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Award Number: DAMD17-01-1-0756 


TITLE: Fourth Annual Program in Neuroscience Symposium, 

Molecular Biology of Neurodegeneration 


PRINCIPAL INVESTIGATOR: Gary Fiskum, Ph.D. 


CONTRACTING ORGANIZATION: University of Maryland, Baltimore 

Baltimore, Maryland 21201 


REPORT DATE: October 2001 


TYPE OF REPORT: Final Proceedings 


PREPARED FOR: U.S. Army Medical Research and Materiel Command 
Fort Detrick, Maryland 21702-5012 


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The Molecular Biology of 



The University of Maryland 
Program in Neuroscience 
Fourth Annual Symposium 
May I, 2001 
8 a.m. - 5 p.m. 
School of Nursing Auditorium 
655 W. Lombard St. 
Baltimore, MD 



Reproduced From 
Best Available Copy 


Copies Furnished to DTIC 
Reproduced From 
Bo\ind Original 




special thanks to our sponsors: 


AstraZeneca Pharmaceuticals 


Guilford Pharmaceuticals 


Sigma-Tau Pharmaceuticals 
Department of Veterans Affairs 







Dear Colleagues: 


It is a pleasure to host the Program in Neuroscience’s fourth annual symposium, entitled, 
“The Molecular Biology of Neurodegeneration.” Benefits of this symposium are twofold. 

It presents a unique opportunity for some of the nation’s leading scientists to share their 
research, discuss major scientific trends, and ultimately, add to our collective knowledge 
of neurodegenerative diseases, as well as underscore the University ’$ investment in excellence. 

The University’s Center for Clinical Trials facilitates the highest-quality clinical studies, 
such as helping Parkinson’s investigators negotiate their projects from pre-study through 
reporting. Moreover, the October groundbreaking for the Health Sciences Facility II has 
set in motion a promising future for innovative studies and approaches to studying and 
treating diseases. We are extremely proud of these efforts and realize that the flow of 
knowledge among research institutions is vital in bringing solutions to these challenging 
and complex diseases. 

Thank you for joining us at this year’s symposium. We trust your visit to our campus proves 
to be enjoyable, rewarding, and encourages future collaborations. 

Sincerely. 


David J. Ramsay. DM. DPhil 



President 

University of Maryland 











Based at the downtown Baltimore campus of the University of Maryland, home to 
the state's health sciences, legal, and social sciences professional schools, the 
Program in Neuroscience, which began in 1996, offers a doctorate in four major 
research areas: behavioral/systems, cellular/molecular, cognitive/computational, 
and developmental neuroscience. Michael T Shipley, PhD, is director of the 
program and chairman of the Department of Anatomy and Neurobiology at the 
University of Maryland School of Medicine. 


ABOUT 


PROGRAM 


N E U R O 


SCIENCE 


t%he p|ogram in Nej^-oscience has collabo&tor^ in basic and clinical departments 
( froSi^veral schooiljpin can^us. The pro^m erpphasizes a multidisciplinary 


approach to neuroscientific problems and broad training in neuroscience for graduate 
students and fellows. Participating faculty members occupy nearly 40,000 square 
feet of laboratory space, equipped with state-of-the-art facilities for all facets 
of contemporary neuroscientific research. The program is affiliated with the 
University of Maryland School of Medicine, School of Pharmacy, Dental School, 
the statewide Program in Neuroscience and Cognitive Sciences at the University 
of Maryland's Baltimore County and College Park campuses. 








The Molecular Biology of 


N E U R O 


DEGENERATION The University of Maryland Program in Neuroscience 


8:15 a.m. 

Welcome 

Michael T Shipley, PhD 
Program Director 


8:25 a.m. 
Introduction— 
Neurodegenerative 
Disease—Shared 
Features and 
Mechanisms 

Paul Fishman, MD, PhD 
University of Maryland 
Baltimore, Veterans 
Administration 
Medical Center 


Molecular Genetic 10:45 a.m. 

Approaches Break 

8:45 a.m. 

Molecular Mechanisms 
of Alzheimer’s Disease 

Sangram S. Sisodia, PhD 
University of Chicago 


9:15 a.m. 

Presinilins and 
Alzheimer’s Disease 

Merwyn Monteiro, PhD 
University of Maryland 
Baltimore 


9:45 a.m. 

