of Pediatrics,
Molecular and Human Genetics, and Neurology
Investigator, Howard Hughes Medical Institute
Cells, flies, and mice: A triune approach to triplet repeats
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE
GIND Seminar Series Guest Lectures
January 17, 2002 May 23, 2002
Mark D. Linder, Ph.D. Christopher Ross, M.D., Ph.D.
Neurocrine Biosciences, Inc. Department of Psychiatry & Division of Neurobiology
San Diego, CA Johns Hopkins University School of Medicine
Behavioral models for drug discovery/development Baltimore, MD
Pathogenesis of Huntington’s disease and related disorders
February 21, 2002
Cori Bargmann, Ph.D. May 30, 2002
Howard Hughes Medical Institute Thomas Wisniewski, M.D.
University of California, San Francisco Department of Neurology
San Francisco, CA New York University School of Medicine
Signaling pathways in neuronal development in C. elegans New York, NY
Therapeutic approach for Alzheimer and prion diseases
March 28, 2002
Ted Dawson, M.D., Ph.D. November 14, 2002
Department of Neurology Marcy MacDonald, Ph.D.
Johns Hopkins University School of Medicine Department of Neurology
Baltimore, MD Massachusetts General Hospital
Animal models, genes, oxidative stress and protein mishandling: Harvard University
Insights into the pathogenesis of Parkinson’s disease Charlestown, MA
April 10, 2002
William H. Frey II, Ph.D. December 12, 2002
Department of Pharmaceutics Huntington Potter, Ph.D.
University of Minnesota College of Pharmacy Department of Biochemistry and Molecular Biology
Minneapolis, MN and Suncoast Gerontology Center
Treating stroke and Alzheimer’s disease with neurotrophins and University of South Florida
antioxidants: Intranasal delivery to bypass the blood-brain barrier Tampa, FL
Beyond beta protein: The essential role of pathological chaperones
April 25, 2002 in Alzheimer amyloid formation and cognitive decline
Jacqueline Crawley, Ph.D.
National Institute of Mental Health December 19, 2002
Georgetown University School of Medicine Alison Goate, Ph.D.
Bethesda, MD Department of Psychiatry
Strategies for behavioral phenotyping of transgenic Washington University
and knockout mice St. Louis, MO
Genetics and the pathogenesis of Alzheimer’s disease
May 9, 2002
Guojun Bu, Ph.D.
Department of Pediatrics
Washington University School of Medicine
St. Louis, MO
Tale of LRP tail
Other Seminar Series at Gladstone, SFGH, and UCSF
Bay Area RNA Club
Members of nearly 40 laboratories working on RNA research gath-
Formal presentations focusing on topics related to cardiovascular dis-
er to hear three informal talks by participating labs; held the first
ease given by guest speakers and candidates for postdoctoral fellow-
Thursday of every month from 6:30–9:30 p.m. in Genentech Hall
ships at the GICD; held from 12:00–1:00 p.m. in SFGH Building 40
Auditorium, Room 106, on the UCSF Mission Bay campus.
conference room (irregular dates). Contact: Aileen Santos (Tel: 695-
Information is available at http://www.ucsf.edu/frankel/Frankel%20
3770; email: firstname.lastname@example.org).
website/RNA_club/RNA_Club_ 2001-2002.html. Contact Jude
Hawley (Tel: 514-2072; email: jhawley@ biochem. ucsf.edu). GIND Seminar
Biochemistry and Biophysics Seminar Presentations focusing on research in disease-related neuroscience
given by graduate students, postdoctoral fellows, or investigators of
Formal presentations by faculty or guest speakers organized by the
the Gladstone Institute of Neurological Disease (GIND) or guest
UCSF Department of Biochemistry and Biophysics; held every
speakers; held every Thursday from 9:00–10:00 a.m. in the 5th floor
Tuesday from 4:00–5:00 p.m. in Genentech Hall Auditorium, Room
library of SFGH Building 3. Contact: Kelley Nelson (Tel: 695-3885;
106, on the UCSF Mission Bay campus. Information is available at
http://biochemistry.ucsf.edu/B&B_seminars03.html. Contact: Judy
Piccini (Tel: 476-1515; email: email@example.com). GIVI Seminar
BMS Journal Club Presentations focusing on research progress in virology and immunol-
ogy, including HIV and AIDS, given by national and international
Journal club of the UCSF Biomedical Sciences (BMS) Program; held
speakers, alternating with presentations by members of the Gladstone
every Wednesday from 12:30–1:30 p.m. in the 4th floor conference
Institute of Virology and Immunology (GIVI); held every Thursday
room of SFGH Building 40. Articles from any area of biomedicine are
from 12:00–1:00 p.m. in the 5th floor library of SFGH Building 3.
discussed in two half-hour presentations by graduate students, post-
Contact: Robin Givens (Tel: 695-3801; email: rgivens@gladstone.
doctoral fellows, and faculty. Contact: Naima Contos (Tel: 695-3729;
Microbiology and Immunology Seminar
Formal presentations by faculty or guest speakers organized by the
Formal presentations by faculty or guest speakers organized by the
UCSF Department of Microbiology and Immunology; normally held
UCSF Cardiovascular Research Institute (CVRI); held on irregular
every Monday from 5:00–6:00 p.m. in HSW-301 on the UCSF
dates from 4:00–5:30 p.m. on the UCSF Parnassus campus (room
Parnassus campus and broadcast to the 5th floor conference room of
varies). Contact: Julie Tom (Tel: 476-1310; email: tomjm@cvri.
