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Parkinson's Fast Track Grants Meeting at ASENT -- Program

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2003 Event Series Calendar

Parkinson's Fast Track Grants Meeting at ASENT -- Participants

Capital Hilton Hotel, Washington DC

March 15, 2003

Introduction

List of Parkinson's Disease R21 Fast Track Award Winners

Executive Summary


Parkinson's Disease Fast Track R21 Program Sponsors

Meeting Sponsors

NINDS' Parkinson's Disease Research Program

Agenda

Abstracts

Participant list


The Parkinson's Disease R21 Fast Track Awards Program

In 2002, the NIH and numerous Parkinson's disease foundation organizations jointly developed a grant announcement requesting research on Parkinson's disease. The purpose of this request was to invite investigator initiated applications focused on advancing our understanding of Parkinson's Disease as relevant to its treatment, cause, prevention, or cure. The granting mechanism was the R21, defined as high-risk, potentially high-impact proposals, designed to generate new preliminary data, or test new ideas which may be of important impact to PD.

Investigators with diverse scientific interests were encouraged to direct their expertise to both basic and applied research; grants were requested that would enhance our understanding of the genetic and environmental causes of PD, develop appropriate models relevant to understanding and treating the disease, or to develop innovative strategies to prevent, limit, or reverse the pathogenesis of PD. An important component of disease cure or prevention is the translation of basic discoveries to their eventual application to the patient, so studies utilizing translational or "bench-to-bedside" approaches were encouraged.

The granting announcement was unique, not only in that it encompassed a large number of supporting organizations (see below), but also that it was "Fast-tracked". Grants were submitted electronically in a shortened form. These files were then sent to the reviewers for their consideration before the review committee met. We received an unprecedented number of applications, over 200, and needed 3 review subcommittees to fully review all the proposals. The highest scoring grants were discussed programmatically with all the sponsors, and because there were additional meritorious grants that warranted additional dollars, the voluntary community generously contributed additional funds to these important projects. Finally the grants received approval and concurrence from the NINDS Council and were awarded within several months of their receipt date.

Today the investigators will be sharing their preliminary data on Parkinson's disease research with the wider community and the RFA sponsors. This booklet highlights their abstracts and findings.

Participating organizations:

  • National Institute of Deafness and Communication Diseases (NIDCD)
  • National Institute of Environmental Health Sciences (NIEHS)
  • National Institute of Mental Health (NIMH)
  • National Institute for Neurological Disorders and Stroke (NINDS)
  • The Michael J. Fox Foundation (MJFF)
  • The National Parkinson's Foundation (NPF)
  • The Parkinson Alliance (PA)
  • The Parkinson's Disease Foundation (PDF)
  • The Parkinson's Unity Walk
  • The Safra Foundation.

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Parkinson's Disease R21 Fast Track Award Winners

Alberto Ascherio, MD, PhD
Harvard University
School of Public Health

Krys Bankiewicz, MD, PhD
University of California at San Francisco

Marie Csete, PhD
University of Michigan

Lee A. Cunningham, PhD
University of New Mexico

Ted M. Dawson, MD, PhD
Johns Hopkins University

Julie Desbarats, PhD
McGill University

Grigori Enikolopov, PhD
Cold Spring Harbor Laboratory

Anthony L. Fink, PhD
University of California at Santa Cruz

Gary M. Fiskum, PhD
University of Maryland at Baltimore

Peter T. Fox, MD
University of Texas Health Science Center at San Antonio

Lawrence I. Golbe, MD
Robert Wood Johnson Medical School/University of Medicine & Dentistry of NJ

Su Guo, PhD
University of California at San Francisco

Theo Hagg, MD, PhD
Dalhousie University

John F. Houde, PhD
University of California at San Francisco

Peng Huang, PhD
Medical University of South Carolina

Lorraine M. Iacovitti, PhD
Thomas Jefferson University

Susan Lindquist, PhD
University of Chicago

Jerilyn A. Logemann, PhD
Northwestern University

Anne Messer, PhD
Wadsworth Center

David G. Morgan, PhD
University of South Florida

George A. Oyler, MD, PhD
University of Maryland at Baltimore

Vincent A. Pieribone, PhD
John B. Pierce Laboratory, Inc

Lorraine O. Ramig, PhD
University of Colorado

Eric K. Richfield, MD, PhD
University of Rochester

David Ron, MD
New York University

Scott J. Sherman, MD, PhD
University of Arizona

Ann L. Smiley-Oyen, PhD
Iowa State University of Science and Technology

Caryl E. Sortwell, PhD
Rush-Presbyterian-St. Luke's Medical Center

Michal K. Stachowiak, PhD
State University of New York at Buffalo

Chantal E. Stern, D.Phil
Boston University

Caroline M. Tanner, MD, PhD
The Parkinson's Institute

Takao Yagi, PhD
Scripps Research Institute

Naoki Yoshimura, PhD
University of Pittsburgh


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Lay Language Summaries of Projects Presented at the 2003 PD Fast Track Awards Meeting
Written by: Susan A. Daniels, PhD and Edited by: Diane Murphy, PhD
NINDS Neurodegeneration Program, NIH

The goal of the Parkinson's Disease R21 Fast Track Awards Grant Program was to stimulate novel or high impact approaches relevant to the cure, cause, prevention, or improved treatments of Parkinson's disease. The Parkinson's disease research community responded enthusiastically to this solicitation, and the resultant awarded grants cover a wide variety of topics within the following broad areas.