Pathobiology of 
Synucleinopathies 

Virginia Lee, PhD 
University of Pennsylvania, 
School of Medicine 


10:15 a.m. 
Pathogenesis of 
Huntington’s Disease 

Marian DiFiglia, PhD 
Massachusetts General 
Hospital 






Fourth Annual Symposium May I, 2001 8 a.m. - 5 p.m. School of Nursing Auditorium 655 W. Lombard St. Baltimore, MD 21201 


Growth Factors 12:00 p.m. 

Mitochondria and 

Animal Models of 

Lunch Break 

Neurodegeneration 

Neurodegenerative 

11:00 a.m. 


Disease 

Trophic Factors and 

1:30 p.m. 


Their Modulators in 

Mitochondrial 

3:00 p.m. 

the Treatment of 

Molecular Targets 

Defective Neurotrophin 

Neurodegenerative 

for the Treatment of 

Signaling in the 

Diseases 

Acute Brain Injury 

Trisomy 16 Mouse 

Dana C Hilt, MD 

Gary Fiskum, PhD 

Bruce K. Krueger, PhD 

Guilford Pharmaceuticals 

University of Maryland 

University of Maryland 

11:30 a.m. 

Baltimore 

Baltimore 

Modifying the Structure 

2:00 p.m. 

3:30 p.m. 

and Function of the 

Mitochrondrial 

Role of Protein 

Nervous System with 

Genes and 

Aggregates in the 

Genomic HSV Vectors 

Neurological Disease 

Pathogenesis of 

David J. Fink, MD 

Salvatore DiMauro, MD 

Neurogenerative 

University of Pittsburgh 

Columbia University 

Disease 

David Borchelt, PhD 


2:30 p.m. 

Johns Hopkins School 


Mitochondria and 
Neurodegeneration 

M. Flint Beal. MD 

Weil Cornell 

Medical College 

of Medicine 





Paul Fishman, MD. PhD 


Dr. Fishman is professor of neurology at the University of 
Maryland School of Medicine, and director of the Division 
for Neurodegenerative Diseases. 

He received his PhD from Yale University, his MD from 
The Johns Hopkins University School of Medicine, and his 
training in neurology was completed at Columbia- 
Presbyterian Medical Center. He is the founder of the 
Alzheimer’s and Parkinson’s Disease Clinic at the 
University of Maryland as well as the Medical Advisory 
Board of the Alzheimer’s Association of Maryland. 

He is the former chair of the Institutional Review Board for 
the supervision of human research at the University of 
Maryland Baltimore. His current research is supported by 
the Department of Veterans Affairs, where he also 
chairs the review group for Neurology and Neurobiology, 
as well as the NIH (NINDS) and the ALS Association. 

He has a longstanding interest in experimental therapies 
for Alzheimer’s disease. Parkinson’s disease, and ALS. 
with a research program devoted to the development of 
vectors to deliver therapeutic proteins to neurons. 


Introduction 

Neurodegenerative Disease—Shared Features and Mechanisms 

Although Alzheimer's disease, Parkinson’s disease, and ALS have widely different 
clinical presentations they share several aspects that suggest common basic underly¬ 
ing mechanisms. All are diseases of older adults, where relatively restricted groups 
of neurons undergo progressive dysfunction and death. All usually occur as sporad.c 
illnesses, but a significant minority of cases have a clear familial basis. Several genes 
have been identified that cause inherited forms of each, almost always on an autoso¬ 
mal dominant basis, resulting in aberrant forms of proteins that appear to have a 
novel toxic gain of function. The sole exception so far is the parkin gene where loss 
of its ubiquitin ligase activity results in an inherited form of Parkinson's disease. 
Understanding the normal function of parkin may provide insight into how mecha¬ 
nisms such as protein processing, degradation, and oxidative injury interact to cause 
the selective neuronal loss of neurodegenerative diseases. 




Sangram S. Sisodia, PhD 


Molecular Mechanisms of Alzheimer’s Disease 


Sangram S. Sisodia, is The Thomas A. Reynolds, Sr., family 
professor of neuroscience and chairman of the Department 
of Neurobiology, Pharmacology & Physiology at the 
University of Chicago. Dr. Sisodia earned his BA in chem¬ 
istry from the College of Wooster (Ohio) in 1977 and his 
PhD in Biochemistry from the University of Georgia in 
1985. He conducted his postdoctoral training in the 
Department of Biological Chemistry at The Johns Hopkins 
University School of Medicine under the aegis of Drs. Don 
Cleveland and Barbara Sollner-Webb. In 1988, Dr. Sisodia 
Joined the Neuropathology Laboratories in the Department 
of Pathology at The Johns Hopkins School of Medicine as a 
Research Associate. In 1990, he was promoted to assistant 
professor of Pathology and rose to the rank of professor of 
Pathology and Neurosciences in 1998. In 1998, he joined 
the University of Chicago as chairman of the Department 
of Neurobiology, Pharmacology and Physiology. Dr. Sisodia 
has been the recipient of several awards, including the 
1997 Potamkin Prize for Alzheimer's Disease Research 
from the American Academy of Neurology, the prestigious 
Metropolitan Life Foundation Award for Medical Research 
in 1998, and selection in 1999 as the annual medical hon- 
oree of the National Alzheimer's Association. He serves on 
the editorial boards of several scientific journals, including 
Cell and Neuron. 


