Where Your Money Goes > Grants Archive
The 2005 Parkinson's Unity Walk Funds Research Grants
Through the generous support of its corporate sponsors, 100% of all donations made to the Parkinson's Unity Walk are distributed among the major U.S. Parkinson's disease foundations for Parkinson's disease research. The foundations include: (1) American Parkinson Disease Association; (2) the National Parkinson's Foundation; (3) the Parkinson's Action Network; (4) the Parkinson's Disease Foundation; (5) the Michael J. Fox Foundation for Parkinson's Research; (6) The Parkinson Alliance; and (7) the Parkinson's Institute.
We are excited to share with you information about the following grants —all of which are made possible from 2005 Parkinson's Unity Walk distributions. We will update the grants with progress reports as they are made available to us. In September 2006, updates to several grants were provided to us. Those updates are indicated below in red.
1. The American Parkinson Disease Associations is using its distribution to fund:
Investigator No. 1: Cyrus P. Zabetian, MD, MS
Location: Geriatric Research Education and Clinical Center VA Puget Sound Health Care System, Seattle, WA
Title of Study: A Comprehensive Screen of the LRRK2 Gene in Familial Parkinson’s Disease and Dementia with Lewy Bodies
Purpose of Study: To perform a comprehensive mutational analysis of the LRRK2 gene by resequencing the entire coding region and all intron/exon junctions in 50 subjects with familial Parkinson’s disease (PD) and 20 subjects with familial dementia with Lewy bodies (DLB). To determine whether newly discovered mutations segregate with disease by genotyping all available family members in each mutation-positive pedigree. To estimate the frequency of any newly discovered putative pathogenic mutations in a large sample of sporadic PD (n=400), DLB (n=200) case, and controls (n=600).
September 2006 Project Update:
1) To perform a comprehensive mutational analysis of the LRRK2 gene by resequencing the entire coding region and all intron/exon junctions in 50 subjects with familial Parkinson’s disease (PD) and 20 subjects with familial dementia with Lewy bodies (DLB).
The sample size for screening in aim #1 was increased to 75 familial PD (fPD) patients and 20 familial DLB (fDLB) cases because (1) additional DNA samples became available, and (2) the PI obtained an ABI 3130 Genetic Analyzer in his lab (through other funding sources), thus reducing sequencing costs by eliminating reliance on core facilities. We have sequenced 49 of the gene's 51 exons, with a first-pass fail rate of approximately 5%. We anticipate completing the remaining 2 exons and all failed samples within the next 3-4 weeks, which is well ahead of schedule.
FPD sample: Thus far, we have identified five pathogenic or putative pathogenic mutations distributed among seven families. Two of these are novel missense mutations that occur in exons not previously known to harbor disease-causing variants. The most common mutation observed was G2019S, which was present in three pedigrees.
FDLB sample: We have discovered three potentially pathogenic mutations among the 20 FDLB index cases. One of these (R1514Q) has previously been implicated as a causative variant in PD. The other two are novel missense mutations that result in nonconservative amino acid substitutions in the leucine-rich repeat (LRR) and WD40 domains of the protein.
We have also observed 82 single nucleotide variants within the combined sample (fPD + fDLB) which we believe are largely nonpathogenic based on location within the gene and/or presence in control samples. Fifty-nine of these are single nucleotide polymorphisms (SNPs) already reported in public databases (e.g., dbSNP). The remaining 23 variants are novel and occur within introns or represent synonymous substitutions (within exons). Though analyses of these latter variants are ongoing, most or all of them are likely SNPs or benign mutations. We hope to use this wealth of SNP data to examine the role of common variation within LRRK2 in modifying susceptibility and age at onset in PD at the conclusion of this project (and will be submitting an application for a second year of funding to do so).
2) To determine whether newly discovered mutations segregate with disease by genotyping all available family members in each mutation-positive pedigree.