  1. Molecular Genetics and Cell Biology of Parkinson's Disease
  2. Environmental Factors, Oxidative Stress and Neuroprotection in Parkinson's Disease
  3. Quality of Life Issues for PD Patients
  4. Experimental Neurotherapeutics and Outcome Measures

The projects from this grant program presented at the 2003 PD Fast Track Awards Meeting are each summarized below.

Molecular Genetics and Cell Biology of Parkinson's Disease

Julie Desbarats et al.
A New Molecular Trigger for Nerve Regeneration

This is an innovative study of the molecule Fas and its ability to promote regeneration of damaged neurons. Fas was previously known to be involved in the process of cell death, but not in regeneration. Recently this group published a paper demonstrating that Fas can induce dramatic regeneration of damaged nerves. Using gene chip technology, they demonstrated that the genes turned on by Fas in its regenerative capacity are different from the genes turned on by other growth molecules - suggesting that the regenerative effects of Fas represent a truly novel neuronal repair pathway. Further study of this pathway may lead to a new set of drugs targets to repair nerve cells in Parkinson's disease.

Lawrence Golbe et al.
Glutathione S-Transferase Polymorphisms in Parkinson's Disease

This group is examining the DNA from several families, including one in which the first PD gene was discovered. They are looking for variations in a gene called glutathione s-transferase (GST), which can help to breakdown environmental toxins within cells. It is thought that variations or mutations in GST could be responsible for increasing or decreasing a person's susceptibility to neurotoxins from the environment, affecting the age of onset of PD. If it is shown that variations in GST are indeed correlated with age of onset in a family with known genetic predisposition to PD, then proteins in the GST toxin-processing pathway may be targets for new drugs to delay or prevent the onset of PD.

Su Guo et al.
Modeling Parkinson's Disease in Zebrafish

These investigators have created a new zebrafish animal model of Parkinson's disease (PD) that can be used to investigate the neurodegenerative process of PD as well as to rapidly screen for new small molecule compounds that can halt neurodegeneration. The zebrafish contain normal and several mutant forms of the human alpha-synuclein transgene. The expression levels of the transgenes are currently being characterized, as well as the degree of neurodegeneration in the brain, and alterations in locomotor behavior. Future work includes taking advantage of the relatively simple but powerful genetics of zebrafish to identify additional genes that may be involved in the process of dopamine neuron degeneration, as well as screening for candidate drugs to stop or reverse the neurodegenerative process.

Eric Murphy et al.
Alpha-synuclein Alters Fatty Acid Uptake but not Targeting in Stably Transfected HEK-293 cells

This basic science study explores the normal physiological function of alpha-synuclein, one of the protein components of the Lewy Body aggregates that are a hallmark of Parkinson's disease. Using a cell culture system, these researchers are examining the possible role of alpha-synuclein in fatty acid metabolism. Fatty acids are molecules required to maintain the integrity of cell membranes, and thus are essential for maintaining the health of all cell types, including brain cells. Results of this study have thus far shown that alpha-synuclein significantly alters the ratios of different fatty acid within cells, but does not alter their spatial distribution in cell membranes as would be expected if alpha-synuclein was a fatty acid binding protein. Studies are ongoing to more clearly define the role of alpha-synuclein in brain lipid metabolism. Understanding of the normal role of alpha-synuclein in the lipid metabolism may clarify if loss of this function in brain cells contributes to the symptoms or progression of Parkinson's disease.

George Oyler et al.
Parkin Facilitates the Elimination of Expanded Polyglutamine Proteins and Leads to Preservation of Proteasome Function

Parkin is the most commonly mutated gene in found in familial Parkinson's disease. This group has shown that normal parkin, in conjunction with stress induced proteins, promotes the "marking" of misfolded proteins so they can be removed from the cell. They have developed a way to visualize the protein degradation machinery (called the proteasome), showing that normal parkin reduces proteasome impairment when exposed to abnormal proteins. This suggests that when parkin is mutated, the proteasome may be less able to perform its function of degrading abnormal proteins, leading to cell dysfunction or cell death. Understanding the function of normal and mutated parkin in brain cells will shed light on the commonalities of the neurodegenerative process in both familial and sporadic PD, and may eventually lead to new targets for drugs to stop or reverse the disease process.

Scott Sherman et al.
Selective Control of Basal Ganglia Output Pathways; Development of a Primary Culture Model System

This group has developed a new cell culture system that models the two opposing pathways that facilitate or suppress motor activity in the basal ganglia of the brain. Imbalances in the regulation of these two pathways are thought to underlie the motor impairments seen in Parkinson's disease. Once the model has been optimized, it will be used to test if gene therapy can be used to control these pathways. If gene therapy is successful in restoring the model to normal activity, similar gene therapy may move to animal models and eventually to human patients to correct the motor deficits caused by PD.

Michal Stachowiak et al.
Reduced FGF Signaling May be an Underlying Cause of Parkinson's Disease

This group is investigating a novel genetic pathway for Parkinson's disease that may be involved in cases of PD unrelated to parkin and alpha-synuclein mutations. Fibroblast growth factor (FGF) had been previously associated with dopamine neuron degeneration. This group introduced a mutated form of the FGF receptor gene into the brains of rats. They found that these rats show progressive loss of neurons in brain regions affected in PD, as well as related behavioral deficits. This suggests that abnormalities in the FGF signaling pathway could lead to PD or PD-like disorders. Further studies of this potential new genetic pathway for PD may lead to enhanced understanding of the cause of non-parkin/alpha-synuclein related PD and reveal novel therapeutic targets for PD.