Mutations in genes encoding the type I membrane protein, amyloid precursor pro¬ 
tein (APP), or the polytopic membrane proteins, termed presenilins (PS I and PS2) 
cosegregate with pedigrees with autosomal dominant, familial Alzheimer's disease 
(FAD). FAD-IInked PS I mutants enhance neuronal vulnerability in vivo and promote 
processing within the APP transmembrane domain leading to increased production 
of highly fibrillogenic AB42 peptides and the acceleration of AB deposition in brains 
of transgenic mice. PS I is essential for intramembranous ("y-secretase”) processing 
of APP and an aspartyl residue at position 385, proposed to reside within the 
membrane bilayer, plays a critical role in the process. We show that a region that 
Includes aspartate 385 does not span the membrane, and that PS I with a mutation of 
aspartate 385 affects the trafficking'of both full-length APP and the carboxyl-terminal 
APP derivatives that serve as the y-secretase substrates. PS I Is also necessary for 
trafficking the BDNF receptor, TrkB, and electrophysiological studies in primary 
neurons indicate that loss of PS I function has profound effects on synaptic trans¬ 
mission. Finally, several in vivo and in vitro studies have indicated that PS I plays a 
role in the Notch proteolysis and signaling. Using a Notch-GFP chimera expressed 
in PS I wt and PS I -deficient cells, we show that PS I plays a critical role in mediat¬ 
ing ligand- or EDTA-dependent intramembranous processing of the Notch chimera 
and nuclear signaling of the cytoplasmic NICD fragment. We have recently developed 
a cell-free assay in which y-secretase processing of APP and Notch is readily 
detected and ongoing studies are aimed at the identification of PS I -associated factors 
responsible for “y-secretase” activity. 




Mervyn ], Monteiro, PhD 


Presiniiins and Alzheimer's Disease 


Dr. Monteiro is an associate professor in the Medical 
Biotechnology Center and Department of Neurology, 
University of Maryland Biotechnology Institute and 
University of Maryland School of Medicine, respectively. 

He is a member of the Neuroscience Program and associ¬ 
ate director of the Molecular and Cell Biology Graduate 
Program. He received a BSc degree in Microbiology from 
Queen Elizabeth College, University of London, and a PhD 
degree from the MRC National Institute for Medical 
Research, Mill Hill. He pursued postdoctoral studies in 
the Department of Developmental Biology at the MRC, 
Mill Hill. After a three-year postdoctoral fellowship with 
Dr. Don Cleveland at The Johns Hopkins University School 
of Medicine, he joined the faculty at UMB. His research 
in aging, with emphasis on Alzheimer's disease, has been 
supported by grants from the American Health Assistance 
Foundation and the National Institutes on Aging. 


Mutations in human presenilin I and presenilin 2 (PS I and PS2, respectively), two 
homologous genes, account for the majority (>50%) of familial Alzheimer’s dis¬ 
ease (FAD). We are studying the funaion of presenilin proteins and the mechanism 
by which FAD mutations cause disease. We have shown that overexpression of 
PS2 in HeLa cells leads to apoptosis and that the FAD PS2(N 1411) mutation causes 
increased apoptosis. Moreover, we have been the first to show that presenilins are 
involved in cell cycle regulation, as overexpression of presenilin proteins in dividing 
cells leads to arrest of cells at the G I/S phase of the cell cycle. Interestingly, cell 
cycle arrest Is potentiated by the FAD mutations in PS I and PS2 proteins. Using 
the yeast 2-hybrid system we have identified several presenilin-interacting proteins. 
One of these is a myristoylated calcium-binding protein, which we named calmyrin 
(for calcium binding myristoylated protein with homology to calcineurin). Calmyrin 
interacts with the PS2-loop region in yeast 2-hybrid assays, in vivo colocalization of 
the two full-length proteins, and by Increased binding of the two proteins by affinity 
chromatography and coimmunoprecipitation. The two proteins when coexpressed 
in HeLa cells induce additive cell death. Ubiquilin is a second and novel presenilin- 
binding protein that we identified. Yeast two-hybrid (Y2H) interaction, GST pull¬ 
down experiments, and colocalization of the proteins expressed in vivo, together with 
coimmunoprecipitation and cell fractionation studies, provide compelling evidence 
that ubiquilin interacts with both PS I and PS2. Ubiquilin is noteworthy for its mul¬ 
tiple ubiquitin-related domains, typically thought to be involved in targeting proteins 
for degradation. However, we have shown that ubiquilin promotes increased 
presenilin protein accumulation. Pulse-labeling experiments indicate that ubiquilin 
facilitates increased presenilin synthesis without substantially changing presenilin 
protein half-life, suggesting that ubiquilin may act as a molecular chaperone. Studies 
on the function of presenilin proteins and the proteins with which they interact are 
likely to provide important insights for the development of rational therapies for AD. 





