Research > Grants Archive

The 2009 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) the American Parkinson Disease Association; (2) the National Parkinson 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 and Clinical Center.

We are excited to share with you information about the following grants —all of which are made possible from 2009 Parkinson's Unity Walk distributions. We will update the grants with progress reports as they are made available to us.


 

1. American Parkinson Disease Association is using its distribution to fund:


Project Title:
The Potential Role of Anti-Oxidant Systems in the Mechanism of DJ-1-Dependent Astrocyte-Mediated Neuroprotection.

Investigator: David A. Hinkle, MD, Ph.D., University of Pittsburgh, Dept. of Neurology, Pittsburgh, PA

Objective: The goal of this project is to identify the mechanism of astrocyte-mediated neuroprotection against rotenone.

September 2010 Project Update:

Background: Occupational pesticide exposure is associated with an increased risk for sporadic Parkinson’s disease (PD). Rotenone is a pesticide that causes experimental PD in animal models. DJ-1 is a cytoprotective protein that is robustly-expressed in sporadic PD reactive astrocytes, but not neurons. DJ-1 mutations that eliminate its expression cause genetic PD. We have found that (i) DJ-1 over-expressing astrocytes are more protective of neurons against rotenone than wild-type astrocytes, and that (ii) DJ-1 under-expressing astrocytes are less protective. The mechanism is not known, but when identified could be targeted for astrocyte-centered disease-modifying therapy against PD.

Design: DJ-1 over-expressing vs. under-expressing astrocytes will be assessed for their relative capacities to (i) express neuroprotective anti-oxidant molecules and enzyme systems and to  (ii) protect neurons against oxidative injury induced by rotenone and other oxidative neurotoxins.

Results: We have developed and validated a novel 96-well plate-based neuronal survival bioassay that will speed our progress. We have not found significant alterations in astrocyte anti-oxidant systems in our model. However, we have found that DJ-1 modulated, astrocyte-mediated neuroprotection is highly-selective for the neurotoxic process of mitochondrial respiratory complex I inhibition. Interestingly, this is the key process through which rotenone and several other environmental pesticides kill neurons, and complex I deficiency is seen in PD brain tissues. Thus, our findings may be highly relevant to sporadic PD, and future investigations regarding the mechanism of astrocyte-mediated neuroprotection in our system may produce novel therapies.


Project Title: Assessing the Pathogenic and Molecular Consequences of FOXO Regulation by Parkin

Investigator: Marc Tatar, Ph.D., Professor, Dept. of Ecology and Evolutionary Biology, Broward University, Providence, RI

Objective:
  Foxo  proteins in mammals and Drosophila ( fruit fly) is a key regulator of the balance between cell survival and apoptosis.  The investigator proposes to establish how parkin regulates foxo, and whether foxo is an intermediary factor that transduces parkin into neuropathology.

September 2010 Project Update:

Background:  FOX (Forkhead box) proteins are a family of transcription factors that play important roles in regulating the expression of genes involved in cell growth, proliferation, differentiation, and longevity. The defining feature of FOX proteins is the forkhead box, a sequence of 80 to 100 amino acids forming a motif that binds to DNA. Foxo has 4 known variants that have different cell regulation effects. Parkin is a protein which in humans is encoded by the PARK2 gene. The precise function of this protein is unknown; however, the protein is a component of a multiprotein E3 ubiquitin ligase or  protein ennzyme complex which in turn is part of the ubiquitin-proteasome system that mediates the targeting of proteins for degradation.. Mutations in this gene are known to cause a familial form of Parkinson’s disease known as autosomal recessive juvenile Parkinson disease.
 

Design: The investigator aimed to understand foxo regulation and its neuronal consequences. In mammals, foxo is broken down prior to degradation, but all of the responsible E3 ligases remain unknown.  It is from this context that the investigator has made a striking observation: over-expression of parkin markedly elevates levels of dfoxO protein.

Results:
  Dr. Tatar has discovered that excessive expression or activity of Parkin results in elevated levels of one of the FOX protein transcription (the process by which the message in DNA results in synthesis of proteins) factors. He showed that Parkin and foxo interact in the fruit fly [Drosophila] to affect the fly’s climbing ability…which is an indication of the fly’s neuromuscular coordination. This model has the potential to shed light on mechanisms in autosomal recessive juvenile Parkinson disease as well as the sporadic non-familial type of PD.



Project Title: Co-transplantation of Human Retinal Pigment Epithelial  Cells to Prevent Immune Rejection of Human Embryonic Stem Cell Xenografts in Parkinsonian Rats

Investigator: Amanda Rowlands,Penn State College of Medicine,Hershey, PA

Objective:  This proposal will examine the usefulness of human retinal pigment epithelial cells (hRPE) co-grafted with human embryonic stem cells (hES) in non-immunosuppressed 6-OHDA striatal lesioned hemiparkinsonian rats to provide functional recovery from Parkinsonism over a period of 3 months. 