We are currently genotyping all available DNA samples from relatives of each index case found to have a pathogenic or putative pathogenic mutation, and attempting to recruit additional family members not previously enrolled in our study. We are also genotyping these mutations (and other variants of less certain relevance to disease) in control DNA samples and performing bioinformatic analyses to better differentiate benign variants from pathogenic mutations.
3) To estimate the frequency of any newly discovered putative pathogenic mutations in a large sample of sporadic PD (n=400) and DLB (n=200) cases, and controls (n=600).
We are in the process of genotyping each of the eight mutations described in aim #1 in a large sample of sporadic PD and DLB cases using TaqMan assays. The size of the sporadic PD group has doubled to approximately 800 cases with the contribution of additional DNA samples from the PI's collaborator, Dr. Haydeh Payami (
Investigator No. 1: Eunsung Junn, Ph.D.
Location: UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ
Title of Study: Effects of DJ-1 and Daxx interaction on Cell Death
Purpose of Study: To determine the role of DJ-1 in the Daxx/ASK1-mediated cell death. Daxx is known to interact with ASK1, causing its activation which subsequently promotes cell death. The role of DJ-1 will be assessed in this context in dopaminergic cells, especially regarding its ability to block cell death and ASK1 activation as well as to regulate Daxx localization. To test if the Daxx/ASK1 pathway is indeed involved in the pathogenesis of a PD model, MPP+ will be employed as a cell death inducer. If the Daxx/ASK1 pathway is activated in this model, and if DJ-1 can block cell death, ASK1 activation and Daxx location induced by MPP+ will be studied.
Reporting procedure: Activity reports will be required before the second (6 month) and third (12 months) payments are made.
September 2006 Project Update:
1.1 Wild-type DJ-1 but not the L166P mutant protects againt Daxx/ASK1 mediated cell death:
Daxx interacts with Apoptosis Signal-Regulating Kinase 1 (ASK1), causing its activation which subsequently promotes cell death demonstrated in non-dopaminergic cells 293, HeLa and DU-145 (prostate adenocarcinoma). First, we tested the effect of these two signaling molecules on the survival of SH-SY5Y dopaminergic neuroblastoma cells by transfecting them with expression vectors of these molecules and with green fluorescent protein (GFP) expression plasmid pEGFP-C1 (Clontech). Cells were plated in 4-well Poly-D-lysine coated slides (Becton Dickinson) and cultured for 24 h. Cell death was assayed using ethidium homodimer-1 (EthD-1) (Molecular Probes). This reagent enters cells that have damaged membranes and undergoes enhancement of fluorescence upon binding to nucleic acids, thereby producing a bright red fluorescence in dead cells, but is excluded by the intact plasma membrane of living cells. Following EthD-1 labeling, at least 200 EGFP-positive cells were counted. The percentage of cell death was calculated as the ratio of double-labeled EthD-1/EGFP-positive cells to single-labeled EGFP-only positive ones. Co-transfection of these cells with both Daxx and ASK1 resulted in death of 25% of transfected cells. Expression of either Daxx or ASK1 alone had no significant impact on cell viability . Based on the interaction between DJ-1 and Daxx as well as their co-localization, we tested the functional role of DJ-1 in Daxx/ASK1 signaling by co-transfecting FLAG-tagged DJ-1. Compared with the 25% cell death associated with the combined expression of Daxx and ASK1, the inclusion of a wild-type DJ-1 expression vector in the transfection mix significantly reduced cell death (Figure 1). On the other hand, the co-expression of the disease associated L166P mutant DJ-1 was unable to protect cells against this death signaling pathway. These observations indicate that wild-type DJ-1 protects against Daxx/ASK1 induced cell death. Of note is the observation that L166P DJ-1 did not aggravate cell death induced by Daxx/ASK1, negating the suggestion that it might act as a dominant negative mutant.