Virginia Man-Yee Lee, /V16A. PhD 


Pathobiology of Synucleinopathies 


Dr. Lee is a senior fellow at the University of Pennsylvania's 
Institute on Aging. Dr Lee earned a PhD in biochemistry 
in 1973 at the University of California, San Francisco. 

In 1984, she received an MBA from the University of 
Pennsylvania. She is co-director of UPenn’s Center of 
Neurodegenerative Disease Research and a professor of 
pathology and laboratory medicine at the School of 
Medicine. Her primary research interests are the neuronal 
cytoskeleton and amyloid beta precursor proteins and their 
roles in the pathobiology of neurodegenerative diseases 
such.as Alzheimer's disease and Parkinson’s disease. 


Since the identification of mutations in the a-synuclein gene in familial Parkinsons’ 
disease (PD), a-synuclein has been implicated as a major component of the abnor¬ 
mal filaments that form Lewy bodies (LBs) in PD, diffuse Lewy body disease (DLB), 
and a Lewy body variant of Alzheimer’s disease, and glial cytoplasmic inclusions 
(GCIs) in multiple system atrophy (MSA). These neurodegenerative diseases are 
collectively known as synucleinopathies. Recent studies have shown that the 
recombinant a-synuclein assembles into 10-nm-diameter filaments that closely 
resemble those found in Lewy bodies. The A53T mutation in a-synuclein increases 
the rate of formation, as well as the amount, of assembled filaments when com¬ 
pared with wild-type a-synuclein. We also identified amino acid residues 71-82 of 
a-synuclein as essential for filament formation. Because oxidative stress has been 
impircated as a pathogenic mechanism for PD, we assessed whether or not 
a-synuclein is a target for oxidation-induced tyrosine cross-linking and tyrosine 
nitration. Using a variety of approaches, we show that a-synuciein is a target for 
tyrosine cross-linking and tyrosine nitration in LBs and GCIs. Our data provide 
evidence to directly link oxidative and nitrative damage to the onset and progres- 
Sion of neurodegenerative synucleinopathies. 








Marian DiFiglia, PhD 


Pathogenesis of Huntington’s Disease 


Dr. DiFiglia is a professor in neurology at Harvard Medical 
School and director of the Laboratory of Cellular 
Neurobiology at the Massachusetts General Hospital. 

After graduating from Queens College in New York, she 
received a PhD in neuropsychology and neurophysiology in 
1973 from the City University of New York. Her postgraduate 
studies in neuroanatomy were completed at the Mount 
Sinai Medical School in New York, and she was subse¬ 
quently appointed to the faculty in neurology there. 

Since 1980, she has been a member of the Harvard 
Medical School faculty and has conducted research at 
Massachusetts General Hospital. 