SFGH Building 3. Information is available at http://itsa.
ucsf.edu/~micro/immunology/seminarseries.html. Contact: Emma
Frontiers in Neurology and Neuroscience Sandoc (Tel: 502-1961; email: firstname.lastname@example.org).
Lectures by faculty or guest speakers focusing on neurological dis- Neuroscience Journal Club
eases and their treatment; organized by the UCSF Department of
Journal club of the UCSF Neuroscience Program; held every Friday
Neurology. Lectures are held every other Wednesday from 5:00–6:00
from 4:00–5:00 p.m. in N-217 on the UCSF Parnassus campus.
p.m. in room N-225 of the School of Nursing on the UCSF Parnassus
Articles from any area of neuroscience are discussed in two half-hour
campus. Contact: Laura Alexander (Tel: 476-1489; email: neurorp@
presentations by graduate students and faculty. Refreshments are
served after the meeting. Information is available at http://www.
GICD Scientists Meeting ucsf.edu/neurosc/jclub2002-2003.htm. Contact: Deb Rosenberg (Tel:
Informal seminars focusing on research progress in cardiovascular 476-1947; email: email@example.com).
disease given by graduate students, postdoctoral fellows, or investiga- Neuroscience Seminar
tors of the Gladstone Institute of Cadiovascular Disease (GICD); held
Formal presentations by guest speakers focusing on basic neuro-
every Friday from 9:00–10:00 a.m. in the 4th floor conference room
science, organized by the UCSF Neuroscience Program; held every
of SFGH Building 40. Contact: Aileen Santos (Tel: 695-3770; email:
Thursday from 4:00–5:00 p.m. in HSW-301 on the UCSF Parnassus
campus. Information is available at http://www.ucsf.edu/neurosc/
seminars02_03.html. Contact: Deb Rosenberg (Tel: 476-1947;
GLADSTONE INSTITUTE OF NEUROLOGICAL DISEASE
Calendar of Gladstone, SFGH, and UCSF Seminars
Monday Tuesday Wednesday Thursday Friday
9:00–10:00 GIND Seminar GICD Scientists Meeting
SFGH B3 SFGH B40
12:00–1:00 GICD Seminar Signaling Club BMS Journal Club GIVI Seminar
(day varies) (monthly) (12:30) SFGH B3
SFGH B40 L-1361 SFGH B40
2:00–3:00 PIBS Journal Club
4:00–5:00 CVRI Lecture Biochemistry and Seminar in Neuroscience Neuroscience
(not held regularly) Biophysics Seminar Biomedical Sciences Seminar Journal Club
(4:00–5:30) Genentech Hall HSW-300 HSW-301 S-217
UCSF (room varies)
5:00–6:00 Microbiology and Frontiers in
Immunology Seminar Neurology and Neuro-
HSW-301 science (2 x/month)
6:00–7:00 Bay Area RNA Club
PIBS Journal Club Wednesday from 4:00–5:00 p.m. in HSW-300 on the UCSF
Journal club of the UCSF Program in Biological Sciences (PIBS); Parnassus campus. Information is available at http://www.ucsf.
held every Wednesday from 2:00–3:30 p.m. in Genentech Hall edu/bms/activities.html. Contact: Monique Piazza (Tel: 476-2189;
Auditorium on the UCSF Mission Bay campus. Articles from any email: firstname.lastname@example.org).
area of biomedicine are discussed in two half-hour presentations by Signaling Club
graduate students and faculty. Information is available at
Informal presentations attended by people from 15–20 laboratories at
http://www.ucsf.edu/pibs/pibs_seminars.html. Contact: Maria
UCSF; held on the first Tuesday of every month from 12:00–1:00 p.m.
Realubin (Tel: 476-6178; email: email@example.com).
in room L-1361 of Long Hospital on the UCSF Parnassus campus.
Seminar in Biomedical Sciences Research on signaling is discussed in two half-hour presentations by
Formal presentations by faculty or guest speakers from any area of graduate students or postdoctoral fellows. Contact: Mark Von Zastrow
biomedicine, organized by the BMS Program; held every (Tel: 476-7855; email: firstname.lastname@example.org).