1.2. Wild-type DJ-1 blocks Daxx-induced ASK1 activation:
To study the impact of DJ-1 on ASK1 activation, COS-7 cells were transfected with Myc-ASK1, HA-Daxx and FLAG-DJ-1. ASK1 was immunoprecipitated using anti-Myc (9E10), followed by in vitro kinase assay in a reaction buffer consisting of 20 mM Tris-HCl (pH 7.5), 20 mM MgCl2, 5 Ci [-32P] ATP for 20 min at 30C using myelin basic protein (MBP) (40 g/ml) as substrate. Samples were resolved in SDS-PAGE and subjected to autoradiography. The presence of wild-type DJ-1 (lane 3) markedly inhibited Daxx-induced ASK1 activation (lane 2), while L166P mutant DJ-1 was incapable of blocking ASK1 (lane 4). As the Daxx/ASK1 pathway kills cells through apoptosis.
Specific Aim 2. To determine the role of Daxx/ASK1 pathway in MPP+-induced cell death.
MPTP impairs mitochondrial complex I activity, depletes ATP and leads to oxidative stress. Cells were plated in 96-well tissue culture plates at a density of 2.5 X 104 cells/well, cultured for 24 hr, followed by treatment with increasing concentrations of MPP+ for 24 hr. The dose-dependent decline in cell survival with this insult was significantly mitigated by DJ-1 over-expression (Figures 3), consistent with recent report. The role of DJ-1 in MPP+-induced cell death was further investigated by experiments using siRNA against DJ-1. SH-SY5Y cells were transfected with different concentrations of wild-type or mutated siRNAs and to DJ-1 using Mirus TransIT-TKO reagent (Mirus). Endogenous DJ-1 expression was analyzed by Western blotting with anti-DJ-1 antibody (Stressgen). Wild-type siRNA significantly reduced DJ-1 expression in a concentration-dependent manner, while mutant siRNA did not. Under these conditions, MPP+-induced death in siRNA-transfected cells accelerated with a profile that correlated with the concentration of siRNA and with the decrease in DJ-1 expression. These results further confirm that DJ-1 confers protection against MPP+-induced cell death.
2.2 Determine if wild-type DJ-1 inhibits ASK1 activity induced by MPP+ treatment.
Firstly, we attempted to investigate if MPP+ can activate the Daxx/ASK1 pathway. SH-SY5Y cells were challenged with 3 mM MPP+ for various durations, according to published treatment paradigms in these cells. Lysates were immunoprecipitated with anti-ASK1 antibody (H-300,
2. The National Parkinson Foundation is using its distribution to fund the following:
Grant Awarded to: Vincenzo Bonifati, M.D., Ph.D, Erasmus Medical Center, Rotterdam Department of Clinical Genetics
Project title: Genetic Determinants of Early-Onset Parkinson’s Disease
By unraveling the genetic defects causing rare forms of PD, the researchers expect to understand better and faster the mechanisms underlying the common, non-hereditary forms of the disease. The researchers aim to analyze the DNA (genetic code) from many patients with PD and their relatives in order to identify novel genes defective in this disease. They believe that this strategy will ultimately open novel avenues for curing and preventing PD.
September 2006 Project Update:
The recent discovery of genes which, when abnormal, cause rare inherited forms of PD has provided tremendous help for the understanding of the mechanisms involved in the degeneration of the dopamine-producing neurons; this might offer clues for the indentification of novel targets to develop a cure for all the patients with PD.
In this project we analyze the genetic code (DNA) from many patients with early-onset PD and their relatives, with the primary aim to identify novel genetic defects causing the disease. At the same time, we perform extensive analysis of the known genes for PD in order to identify novel mutations and study the associated clinical features. Unraveling novel genetic defects causing rare, inherited forms of PD will facilitate understanding the mechanisms underlying the common, non-hereditary forms. We believe that this is the way for the identification of novel strategies for the cure and prevention of PD.