The expansion of a polyglutamine tract in the N-terminal region of huntingtin causes 
dysfunction and death of striatai and cortical neurons in Huntington’s disease (HD). 
How this occurs is unclear. A gain of function by the mutant protein has been pro¬ 
posed to explain pathogenesis. Polyglutamine expansion in huntingtin may change 
its solubility, metabolism, and/or its binding properties with interacting proteins, 
thereby leading to changes in function of the mutant protein in cells. Another 
possibility is that mutant huntingtin attenuates a function of wild-type huntingtin. 
The roie of wild-type huntingtin in neurons is unknown. Endogenous wild-type 
huntingtin localizes mainly to the cytoplasm in neurons. We showed the wide¬ 
spread distribution of huntingtin in secretory and endocytic pathways. In brain and 
fibroblasts, wild-type huntingtin associates with vesicle membranes, endosomes, 
the trans Golgi network, and plasma membranes and appears in synaptosomal 
membrane fractions by Western blot. We found clathrin, a protein required for 
membrane budding, co-localized with huntingtin on vesicles in the cytoplasm, the 
trans Golgi network, and at the plasma membrane. In axons, the huntingtin associated 
with vesicle membranes moves anterogradely by fast transport and is retrogradely 
transported, consistent with a presence on endosomes. Huntingtin’s association with 
endosomes is altered after stimulation with forskolin, which activates cyclic AMR 
or by a dopamine DI receptor agonist. This suggests that huntingtin’s role in intra¬ 
cellular membrane trafficking is linked to receptor activation at the cell surface. 
Some proteins that interact with huntingtin are localized to vesicle membranes and 
function in membrane trafficking and cytoskeleton stability. These interactors bind 
differently to mutant huntingtin than to wild-type huntingtin, thereby favoring the 
possibility that there is altered membrane trafficking in HD. 


Dana C HHt, MD 


Trophic Factors and Their Modulators in the Treatment of 
Neurodegenerative Diseases 


Dr. Hilt is vice president of Clinical Research at Guilford Trophic factors are taf^get-derived proteins that promote the growth, survival, and 

Pharmaceuticals. Inc. in Baltimore. He received his MD function of specific neurons. Originally described as essential factors for the develop- 

from Tufts University and trained in Internal Medicine at ment of the nervous system, trophic factors have been described recently as having 

Harvard University and Neurology at The Johns Hopkins the ability to promote the function and survival of adult neurons in both the normal 

University. After postdoctoral training at the NIH with adult nervous system and in specific neurodegenerative disease models. Both protec- 

Marshall Nirenberg in molecular neurobiology he joined th/e and restorative actions have been observed in preclinical animal models. For 

the Department of Neurology. University of Maryland example, nerve growth factor (NGF) can prevent cholinergic neuronal cell death and 

School of Medicine. In 1993 he was appointed director of dysfunction in models of Alzheimer’s disease. Glial-cell line derived neurotrophic fac- 

clinical neuroscience at Amgen. Inc., in Thousand Oaks. tor (GDNF) has similar trophic actions on dopaminergic neurons In Parkinson’s dis- 

Calif. where he was involved in the clinical trials ofBDNT ease models including the primate MPTP model. The activities of a number of neu- 

NT~3. and GDNF In 1998 he assumed his present position rotrophic protein growth factors in neurodegenerative disease models will be 

and is responsible for ongoing clinical development trials reviewed. In addition to protein growth factors, a number of small-molecule trophic 

testing neuroprotective small molecules in a number of factors/modulators have been developed recently. In some cases, these compounds 

neurodegenerative diseases. have actions similar to neurotrophic protein factors in disease models. For example, 

neuroimmunophylin ligands are small molecular compounds that possess neurotrophic 
activities similar to those described with FK-506 on a variety of neuronal cell types, 
but lack the immunosuppressant actions. These agents have had significant trophic 
actions In Parkinson’s disease models (MPTP protective and restorative rodent and 
primate models) and Alzheimer’s disease models (aged rodent memory function and 
biochemical enhancement of cholinergic neuron function). The small-molecule neu¬ 
rotrophic factors/modulators offer the potential benefits of oral dosing and easier pas¬ 
sage through the blood brain barrier. These problems have impaired the ability to 
study protein neurotrophic factors. Early clinical trials have been conducted with pro¬ 
tein neurotrophic factors in a number of neurodegenerative diseases including 
Alzheimer’s and Parkinson’s disease, peripheral neuropathy, and amyotrophic lateral 
sclerosis. These clinical results will be reviewed. Another review will cover early clini¬ 
cal trials initiated recently with small molecule trophic factors/modulators. 






David / Fink, MD 


Dr. Fmk is professor of neurology, molecular genetics and 
biochemistry at the University of Pittsburgh, chief of the 
Neurology Service and Director of the Geriatric Research 
Education and Clinical Center (CRECC) at the VA Pittsburgh 
Healthcare System. He received a BA from Yale College 
an MD from Harvard Medical School, completed a residen¬ 
cy in internal medicine at the Massachusetts General 
Hospital, a residency in neurology at the University of 
California San Francisco (UCSF). and trained in research 
with Dr. Harold Gainer in the laboratory of neurochemistry 
at the NIH. After 12 years at the University of Michigan, 
he moved to the University of Pittsburgh where he has 
been since I99S. Dr. Fink's research, funded by the NIH 
the Department of Veterans Affairs, and several private ’ 
foundation grants, is focused on the development of recom¬ 
binant herpes simplex virus vectors for the treatment of 
neurologic conditions. 