Environmental Factors, Oxidative Stress and Neuroprotection in Parkinson's Disease

Gary Fiskum et al.
Regulation of Reactive Oxygen Species Generation by Brain Mitochondria

In this study, the processes by which mitochondria function normally in cells to produce energy have been examined. They have found several independently functioning pathways that, when disrupted, may lead to cell death in response to genetic defects or environmental factors. Certain threshold levels of molecules called free radicals, generally known to cause cell damage, may also be able to trigger neuroprotective responses within cells that counteract oxidative stress. If these beneficial responses can be triggered without damage, these pathways may provide new targets for dietary interventions or drugs to treat or prevent PD.

Eric Richfield et al.
Genetic Loci Contributing to Mouse Neurotoxicant Parkinsonism

Most cases of Parkinson's disease are thought to be caused by a complex combination of genetic and environmental risk factors. This group hopes to identify genes that may play a role in susceptibility to environmental toxins implicated in PD. By comparing physical and behavioral effects of chemical exposures in normal vs. inbred mice, then correlating these effects to changes in specific regions of DNA, this group has identified regions of chromosomes associated with brain changes caused by chemical exposures. They plan to narrow down the regions to identify new genes that may be responsible for susceptibility to or protection against chemical exposures that cause Parkinson's-like disorders. This study may identify new genes that are responsible for cases of Parkinson's disease not associated with already known PD genetic pathways.

David Ron et al.
ER Stress in Cellular Models of Parkinson's Disease

This study examines the pathways that regulate response of brain cells to environmental stressors such as the neurotoxins that may play a role in some forms of Parkinson's disease. This group has shown in a cell culture model that the integrated stress response (ISR) pathway is activated to promote cell survival against stressful stimuli. Data obtained suggest that changes in gene expression in response to the activation of this pathway by drugs can sustain the neuroprotective effects over time. Better understanding of this neuroprotective pathway may lead to new targets for drugs that could stimulate neuroprotection to prevent progression of PD-related neurodegeneration.

Yagi et al.
Use of a Single Subunit NADH Dehydrogenase (NDI1) to Retard Parkinson's Disease

Environmental chemicals that are known to cause Parkinson's-like neurodegeneration and symptoms are known to inhibit a group of proteins called Complex I within the mitochondria in brain cells. Complex I is required for the normal functioning of mitochondria, which provide energy to power the cells. In vitro studies by this group have suggested that a component of Complex I, called NDI1, may exert neuroprotective effects on cells exposed to neurotoxic chemicals. This group has been developing a method for delivering NDI1 to brain cells in mice; they will then expose the mice to neurotoxic chemicals and determine if the introduced NDI1 was able to protect neurons from damage. If this method is validated and optimized, it may lead to the development of an analogous tool for use in human patients to retard PD.

Quality of Life Issues for PD Patients

John Houde et al.
The Neural Substrates of Impaired Feedback Control in Parkinsonian Speech

Parkinson's patients suffer from a variety of speech disorders that have a significant impact on their quality of life. This study focuses on the ability of PD patients to alter their speech in response to changes in pitch or volume of sounds they hear. A clinical study is underway using psychological and imaging methods to determine which conditions give PD patients the most difficulty. fMRI will identify the brain regions involved in speech responses and the sequence of neural activities that result in parkinsonian speech deficits. Better understanding of the mechanisms of speech pathology in PD will promote refined targeting of treatments for those disorders.

Lorraine Ramig et al.
PDA-Enhanced Speech Treatment for Parkinson's Disease

This group is doing a clinical study to examine the feasibility and effectiveness of using a specially programmed personal digital assistant (PDA) device to supplement and enhance the benefit of the Lee Silverman Voice Treatment (LSVT), a regimen of motor exercises and sensory training to treat speech disorders caused by Parkinson's disease. The PDA can serve as a surrogate "trainer" for the patient, making the benefits of LSVT available even to patients who are unable to travel to medical centers that offer LSVT training with clinicians. Pilot data has been obtained from two individuals thus far which support the use of this device to deliver and enhance LSVT. In addition to making LSVT available to a much larger proportion of PD patients currently affected by speech disorders, the PDA could be used to collect data from patients nationwide to document and enhance the clinical effectiveness of speech treatments such as LSVT.

Ann Smiley-Oyen et al.
Changes in Motor Skills with Practice in Parkinson's Disease: Preliminary Results in Rapid Aiming and Buttoning

Improvement of motor capabilities is an important quality of life issue for patients living with Parkinson's disease. This group is doing a clinical study to determine whether motor behaviors in PD patients can be improved by extensive practice of motor tasks. Preliminary data suggest that movements requiring high accuracy are more readily enhanced by this therapy than movements that require speed and strength. Other experiments are planned to discover which types of movement therapy are most effective in improving sensory-driven tasks, repetitive motor tasks and tasks that require a sequence of movements with shifts in weight.

Naoki Yoshimura et al.
Therapeutic Effect of Dopaminergic D1 Agonist in Lower Urinary tract Dysfunctions in 6-hydroxydopamine Lesioned Parkinsonian Rats

Lower urinary tract dysfunction is a commonly observed problem in Parkinson's disease patients that adversely impacts quality of life. This study has examined the effects of dopamine D1 agonist drugs on bladder overactivity in a rat model of PD, and found that dopamine D1 agonists only affect a specific subset of muscle activities related to overactive bladder. This refined information, if verified, may lead to more effective use of dopamine D1 agonists to treat bladder dysfunction experienced by PD patients.