Grant awarded to: Jonathan Sebat, Ph.D, Cold Spring Laboratory
Project Title: Determining Genetic Causes of Parkinson’s Disease by ROMA
ROMA is a DNA microarray chip methodology that enables the researchers to scan the entire genome at high resolution and to identify the disease-causing mutations directly, greatly accelerating the process of identifying disease genes. The initial efforts in the use of ROMA to study a neurological genetic disorder have proven successful. By scanning the genomes of 80 patients with autism, the researchers succeeded in identifying mutation in several disease-causing genes. This project will be a pilot study to analyze the DNA of 100 patients diagnosed with Parkinson’s disease by ROMA in order to ﬁnd gene copy number polymorphisms speciﬁcally associated with Parkinson’s disease (PD), and to identify candidate PD genes in these regions.
September 2006 Project Update:
The primary goal is to evaluate the potential role of genes and their protein products in the pathology of Parkinson’s disease (PD), and to identify based on genetic and functional data the best candidates for further study. This project is a very important complement to our ongoing genetic studies of PD. The specific aims of this project are to perform computational analysis of gene function, to predict functional interactions between the known genetic risk factors and the candidate disease genes identified in our study, and to determine how genetic variants identified in our study may alter protein function. Lastly, we propose to confirm these functional effects experimentally. This research is a collaboration involving Dheeraj Malhotra (CSHL) Xiayoue Zhao (CSHL), Lilia Iakoucheva (
3. Parkinson's Action Network (PAN), founded in 1991, is the unified education and advocacy voice of the Parkinson's community, fighting for a cure. Through education and interaction with the Parkinson's community, scientists, lawmakers, opinion leaders, and the public at large, PAN works to increase awareness about Parkinson's disease and advocates for increased federal support for Parkinson's research.
PAN also provides the information and resources necessary to empower people with Parkinson's disease to act on their own behalf and gain a greater sense of control over their health and their future. For more information on PAN, please see its website at www.parkinsonsaction.org.
4. Parkinson’s Disease Foundation is using its distribution to fund the following:
Investigator: Dr. Ying Tan
Project Title: Interaction between PD and Nrdp1 in drosophila models
Parkin is a gene that was very early discovered to, when deficient cause familial parkinsonism of the young-onset of “juvenile” type. It too, is autosomal-recessive, thought to be a “loss-of-function” PROBLEM. Since drosophila (fruit fly) models are now available, many scientists are using these models to investigate genetic functions (they are easily bred and inexpensive). Dr. Ying Tan at the University of Massachusetts (Worcester) will use his funds to generate yet another drosophila model that overproduces a protein called Nrdp 1 that destabilizes the production of parkin in cultured animal cells. His hypothesis is that this protein may modify the features observed in the model with either elevated or reduced levels of the parkin protein. He and his colleagues will also cross these models with those over-and under-expressing other proteins such as alpha-synuclein in an attempt to determine how multiple modifications can affect the molecular pathogenesis of human Parkinson’s disease.
Investigator: Dr. Nicholas Hallworth
Project Title: Mechanisms Underlying Pathological Rhythmic Burst Firing in Subthalamic Nucleus Neurons Following Dopamine Dennervation
Over the nearly twenty years of studying the use of deep-brain stimulation for symptomatic control of PD symptoms, most surgeons agree that regulating the subthalamic nucleus in this manner provides the best relief. This brain area’s irregularity seems to be caused by the degeneration of mid brain dopaminergic (DA) neurons. Dr. Nicholas Hallworth will use his PDF funds to continue working with his mentor. Dr. Mark Bevan, at Northwester University (Chicago). He will compare electrical activities of brain tissue obtained from healthy animals with that of reserpinized (lesioned) mice, in an attempt to clarify both the mechanism(s) underlying and the site of origin of the abnormal activity within the brain. He concluded that his study’s results could contribute to the refinement of high-frequency stimulation procedures and may provide a foundation for the rational development of even better symptomatic treatments.