September 2010 Project Update:

Background: 
Transplanting dopaminergic cells into the striatum of Parkinson patients has shown promise in experimental trials.  However, most dopaminergic transplants including newly defined hES have the disadvantage to requiring continuous systemic immunosuppression for successful engraftment.  Systemic immunosuppression comes with the risk of opportunistic infections and medication induced side effects.  hRPE are the exception to this requirement as they have been successfully transplanted into the striatum of non-immunosuppressed parkinsonian animals and in patients with Parkinson’s disease (PD) with no evidence of a deleterious host immune response.  Preliminary studies suggest that hRPE cell transplants are immune protected from host inflammatory cells via the secretion of immunosuppressive substances from the grafted hRPE cells and that they protect cografts of hES cells for up to 3 weeks from immune rejection.  

Design: 
Five groups of rats will receive the following treatments:
Group 1.  hRPE mixed with hES cells attached to microcarriers
Group 2.  hES attached to microcarriers.
Group 3.  hES without microcarriers
Group 4.  hES xenografts (nomicrocarriers) with chronic daily cyclosporine injections
Group 5.  hES xenografts attached to microcarriers with daily injections of cyclosporine.

September 2011 Project Update:

The investigators have shown that hRPE cells transplanted with hES cells provided immune protection from rejection for 15 days in a rat model. The completion and success of this project will be a basis for future studies.


Project Title:  Identification of Proteins Interacting with GIGYF2, the Product of a Putative Parkinson’s Disease Gene.

Investigator:  Dustin Freckleton, Baylor College of Medicine, Houston, TX

Objective:  The long term goal of this project is to identify intracellular pathways of GIGYF2 protein interactions that may help to shed light on Parkinson’s disease (PD) pathogenesis.

September 2010 Project Update:

Background: Mutations in the gene, known as GIGYF2, appear to be directly linked to the development of Parkinson's in people with a family history of the disease. Evidence suggests however, the effect of these mutations on protein function is unknown.  Mutations in the GIGYF2 gene appear to be directly linked to the development of Parkinson's in people with a family history of the disease. Therefore, in addition to studying normal protein interactions, the investigator will generate expression clones containing point mutations identified in PD patients.

Design: The interaction of gene known as GIGYF2 was tested against the protein products of other known PD genes as well as the known interactors of these genes (a total of over 70 different gene products). A brain cDNA library was screened to identify new GIGYF2 protein interactors.

Results: The investigators developed molecular methods to study different point mutations within the GIGYF2 gene complex, and work is underway to discover what interactions occur with the protein products of these mutations within the cell.

September 2011 Project Update:

The investigators developed molecular methods to study different point mutations within the GIGYF2 gene complex.  This method will help for future studies on the interactions that occur with the protein products of these mutations within the cell.  The project is complete and the method was successfully developed.


Project Title: Elucidating Relationships between Uricemia and Clinically Relevant Outcomes in Parkinson’s Disease:  Secondary Analysis of Data from a Randomized Trial.

Investigator: Daniel A. Lieberman, Massachusetts General Hospital, charlestown, MA

Objective: Studies supporting the hypothesis that serum urate levels are predictive of Parkinson’s disease (PD) risk and progression raise the possibility that elevating serum urate might be useful to prevent and/or treat PD.  A major clinical trial is currently underway to examine the effectiveness of the urate precursor inosine as a treatment to slow the progression of PD.

September 2010 Project Update:

Background:  Parkinson’s disease is a neurodegenerative movement disorder affecting approximately one percent of individuals over 55.  Treatment with dopaminergic agents such as levodopa is usually initiated when symptoms begin to interfere with quality of life and primarily control the symptoms of the disease.  Prolonged treatment with dopaminergic agents often leads to significant, disabling side effects, such as dyskinesia.
Oxidative damage is thought to play a role in the neuronal degeneration in PD.  Several studies have shown that healthy individuals with higher blood uric acid levels (within the upper limit of the laboratory normal range) have a reduced risk for developing PD.  

Design: 
Data from a randomized clinical trial, in which some study subjects received a uric acid precursor, was used to examine the hypothesis that blood urate (uric acid) levels are predictive of treatment related dopaminergic motor complications and quality of life measures in PD patients.

Results: 
The investigators found that blood urate (uric acid) levels did not predict the timing of onset of complications associated with the standard dopaminerigic drug therapy for PD.
 

September 2011 Project Update:

The investigators found that blood urate (uric acid) levels did not predict the timing of onset of complications associated with the standard dopaminerigic drug therapy for PD.  The project is complete and there is no further progress to report.