Modifying the Structure and Function of the Nervous System with 
Genomic HSV Vectors ^ 

There are three different strategic approaches to use HSV-based vectors to treat 

r "T"' stereotactic inoculation 

to specific sites in the central nervous system; (2) peripheral inoculation, using 

retro^de ^onal transport to deliver the vector from the skin to sensoiy neurons 
o^ root ganglion; and (3) infection of other tissue (e.g., fat) for the contin¬ 
uous production and release of the transgene product resulting in systemic delivery. 
A recombinant genomic HSV vector containing the coding sequence for the human 
anti-a^ptotic peptide bcl-2 protects dopaminer^c neurons of the rat substantir 
nigra from cell death caused by injection of the neurotoxin 6-hydroxydopamine into 

lelnT"!’ hydroxylase expression in 

esioned cells. A vector expressing glial-derived neurotrophic factor (GDNF) acts 

a ditively to improve both cell survival and neurotransmitter expression A recom- 

3o f T development of sensoo. neuropathy caused by 

overdose of pyridox.ne, and is also effective in preventing the development of dia- 

vation ^finding has been the obser- 

ImdZ-TfLrn 7 ‘he blood of at least one transgene 

product (NGF). In the streptozotocin-diabetes model in the mouse, inoculation of 

the NGF-expressing vector into fat prevents the reduction in foot sensoiy nen,e 
r^rnTr^ of neuropathy in that model. By exploiting the natural neu- 

usIZ ! ’ “ostructed a series of recombinant vectors that can be 

used to protect neurons of the central nervous system from the degeneration 
rnduced by toxins or trauma, and which may also be used to modify the physiology 
of the nervous system through synthesis and release of neurotransmitters 



Gary Fiskum, PhD 


Mitochondrial Molecular Targets for the Treatment of Acute Brain Injury 


Dr. Fiskum is a professor and research director of anesthe¬ 
siology, professor of biochemistry and molecular biology, 
and professor of pharmacology and experimental therapeu¬ 
tics. He earned his bachelor's degree from the University 
of California, Los Angeles and his doctorate in biochemistry 
from St, Louis University. As a postdoctoral fellow, he stud¬ 
ied with Professor Albert L. Lehninger at The Johns Hopkins 
University. He then served on the biochemistry faculty of 
George Washington University for 16 years before moving 
to the University of Maryland Baltimore. Dr. Fiskum is a 
regular grant reviewer for the NIH and is the recipient of 
research grant awards from the NIH. the Department of 
Defense, and several pharmaceutical firms. He has 
received international recognition for his work on mito¬ 
chondrial dysfunction and acute brain injury and serves as 
the convener of the neuroprotection research focus group 
at the University of Maryland Baltimore. 


Alterations in a few key parameters appear to be responsible for inducing both 
necrosis and apoptosis in experimental models of both acute and delayed neural 
cell death following stroke, cardiac arrest, head trauma. These alterations include 
an increase in intracellular Ca», a decrease in pH, and an increase in reactive 
o^gen specif. If the extent or the duration of these aiterations is sufficiently great 
• for prolonged periods and cellular membranes become 

irreversibly damaged, necrosis and secondary inflammatory tissue injury ensue 
f howler, cellular ATP levels can be maintained, celis may recover or proceed 
through a programmed” series of steps toward apoptotic cell death. Elevated 
ntracellular Ca ^ caused by massive influx through both ligand-gated channels e g 

mtrr r'” Kr ' ^e level of mitochondria ' 

to cripple their ability to respire and generate ATP and to stimulate the production 

of toxic reactive oxygen species. Alternatively, Ca’* may trigger the release of the 
protein cytochrome c from its normal location between the inner and outer mito¬ 
chondrial membranes into the cytosol where it acts together with other factoid to 
activate a class of proteases known as caspases that mediate the process of 
apoptosis^The release of cytochrome c and other apoptogenic proteins from 
mitochondria IS also triggered by specific pro-apoptotic intracellular proteins e g 
Bax, and inhibited by others, e.g., Bcl-2. Our work has provided insight into the 
-nechanisms of mitochondrial dysfunction that compromise cerebral energy metab¬ 
olism and promote apoptosis. This knowledge has led to the development of 
neuroprotect,ve interventions, e.g., intravenous administration of acetyl-t-carnitine 
or cyclosporin A. and exposure to hyperbaric oxygen, that are being tested in 
animal models or clinical trials for ischemic or traumatic brain injury. 