Experimental Neurotherapeutics and Outcome Measures

Peng Huang et al.
Global Statistical Tests for Parkinson's Disease Clinical Trials

Clinical trials to test new therapies that may benefit PD patients is an essential component to developing the best treatments or cures. However, clinical trials are only meaningful when the results are appropriately interpreted. A variety of statistical tests can be used to evaluate results, but it is difficult to compare results of one trial with another. This group is developing Global Statistical Tests that could be used to provide better, more objective analysis of data from Parkinson's disease clinical trials. These tests measure and weight multiple variables in a single test battery, and if used as a standard in PD clinical trials, would increase the ability to compare results from different trials. Use of such tests will maximize the utility and benefit of these trials to Parkinson's patients.

Anne Messer et al.
Intrabody Therapy of Parkinson's Disease

Intrabodies are a special type of antibodies that are engineered specifically to adhere to proteins. In this study, investigators propose to develop an intrabody to counteract the misfolding of alpha-synuclein protein in cells (the accumulation of which is an early step in PD pathology) to attempt to correct the misfolding. If effective, an intrabody could be introduced into the diseased brain cells of Parkinson's patients via gene therapy to reverse the damaging effects of misfolding and subsequent accumulation of misfolded proteins such as alpha-synuclein in PD.

David Morgan et al.
Vaccination with Recombinant Alpha-Synuclein

This group is developing a vaccine approach to treating Parkinson's disease that also targets the abnormal aggregates of proteins found in the neurons of PD patients. These aggregates, called Lewy bodies, play a role in the disease process and eventual death of dopamine neurons. Alpha-synuclein is one of the major protein components of Lewy bodies. In this study, mutant forms of alpha-synuclein are injected into rodents to raise antibodies that bind the alpha-synuclein and allow the cells of the immune system to remove abnormally aggregated alpha-synuclein from brain cells. Once a molecular construct of alpha-synuclein is developed that is effective in raising a strong immune response against alpha-synuclein (a vaccine), this vaccine may be tested for safety and efficacy in rodent models of PD and, if successful, eventually in human clinical trials.

 


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PARKINSON'S DISEASE FAST TRACK AWARDS PROGRAM SPONSORS

NINDS' Parkinson's Disease Research Program

For more than three decades, NINDS has been heavily invested in PD research. We have supported early studies of L-dopa, fundamental research on the brain circuitry affected by PD, the development of critical animal models, and important advances in understanding the genetic basis of parkinsonism. In the late 1990s, NINDS expanded these efforts by establishing the Morris K. Udall Centers of Excellence for Parkinson's Disease Research. Today's meeting, co-sponsored by NINDS along with other PD community partners, is the first gathering of researchers who were awarded funds for Parkinson's disease research under the innovative Parkinson's Disease R21 Fast Track Awards Program. This recent grant program on PD was successfully co-funded by NIH and private research agencies and serves as a model for productive collaboration between these groups.

Useful websites:

NINDS Home Page

NIH Clinical Trials Website

 

NINDS Parkinson's Research Web

NINDS Translational Research on Cognitive Sequelae of PD


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AGENDA

Capitol Hilton, Washington DC
March 15, 2003

12:00-1:00 pm Luncheon
Introduction/Welcome from Meeting Sponsors:
  • Diane Murphy, NINDS/Neurodegeneration Program
  • Ira Shoulson, ASENT
  • Robin Elliot, Parkinson's Disease Foundation
  • Carol Walton, Parkinson Alliance

1:00-1:45 pm "Data Blitz"
Slide Presentations
  • Alberto Ascherio
  • Julie Desbarats
  • Su Guo
  • Peng Huang
  • Anne Messer
  • Lorraine Ramig
  • Scott Sherman
  • Ann Smiley-Oyen
  • Takao Yagi
  • Naoki Yoshimura

1:45-2:45 pm Gene/environment interactions in PD (Discussion Moderator: Cindy Lawler, NIEHS)
  • Gary Fiskum - Overview of oxidative mechanisms
  • Eric Richfield- Gene/environment interactions

2:45- 3:00 pm Break

3:00-4:00 pm Cell biology studies of PD (Discussion moderator: Ted Dawson)
  • David Ron: ER stress in PD
  • Susan Lindquist: Yeast models of PD

4:00-5:00 pm General discussion, Poster sessions

Alberto Ascherio

Julie Desbarats

Su Guo

Peng Huang

Anne Messer

Lorraine Ramig

Scott Sherman

Ann Smiley-Oyen

Takao Yagi

Naoki Yoshimura

1:45-2:45 pm Gene/environment interactions in PD

Gary Fiskum - Overview of oxidative mechanisms

Eric Richfield- Gene/environment interactions

Discussion Moderator: Cindy Lawler, NIEHS

 

2:45- 3:00pm Break

3:00-4:00pm Cell biology studies of PD:

David Ron: ER stress in PD

Susan Lindquist: Yeast models of PD

Discussion moderator: Ted Dawson

4:00-5:00pm General discussion, Poster sessions


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ABSTRACTS

Epidemiology of Cognitive Dysfunction and Parkinson's Disease
PI: Alberto Ascherio
Institution: Harvard University School of Public Health