October 2006 Project Update:
Summary of completed research – Dr. Nicholas Hallworth – IRGP 2005-2006
Parkinson’s Disease is associated with the progressive degeneration of a subpopulation of dopamine-containing neurons within the midbrain. In both Parkinson’s Disease patients and animal models of the disease a proportion of neurons within the subthalamic nucleus, a structure targeted by midbrain dopamine neurons, exhibit abnormal activity. In particular, some subthalamic neurons fire in rhythmic bursts, the frequency of occurrence of which can be phase-related to parkinsonian resting tremor. High-frequency stimulation of the subthalamic nucleus through surgically-implanted electrodes has emerged as a valuable therapy for Parkinson’s Disease patients for whom dopamine replacement medications have proved ineffective. However, this relatively invasive intervention is not without risks, and the manner by which high-frequency stimulation exerts its remedial effects is still not entirely understood. The general objective of work performed during this period of funding was to elucidate how the degeneration of midbrain dopamine neurons leads to pathophysiologically-relevant rhythmic burst firing within the subthalamic nucleus. The electrophysiological properties of subthalamic neurons within brain slices obtained from normal animals were compared to the properties of neurons within slices from animals having an acute dopamine depletion or a stable lesion of midbrain dopamine neurons. This analysis revealed that, while the intrinsic properties of subthalamic neurons are unchanged by dopamine loss, the impact of a major inhibitory input to the subthalamus is enhanced. Given that this enhancement may underlie the abnormal rhythmic burst firing observed in vivo, further characterization of the cellular and molecular mechanisms that mediate this change is warranted. An understanding of these mechanisms may be critical for the refinement of high-frequency stimulation therapies and may provide a foundation for the rational development of novel anti-parkinsonian treatments.
Investigator: Dr. Mihaela A. Stavarache
Project Title: In vivo silencing of PINK1 expression using Adeno-Associate Virus vectors
Yet another gene responsible for a hereditary form of early-onset parkinsonism is PINK 1 (PTEN-induced putative kinase 1). It is known to be an autosomal recessive mutation. This suggests that the problem is caused by a loss of function rather than an overdose of genetic material. Dr. Mihaela S.A. Stavarache will continue her post-doctoral work at Weill Medical College (Cornell University, New York) using a genetic technique known as RNA interface (RNAi) that shuts down or “silences” a disease gene while leaving others untouched. An adeno-associated virus of the type used in inserting, for example, growth factors into a laboratory animal will be used to take the RNAi molecule into the brains of rodents so that the gene’s function can be studied in the dopamine-producing neurons of the substantia nigra, a brain area affected in classic Parkinson’s disease. Dr. Stavarache and her colleagues hope to prove that loss of PINK 1 can cause neuronal death and that the obverse, or over expression of the gene, might protect nigral dopaminergic neurons during life. As with all of the genetic studies ongoing, the goal of this study is to learn the mechanisms of the classic disease that, to date, remain unknown.
5. The Michael J. Fox Foundation for Parkinson's Research will use its grant toward the Community Fast Track initiative and Biomarkers II program.
Under the Community Fast Track initiative, researchers are invited to submit investigator-initiated grant application to conduct new, novel or innovative research relevant to the cure, cause, prevention, or improved treatment of PD and its complications. Biomarkers II is a two-year research program designed to accelerate the development and validation of a biomarker of Parkinson’s disease. For more information see its website at website at www.michaeljfox.org.
6. The Parkinson Alliance is committed to funding the most promising and scientifically validated Parkinson's disease research that will help find the cure to Parkinson's. Funding from the Parkinson's Unity Walk helped finance the Michael J. Fox Foundation for Parkinson's Research "Community Fast Track." The annual program seeks out proposals that represent a new approach or new concept, and have the potential to significantly advance the field of Parkinson's research.
In addition, the funds from the Parkinson's Unity Walk will support 16 specific research grants. Selection of a grant is on the basis of scientific merit of the project. This is determined by scientific advisory boards of the major Parkinson's foundations. For more information please visit its website at www.parkinsonalliance.org.