Salvatore DiMouro, MD 


Mitochrondrial Genes and Neurological Disease 


Dr. DiMauro graduated from the University of Padova in 
Italy with a specialization in neurology in 1967. He came 
to the University of Pennsylvania in 1968 and began 
receiving MDA research grants in 1969. DiMauro has 
served as an MDA scientific adviser and has written hun¬ 
dreds of articles and book chapters on muscle disorders. 
Since 1991, he has been the Lucy G. Moses Professor of 
Neurology at Columbia University. 


The small, maternally inherited mitochondrial DNA (mtDNA) is a veritable 
Pandora's box of pathogenic mutations. Thirteen years into the era of "mitochon¬ 
drial medicine," more than 100 point mutations and innumerable rearrangements 
(deletions, duplications, or both) have been associated with a bewildering variety 
of multisystemic, as well as tissue-specific, human diseases. After reviewing the 
principles of mitochondrial genetics, comparisons will be made between the clinical 
and pathological features of disorders due to mutations affecting mitochondrial 
protein synthesis and disorders caused by mutations in protein-coding genes. In 
contrast with the remarkabie progress in understanding etiology, pathogenesis is 
not completely explained by the rules of mitochondrial genetics. For example, 
we do not understand why mutations in two tRNA genes, both impairing protein 
synthesis to similar extents, should give rise to different syndromes, MERRF and 
MELAS. Nor is it understood why epilepsy is invariably present in both MERRF 
and MELAS, but only rarely in Kearns-Sayre Syndrome, another defect of mito¬ 
chondrial protein synthesis. There has been some recent progress in epidemiology 
and genetic counseling, but therapy is woefully inadequate. However, several 
therapeutic approaches are being explored and will be reviewed. 








M. Flint Beal, NiO 


Mitochondria and Neurodegeneration 


Dr Ad. Flint Beal is an Anne Parish Titzel professor of neu¬ 
rology and neuroscience and chairman of the Department 
of Neurology and Neuroscience at Weil Cornell Medical 
College in New York. Dr. Beal received his BA in 1975 from 
Colgate University and his MD in 1976 from the University 
of Virginia. Dr. Beal came to New York Weil Cornell from 
Harvard Medical School and Massachusetts General 
Hospital, where he was chief of the Neurochemistry 
Laboratory and director of the Clinical Trials Unit in the 
Department of Neurology. 


There is substantial evidence implicating mitochondria as playing a crucial role in 
both necrotic and apoptotic cell death. Mitochondria are essential in controlling 
specific apoptosis cell death pathways. There is increasing evidence implicating 
mitochondrial dysfunction in the pathogenesis of neurodegenerative diseases such 
as ALS and Huntington’s disease. We and others have shown that lactate is elevated 
in the cortex of HD patients, that there is reduced phosphocreatine to inorganic 
phosphate ratio in resting muscle of HD patients, that the maximum rate of mito¬ 
chondrial ATP generation In muscle is reduced in both symptomatic HD patients 
and presymptomatic gene carriers and that HD lymphoblast mitochondria show 
increased susceptibility to depolarization that directly correlates with CAG repeat 
lengths. Furthermore, mitochondrial toxins produce striatal lesions, which closely 
resemble the histopathoiogy of HD. In ALS, muscle biopsies show increased 
mitochondrial volume and calcium levels. There is reduced cytochrome oxidase 
activity In the anterior horn motor neurons in patients with sporadic ALS. We have 
found increased mitochondrial DMA point mutations in spina! cord tissue. A study 
of ALS cybrids show a significant decrease in complex I activity as well as trends 
towards reduced complex III and IV activities. We found increased levels of 8- 
hydroxy-2-deoxyguanosine, a marker of oxidative damage to DNA in the plasma, 
urine and CSF of sporadic ALS patients. There is also evidence of mitochondrial 
dysfunction in transgenic mouse models of both HD and ALS. We found that 
administration of creatine, which may Increase cellular phosphocreatine levels, can 
significantly improve survival and motor function in transgenic mouse models of 
both HD and ALS. We have also found that a number of other agents that improve 
mitochondrial function also show efficacy. It is, therefore, possible that therapeutic 
strategies aimed at improving mitochondrial dysfunction may be beneficial in the 
treatment of neurodegenerative diseases. 