Cognitive function is affected early in Parkinson's disease (PD), but few studies have addressed the determinants of this cognitive decline. We are conducting a case-control study of cognitive function in PD nested within two large ongoing cohorts, the Health Professional Follow-up Study (HPFS - comprising over 50,000 men followed since 1986) and the Nurses' Health Study (NHS - comprising over 120,000 women followed since 1976). Participants in these studies have provided detailed, validated, and updated information on diet and several aspects of lifestyle. Further, blood samples or cheek-cells have been collected from most participants for genetic analyses. We have confirmed 470 incident cases of PD in these cohorts, and we are now conductive telephone interviews to assess cognitive function among these cases and age- and sex- matched controls. Administered tests include the Telephone Interview for Cognitive Status (TICS) plus additional tests of memory and verbal fluency. We have so far completed the interviews in 86 men with PD and 169 controls. Individuals with PD had lower scores than controls in each of the tests. The mean TICS score was 32.1 among cases (range 21 to 39) and 33.6 among controls (range 24 to 40) (P=0.001). This difference in mean TICS score between cases and controls is equivalent to10 years of aging among people without PD in our study. In future analyses we will take into account the duration of disease and explore the associations of several lifestyle factors with cognitive function. Further, we will determine the APOE genotype of participants and determine its role in predicting cognitive decline and its interactions with other risk factors.

A New Molecular Trigger for Nerve Regeneration
Authors: J.-S. Palerme, M. Rongy-Mimouni, A.M. Landau, and J. Desbarats
Institution: McGill University

Fas (CD95) is a member of the tumor necrosis factor receptor (TNF-R) superfamily, best known for its role as a "death receptor". Fas can trigger neuron apoptosis in animal models of stroke in vivo, and in motoneurons in vitro. However, we have previously shown that Fas can also promote growth, proliferation, and differentiation in a variety of cell types.

Here we show that Fas engagement on sensory neurons can induce a dramatic regenerative response, resulting in prolific axon growth in vitro and accelerated recovery after nerve damage in vivo (Nature Cell Biol. 2003, 5:118-125). We demonstrate that Fas-mediated axon regeneration is independent of the Fas apoptotic pathway, but requires activation of the Extracellular-signal Regulated Kinase (ERK) pathway. Furthermore, our data indicate that Fas engagement can promote neuron survival and preserve neuritogenic potential, as well as stimulate new axon growth. Fas engagement triggered axon regeneration that was morphologically and kinetically similar to that induced by the neurotrophin nerve growth factor (NGF). However, DNA microarray analysis revealed a family of genes uniquely upregulated during Fas-stimulated neural regeneration, that was not induced during NGF-stimulated regeneration.

Our data show that Fas is a trigger for axon regrowth, and suggest that Fas may engage an as yet unstudied intracellular program of neural regeneration. This new mechanism for promoting neuron survival and axon regrowth may lead to a novel approach in the treatment of neurodegenerative diseases such as Parkinson's Disease.

Regulation of Reactive Oxygen Species Generation by Brain Mitochondria
Authors: Gary Fiskum1, Anatoly Starkov2, and Alicia Kowaltowski3
Institution: 1Univ. of Maryland School of Med., 2Weill Medical College Cornell Univ., Univ. San Paolo, Brazil

Mitochondrial dysfunction, either due to environmental or genetic factors, can result in excessive production of reactive oxygen species (ROS), triggering the apoptotic death of dopaminergic cells in Parkinson's disease. Mitochondrial ROS generation is promoted by the inhibition of electron transport at any point distal to the sites of superoxide production. Neurotoxins that induce Parkinsonian neuropathology, e.g., MPP+ and rotenone, stimulate superoxide production at Complex I of the electron transport chain. We tested the hypothesis that TCA cycle dehydrogenases also contribute to mitochondrial ROS production. Amplex Red fluorescent measurements of H2O2 formation by isolated forebrain mitochondria were performed in the presence of different NADH-linked respiratory substrates. H2O2 production varied with the type of respiratory substrate, with rates supported by a -ketoglutarate > pyruvate > malate @ glutamate during physiological, State 3 (phosphorylating) respiration. This order is not related to relative rates of respiration, as would be expected if the electron transport chain was the sole source of H2O2. Experiments performed with purified a -ketoglutarate dehydrogenase demonstrated substrate-dependent ROS production that was greatest at high NADH/NAD redox state, as exists when Complex I is even partially inhibited by agents e.g., rotenone.

Mitochondrial ROS production is accelerated by events other than inhibition of Complex I, including the release of cytochrome c mediated by the pro-apoptotic protein Bax, or by Ca2+ (1). The anti-apoptotic mitochondrial protein Bcl-2 inhibits both these pathways but by different mechanisms (2,3). Bcl-2 inhibition of cytochrome c release caused by osmotic swelling due to Ca2+ and pro-oxidant activation of the mitochondrial permeability transition pore is due to resistance of Bcl-2-containing mitochondria to NAD(P)H oxidation (3). We tested the hypothesis that the resistance is due to lower rates of ROS production. Overexpression of Bcl-2 in PC12 and GT1-7 neural cells resulted in a 40-100% increase in the rate of mitochondrial ROS production. We now hypothesize that Bcl-2 affects mitochondrial structure and function in a way that actually increases basal oxidative stress, and that this sub-toxic stress results in the overexpression of anti-oxidant systems that increase cellular resistance to potentially lethal oxidative stress.