7. The Parkinson’s Institute is using its distribution to fund the following:
Project Title: The cause and consequence of abnormal a-synuclein aggregation
Principal Investigator: Seung-Jae Lee
Deposition of amyloid-like fibrillar protein aggregates is a common pathological feature of many human neurodegenerative diseases, including Parkinson's disease (PD). While the direct role of these fibrillar inclusion bodies in disease progression is the subject of intense debate, it has become evident during recent years that protein conformational defects that lead to fibrillation are an integral part of the pathogenic mechanism. Therefore, understanding the molecular mechanisms underlying protein misfolding and aggregation might hold the key to unraveling common pathogenic mechanisms for these devastating neurologic disorders. One of the main goals of my research group is to elucidate the pathophysiological function of a-synuclein, a neuronal protein that is implicated in several human neurodegenerative diseases, including PD, dementia with Lewy bodies, and multiple system atrophy. The objective of the current project is to understand the cause and consequence of abnormal a-synuclein aggregation at the molecular and cellular levels and to use this information to further understanding of the pathogenesis of PD. Knowledge we obtain from this study will eventually lead to the development of new strategies that are designed to prevent the progression of a-synuclein pathology, something which would be most welcome in the current research climate where new strategies for neuroprotection are being searched for intensively.
September 2006 Project Update:
Background: Deposition of amyloid-like fibrillar protein aggregates is a common pathological feature of many human neurodegenerative diseases, including Parkinson’s disease (PD). While the direct role of these fibrillar inclusion bodies in disease progression is the subject of intense debate, it has become evident during recent years that protein conformational defects that lead to fibrillation are an integral part of the pathogenic mechanism. Therefore, understanding the molecular mechanisms underlying protein misfolding and aggregation might hold the key to unraveling common pathogenic mechanisms for these devastating neurologic disorders.
Project Goals: The primary aim of the research carried out with funding from the Parkinson’s Unity Walk during this past year was to elucidate the pathophysiological function of a-synuclein, a neuronal protein that is implicated in several human neurodegenerative diseases, including PD, dementia with Lewy bodies, and multiple system atrophy. More specifically, we wished to understand the cause and consequence of abnormal a-synuclein aggregation at the molecular and cellular levels and to use this information to further understanding of the pathogenesis of PD. Success in this area has a high probability of leading to the development of new strategies that aimed at the altering progression of a-synuclein pathology, something which would be most welcome in the current research climate where new strategies for neuroprotection are being searched for intensively.
Results: The work carried out during the last year focused on understanding the conformational properties of a-synuclein to further define the role of this protein in these diseases. We found that, in vitro, a-synuclein can stably bind synthetic phospholipid vesicles through its N-terminal repeat region, which is devoid of stable structure. Upon binding to lipid membranes, this region forms an elongated a-helix. However, it has not been demonstrated whether the helix-mediated membrane interaction of a-synuclein occurs in cells, nor has it been understood how the membrane interaction is regulated. To address these issues, we have determined the membrane binding properties of a-synuclein to biological membranes by using bi-functional chemical crosslinkers, which allow the detection of transient, but specific, interactions. By utilizing various point mutations and deletions within a-synuclein, we found that the membrane interaction of a-synuclein in cells is also mediated by a-helix formation in the N-terminal repeat region. Moreover, the PD-linked A30P mutation causes reduced membrane binding, which is concordant with the artificial membrane studies. However, contrary to the interaction with artificial membranes, the interaction with biological membranes is rapidly reversible and is not driven by electrostatic attraction. Furthermore, the interaction of a-synuclein with cellular membranes only occurs in the presence of non-protein and non-lipid cytosolic components, which distinguishes it from the spontaneity of the interaction with artificial membranes. These results suggest that in cells, a-synuclein is engaged in a fundamentally different mode of membrane interaction than the charge-dependent artificial membrane binding, and the mode of interaction is determined not only by the intrinsic properties of a-synuclein itself but also by the cytoplasmic context.
Significance: These observations are hugely important for the investigators carrying out research in this field as they suggest that studies using artificial membranes cannot be generalized to living organisms. Furthermore, chemical cross-linker mediated stabilization of otherwise transient membrane-bound helical a-synuclein provides a valuable tool to analyze the dynamic membrane interaction. We believe that this tool will prove invaluable in determining the role of this enigmatic protein both in health and in disease.