Bruce K. Krueger. PhD 


Defective Neurotrophin Signaling in the Trisomy 16 Mouse 


Dr. Krueger is a professor of physiology at the University of 
Maryland School of Medicine and a member of the Brain 
Injury and Neuroprotection focus group of the UMB 
Program in Neuroscience. He received his BS and PhD 
degrees from Yale University. After postdoctoral training at 
Washington University School of Medicine, he Joined the 
faculty of the UMB School of Medicine in 1979. Dr. Krueger 
was the recipient of an Alfred P Sloan Fellowship in neuro¬ 
science and was awarded Fogarty and Guggenheim fellow¬ 
ships for his research while on sabbatical leave with Martin 
Raff, University College London. His current research is 
supported by the National Institutes of Health (NIA. 
NINDS) and focuses on the molecular mechanisms underly¬ 
ing neurodegeneration and abnormal brain development in 
animal models of Down syndrome and Alzheimer's disease. 


Two of the hallmarks of Down syndrome (DS; trisomy 21) are mental retardation 
and the early occurrence of Alzheimer’s disease. We have observed accelerated 
death (apoptosis) of neurons in vitro and in vivo in the hippocampus of the trisomy 
16 (Tsl6) mouse, an animal model of DS. Cultured Is 16 hippocampal neurons 
undergo accelerated apoptosis due to their failure to respond to the neurotrophin, 
BDNF, which is produced by the neurons themselves and promotes their survival. 
Is 16 neurons have elevated levels of the inactive, truncated isoform of the BDNF 
receptor, trkB, leading to the hypothesis that truncated trkB overexpression causes 
the BDNF signaling failure and accelerated death. Adenovirus-mediated introduc¬ 
tion of exogenous full-length trkB into Tsr6 neurons fully restored BDNF-mediated 
survival, whereas exogenous truncated trkB expression in normal, euploid neurons 
reproduced the Tsl6 BDNF signaling failure. Thus, aberrant expression of trkB 
isoforms selectively eliminates the ability of BDNF to promote the survival of TsI 6 
neurons and this defect can be corrected by genetic manipulation of trkB expres¬ 
sion. A general failure of BDNF signaling could contribute to neurological disorders 
not only by increasing neuron death, but also by altering the modulation of neural 
connectivity and synaptic plasticity by BDNF thereby compromising cognitive function. 




David Borchek, PhD 


Role of Protein Aggregates in the Pathogenesis of Neurogenerative Disease 


Dr. Borchek is an associate professor of pathology and 
neuroscience at The Johns Hopkins University School of 
Medicine. He received his BS, MS, and PhD degrees from 
the University of Kentucky and his postdoctoral training at 
the University of California, San Francisco, in the laboratory 
of Dr. Stanley Prusiner. In 1992 Dr. Borchek Joined the 
faculty of Johns Hopkins. Dr. Borchek is a cellular I molecular 
biologist who has worked on a variety of in vitro and 
in vivo models of genetic neurodegenerative diseases, 
including Alzheimer's disease, ALS, and Huntington's 
disease. His current work is supported by the National 
Institutes of Neurologic Disease and Stroke, National 
Institutes of Aging, the Huntington's Disease Society of 
America, and the Hereditary Disease Foundation. 


In familial Alzheimer’s disease, familial amyotrophic lateral sclerosis (FALS), and 
Huntington’s disease, mutations in specific proteins (e.g., presenilins and amyloid 
precursor protein in familial Alzheimer’s disease, superoxide dismutase I in familial 
ALS, and huntingtin in Huntington’s disease) create molecules that are toxic to 
specific populations of neurons. The mechanisms by which these mutant proteins 
injure and destroy subsets of neurons remains elusive. However, a common theme 
in each of these disorders is the extracellular, intracellular, or intranuclear accumula¬ 
tion of aggregated proteins in neurons and astroglial. Primary interest is to under¬ 
stand how these aggregates of protein lead to the dysfunction and death of selected 
populations of neurons. The mechanisms by which mutations in the enzyme 
superoxide dismutase I (SODI) cause FALS have remained elusive. In the present 
study of mice expressing three different mutant proteins (G37R, G85R. and G93A), 
we demonstrate an age-dependent aggregation of SOD I monomers into macro- 
molecular structures that are retained after filtration through cellulose acetate. 
Further, we demonstrate similar aggregates of B-amyloid peptides and tau in the 
brains of Alzheimer’s patients. We suggest that the mechanisms of motor neuron 
degeneration in FALS, caused by mutations in SOD I, may overlap with those of 
other neurodegenerative disorders where the accumulation of aggregated protein 
plays a pivotal role in disease pathogenesis. 




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