These results have implications in the possible treatment of Parkinson's disease through dietary regulation of fuel for aerobic metabolism, and through the use of agents that possess one or more of the anti-death mechanistic properties of Bcl-2.

  1. Starkov, A.A., Polster, B.M., and Fiskum, G., Regulation of mitochondrial reactive oxygen species generation by calcium and Bax., J. Neurochem. 83, 220-228 (2002)
  2. Polster, B.M., Kinnally, K.W. and Fiskum, G., BH3 domain peptide induces cell-type selective mitochondrial outer membrane permeability, J. Biol. Chem. 276, 37887-37894 (2001).
  3. Kowaltowski, A.J., Vercesi, A.E. and Fiskum, G., Bcl-2 prevents mitochondrial permeability transition and cytochrome c release via maintenance of reduced pyridine nucleotides, Cell Death Diff. 7, 207-214 (2000).

Glutathione S-Transferase Polymorphisms in PARK1 Parkinson's Disease
Author: Lawrence I. Golbe, MD
Institution: UMDNJ- Robert Wood Johnson Medical School, Department of Neurology

This project tests the hypothesis that mutations in glutathione S-transferase (GST) isozymes influence onset age in Parkinson's disease (PD) caused by the PARK1 mutation. Environmental toxins, some of which are degraded by GST-dependent pathways, have been implicated in the cause or onset age of PD. This work takes advantage of the PARK1 "genetic model" of PD to seek insight into genetic and environmental bases for the wide variance in onset age of all PD.

In 1997 our group and collaborators reported PARK1, a G209A nucleotide (A53T amino acid) substitution in -synuclein at 4p21 in one Italian and two Greek families. Since then several other Greek families have been reported with PARK1 PD. Despite this unitary etiologic factor, the onset age of PARK1 PD ranges from 22 to 86. Intraneuronal -synuclein aggregation in the form of Lewy bodies occurs as the pathological hallmark of sporadic and most familial PD, including PARK1 PD. Therefore, explaining the variation in onset age could offer a means of usefully delaying the onset of all PD.

We have preliminary data from 14 affected members of the Italian family (the "Contursi kindred") showing a strong trend toward a relationship of younger onset age with homozygosity for the A313G allele of glutathione-S-transferase P1 (GST-P1) gene (p=.051, Mann-Whitney rank sum test; p=.079, 2-sample t-test). We did not examine GST genes other than P1.

The protocol calls for enlarging our N by adding at 20 samples from affected members of Greek PARK1 PD families. Our aims are to 1) Analyze our enlarged set of DNA samples for polymorphic alleles of GSTM1, -M3, -P1, -T1 and -Z1 and to determine their effect, alone or in combination, on PD onset age; and 2) Assay GST activity in lymphoblasts from some of the affected individuals to determine the functional significance of the mutations we may associate with PD onset age.

Since initial funding, we have made fresh contact with all three US branches of the Contursi kindred that are at risk, ascertaining and confirming the diagnosis in one newly affected individual. We have also requested, received and processed DNA and blood samples from the consultants outside the PI's institution, augmenting our collection of DNA samples from affected carriers of the mutation to 37. This will be ample to test our hypothesis and we may yet receive more samples from our European consultants.

We have also refined the typing procedures for the various GST polymorphisms and have applied those assays to a control population. The validity of our assays is excellent as measured by the close resemblance of our allelic distributions of the four GST isoenzyme genes to those in the literature.

Modeling Parkinson's Disease in Zebrafish
Authors: Kayta Kobayashi and Su Guo*
Institutions: Department of Biopharmaceutical Sciences, Programs in Pharmaceutical Sciences and Pharmacogenomics, *Biological Sciences, and *Human Genetics, University of California, San Francisco

Parkinson's Disease (PD), the 2nd most common neurodegenerative disorder, is characterized by an age-dependent loss of dopaminergic neurons leading to disabling motor impairments including difficulty initiating movements, resting tremors, and slowness of movement. We hope to understand the molecular basis of PD pathogenesis and progression through a zebrafish model of PD. We have generated a transgenic zebrafish model of PD that express human alpha-synuclein ( a -syn), a protein believed to be central to PD, in neurons of the fish. So far, ten different lines that express either the wildtype or the mutant forms (A53T or A30P) of a -synuclein have been created. We have begun to characterize these lines at the cellular and molecular level by Western blot analysis to determine the expression level of a -syn and the presence of oligomerized a -syn, which is believed to have toxic properties. We will further determine the neurotoxicity of a -syn expression by examining the states of dopamine neurons and the locomotor behavior of zebrafish. Later, we plan to utilize the strength of the zebrafish model to conduct genetic screens to further elucidate the process of dopaminergic neuron degeneration seen in PD. Furthermore, these fish can be used to screen for small molecule compounds that can halt dopamine neuron degeneration.

The Neural Substrates of Impaired Feedback Control in Parkinsonian Speech
Authors: John F. Houde, Robert S. Turner, William J. Marks
Institution: University of California, San Francisco

Patients with Parkinson's disease (PD) have a variety of difficulties with speech production. These deficits may result in part from a reduced ability of PD patients (PDs) to use sensory feedback to control movement - a hypothesis consistent with studies showing that PDs have impaired vocal responses to changes in the pitch frequency or amplitude (volume) of auditory speech feedback. We seek to determine the neural substrates of these impairments. In our experiments, PD and normal subjects vocalize while listening to perturbations of the frequency or amplitude (loudness) of the pitch in the auditory feedback of their speech. These perturbations cause pitch-perturbation responses (PPRs) - compensatory changes in pitch frequency or amplitude that are well characterized for normal subjects but only minimally characterized for PDs. Currently, we are conducting psychophysical experiments to more fully describe how PPRs of PDs and normals differ, and to determine what pitch perturbation parameters cause the clearest differences. We then plan to identify the neural systems involved in PPRs in PDs and normals. This will be done using fMRI scanning of PDs and normals producing or passively listening to PPRs. We will find CNS regions that are more active after PPRs than after unaltered vocalizations, but show no activity when the subject passively listens to a normal and pitch-perturbed speech. After finding areas of the CNS that appear involved in PPRs, the third aim of the study will determine the order of their neural activities resulting in a PPR. To examine this, we will induce PPRs in vocalizing PD and normal subjects while using magnetoencephalography and electroencephalography to record the sequential activation of different brain regions following perturbation but preceding the PPR. Better understanding of the neural substrates of impaired speech feedback control in PDs will elucidate the pathophysiology of speech disorders associated with PD and promote refined targeting of treatments for those disorders.

Global Statistical Tests for Parkinson's Disease Clinical Trials
Authors: Peng Huang, Ph.D.; Barbara C. Tilley, Ph.D.; Yuko Palesch, Ph.D.; Jordan E. Jaskwhich, M.A; Kenneth Bergmann, MD; Paulo Guimaraes, Ph.D.
Institution: Medical University of South Carolina

Comparing treatment efficacy in Parkinson's disease studies often involves multiple correlation outcomes to obtain an objective assessment due to the lack of known biomarkers for the disease. Several types of statistical tests have been proposed in the literature for treatment comparisons with multiple outcomes. However, which test is appropriate depends on how we define a beneficial treatment. The advantages and disadvantages of these tests and the situations where these tests are appropriate to use are discussed here. We then focus on the case when treatment efficacy requires that the majority of outcomes have shown improved results. We review the principles of available nonparametric global statistical tests and illustrate their applications using data from previously conducted trials on Parkinson's Disease.

Objective. To compare Parkinson's disease treatment when treatment efficacy requires evidence of treatment benefit from a majority of correlated outcomes.

Background. Clinical trials for Parkinson disease typically have multiple outcomes. However, most trials are designed based on a single primary outcome, such as some weighted linear combination of several outcomes. When no single outcome is accepted as a gold standard for treatment comparison, results based on a single outcome often leave findings from other outcomes unclear. On the other hand, in order to examine several outcomes simultaneously, one often makes sample size unnecessarily large in order to preserve the overall statistical significance level. A global statistics test (GST) is a single statistical test that can consider all outcomes simultaneously with a prespecified significance level and combine evidence from each single outcome to yield an assessment of the overall treatment difference. This provides a more objective conclusion about treatment efficacy than Bonferroni adjustment and Hotelling's T2 test when treatment efficacy requires improvement from a majority of outcomes.

Methods. The applications of two types of GSTs are illustrated using data from the Multicenter Randomized Controlled Trial of Remacemide Hydrochloride as Monotherapy for PD (RAMP) and data from the multicenter controlled clinical trial Deprenyl and Tocopherol Antioxidative Therapy of Parkinsonism (DATATOP). We define a parameter that measures the overall treatment effect based on multiple correlated outcomes. Subject's total ranks from different outcomes are used in nonparametric GST. The advantage of using GSTs is shown through the comparisons of power and the length of confidence intervals between GST and univariate test.

Conclusions. GSTs are more powerful tests for comparing treatment effects with multiple correlated outcomes, especially when the preference of new treatment requires evidence from several outcome measures. GSTs could be considered for use in Parkinson's disease clinical trials.

Intrabody Therapy of Parkinson's Disease
Authors: Anne Messer, Chun Zhou, Yangsheng Zhou, William Wolfgang and Michael Sierks*,
Institution: Wadsworth Center, NY State Dept. of Health and Univ. at Albany, Albany, NY and *Arizona State Univ., Phoenix. AZ

This project is developing engineered intracellular single-chain antibodies (intrabodies) as novel potential clinical reagents and drug discovery tools for the treatment of PD. Intrabodies utilize the specificity of antibodies to form complexes with intracellular proteins. Once complexed, the properties of the target protein can be changed. The capacity to alter the solubility, or to redirect a-synuclein within cells, should allow amelioration of the first step in Parkinson's Disease pathology, a-synuclein misfolding. We have recently shown that this approach leads to functional correction in brain slice cultures for a model of Huntington's Disease. The intrabodies used in the PD studies are being selected from a human phage display library representing the diversity present in a normal human spleen. The target sequences include full-length synuclein in both its native monomer and fibrillary forms, as well as peptides chosen for their putative involvement in the pathogenic process. All single-chain Fv genes are transferred to mammalian expression vectors, and tested for specificity and stability using a nuclear localization assay. The successful ones are then assayed for their capacity to protect against overexpression of wild-type and mutant a-synuclein in cell lines, and in a Drosophila model of PD. Long-term goals include administering these antibody reagents using gene therapy vectors, or as stable, multi-functional proteins.

Vaccination with Recombinant alpha-synuclein
Authors: C. Cao, Q. Li, Y. Bai, M. Gordon, R. Hauser, D. Morgan and K. Ugen
Institution: University of South Florida

Abstract: Parkinsons Disease (PD) is a serious neurodegenerative disorder in which novel therapies are needed. The protein alpha synuclein (ASyn), a component of neuronal inclusions called Lewy bodies, has a pathogenic role in PD. We decided to develop a vaccine strategy against PD which targets ASyn. This approach was by analogy to our work on the beta amyloid 1-42 peptide against Alzheimers Disease. The ASyn gene was amplified from a brain cDNA library, cloned into a GST-fusion prokaryotic vector and its identity was confirmed by nucleotide sequencing. Furthermore, the recombinant ASyn demonstrated good immunoreactivity by ELISA and Western Blotting methods using commercial monoclonal and polyclonal antibodies. Recombinant ASyn was inoculated into mice and rats and demonstrated considerable anti-ASyn antibody titers after only one vaccination (approximate titer of 5,000). The ASyn gene was also subcloned into pcDNA3.1(+) to develop a DNA based vaccine. Further comprehensive characterization of the nature of the immune responses to ASyn is ongoing. Our results indicate that the recombinant ASyn protein generated is very immunogenic in rodents in terms of the generation of specific antibody responses. Also, preliminary results indicate that a DNA plasmid based vaccine against ASyn can, at least in some animals, result in the generation of antibody responses. These results lay the foundation for testing of these anti- ASyn preparations in rodent models of PD

Alpha-Synuclein Alters Fatty Acid Uptake but not Targeting in Stably Transfected HEK-293 Cells
Authors: Nelson Colea, Robert K. Nussbauma, and Eric J. Murphyb
Institution: aGenetic Disease Research Branch, NHGRI, NIH, bDepartment of Pharmacology, Physiology, and Therapeutics, University of North Dakota,

Abstract: Alpha-synuclein is a protein found in the CNS that is associated with familial Parkinson disease, but for which a physiological role is poorly defined. Recently, alpha-synuclein has been proposed to function as a fatty acid binding protein (FABP) in the CNS based upon fatty acid binding studies in vitro (Sharon et al, 2001). However, binding studies in vitro do not necessarily correlate with function within a more physiologically relevant system. Further, fatty acid association with fatty acid binding proteins does not obligate an increase in fatty acid uptake into cells, as demonstrated by intestinal (I) and liver (L) FABP expression in L-cell fibroblasts (Murphy, 1998, Prows et al, 1996, Murphy et al, 1996, Prows et al, 1995). Thus, to better define the physiological role of alpha-synuclein, we examined the influence of alpha-synuclein expression on fatty acid uptake and trafficking in stably transfected HEK-293 cells. Fatty acid tracers were dissolved in ethanol and cells were incubated with 2 µM fatty acid (below the CMC) in the absence of serum albumin. Ethanol concentration in the medium was 0.2%. Tracer specific activities were 5.35 nCi/nmol for [1-14C] arachidonic acid; 4.4 nCi/nmol for [1-14C] palmitic acid; and 4.42 nCi/nmol for [1-14C]docosahexaenoic acid. Our results demonstrate a dramatic reduction in initial rate of fatty acid uptake in alpha-synuclein expressing compared to control cells. The initial rates were reduced 65%, 50%, and 65% from control for [1-14C] palmitic acid, [1-14C] arachidonic acid, and [1-14C] docosahexaenoic acid, respectively. However, only palmitic acid demonstrated an alteration in fatty acid targeting, with more palmitate being esterified into the phospholipid compartment and less into the neutral lipid compartment in alpha-synuclein expressing cells relative to control. Each of the fatty acid tracers demonstrated rapid esterification into stable lipid compartments, with >90% of the fatty acid esterified within 15 min. This is consistent with the rapid esterification of fatty acids demonstrated in other cell culture systems as well as in tissues in vivo. FABP also influence phospholipid synthesis and steady-state phospholipid levels, with expression in cellular systems resulting in an increase in steady-state phospholipid levels (Jolly and Murphy, 2003). In contrast, in alpha-synuclein expressing HEK cells, the total phospholipid level was significantly (p<0.001) reduced 20%, while the cholesterol content remained unchanged. This resulted in an elevation in the cholesterol to phospholipid ratio, a major determinant of membrane order. Cardiolipin, phosphatidylcholine, and sphingomyelin masses were also significantly reduced in the HEK expressing cells, suggesting alpha-synuclein expression altered the basal phospholipid metabolism. These data are inconsistent with alpha-synuclein functioning as a FABP; however these data do not negate such a hypothesis. Nonetheless, alpha-synuclein expression appears to alter lipid metabolism in stably transfected HEK cells. Studies are on-going to determine the role of alpha-synuclein in brain lipid metabolism, using gene-abated mice as well as defining the potential affect of the mutant forms of alpha-synuclein on lipid metabolism.

Parkin Facilitates the Elimination of Expanded Polyglutamine Proteins and Leads to Preservation of Proteasome Function.
Authors: Oyler, G.A., Tsai, Y.C., Thakor, N.V., and Fishman, P.S.
Institution: University of Maryland, VA Medical Center Baltimore, Johns Hopkins University

Parkin, the most commonly mutated gene in familial Parkinson's Disease, encodes an E3 ubiquitin ligase. A number of candidate substrates have been identified for parkin ubiquitin ligase action including CDCrel-1, o-glycosylated a -synuclein, Pael-R, and synphilin-1. We now show that parkin