Project Title: Consolidation of Motor Skills and Sleep Homeostasis in Parkinson’s Disease

Investigator: April Pruski, Sophie Davis School of Biomedical Education, New York City, NY

Objective:  Little is known about the neural mechanisms that may be responsible for impairment in the execution of automatic routine movements. This study will be the foundation to understanding Parkinson disease (PD) and the role of sleep and its importance to determine the effects on neurologically normal subjects. 

September 2010 Project Update:

Background: 
Recent studies have suggested that one of the problems in performing daily motor tasks in PD patients may be due to an impairment of motor memory consolidation. In patients with PD, the execution of automatic tasks is already significantly impaired in the early stages of the disease. This impairment, which becomes progressively more severe, exacerbates the motor symptoms characteristic of PD and adds a significant burden to the execution of daily activities and to the patient’s and caregiver’s quality of life.

Design: 
The investigators studied the effects on brain wave activity of eight healthy subjects using high-density EEG recordings while performing two motor tasks. The subjects were then tested at night on their motor skills while sleeping and retested in the morning in order to understand, if consistent in PD patients.

Results:
The investigator found motor tasks did not occur at the same time, but occurred near the onset of the movement and were more evident during the task execution. The study is ongoing and will help shed light on the mechanisms underlying the problem of inability to perform daily tasks, particular those of automatic nature. 

September 2011 Project Update:

The investigator found motor tasks did not occur at the same time, but occurred near the onset of the movement and were more evident during the task execution. The project is complete and there is no further progress to report.

2. National Parkinson Foundation is using its distribution to fund the following:

Project Title:  NPF Quality Improvement Research Initiative

Investigator(s):

Jay Nutt, MD, Medical Director, NPF Center of Excellence at Oregon Health Sciences University.

Andrew Siderowf, MD, MSCE, Fellow at the University of Pennsylvania Institute on Aging and Medical Director of the University of Pennsylvania Parkinson’s Disease and Movement Disorders NPF Center of Excellence.
 

Project Description:  NPF, in partnership with 43 top medical centers (NPF's Centers of Excellence), has launched the first data-driven quality improvement research initiative to systematically improve care for every patient diagnosed with Parkinson’s disease. With a goal of tracking every patient seen in every NPF Center of Excellence (35,000-50,000 patients worldwide), this research initiative will generate the largest outcomes database in Parkinson’s disease.  Our ultimate aim is to create and share models of excellent care, so that every Parkinson’s patient receives the most effective treatment options available, whether they are seen by a specialist at a NPF Center of Excellence, a general neurologist or their primary care physician.  Financial support from the Unity Walk will support the initial pilot test of NPF's Quality Improvement Research Initiative, which involves collection and analysis of 600 patient records to be completed by January 2010.

September 2010 Project Update:

Objectives:
1)  Identify correlations between therapeutic choices and improved outcomes
2) Establish an evidence base as a foundation for future innovation
3) Improve the body of research to support evidence-based medicine decisions

Background:  With a goal of tracking a comprehensive sample of the patients seen in the NPF Centers of Excellence (35,000-50,000 patients), NPF’s QII already represents the largest international outcomes registry in Parkinson’s disease.  Our ultimate aim is to quantify and evaluate care as practiced at NPF’s Centers of Excellence in order to identify and share models of “best practices”, with the goal of providing every Parkinson’s patient the most effective treatment options available, whether they are seen by a movement disorder specialist, a general neurologist or their primary care physician.

Update/Results:  NPF launched the project in early 2009, and expanded clinical data collection from six to seventeen centers in 2010.  Components of this initiative include:

  • Quality Improvement Initiative (QII) Workshop: In early 2009, NPF convened a small group of scientist-practitioner thought leaders in quality indicator development in Parkinson’s disease.  This Quality Improvement Task Force led the development of the initial data set and set in place strategies for data collection.
  • QII Pilot Research Study: During the second half of 2009, NPF beta-tested a “one page per patient” data form, collecting care quality data from six centers.  Close to 700 patient records were collected and analyzed.  This phase established feasibility of collection and relevance of the data.
  • QII Summit: Findings from the pilot phase were shared with representatives from NPF’s 43 Centers of Excellence in December 2009.  Based on this initial pilot, we observed: a high incidence of psychiatric and cognitive issues; high degree of comorbidities, with arthritis and heart disease the most common; early indications of the positive effects of exercise programs; and distinct variations in physician treatment patterns and preferences.
  • QII Launch: In early 2010, the six pilot centers met to review the form and discuss revisions to the data set.  The group added data on falls and hospitalization, additional detail on DBS surgeries, and race and ethnicity data in patient demographics.  After making these changes, the six pilot sites and the eleven launch sites submitted the form and protocol to their institutional review boards and, upon approval, began data collection.


2010-2011 Planned Activities: In the Fall of 2010, each of the 17 participating Centers of Excellence’s located in the United States, Canada, Israel, and Europe will complete the collection of approximately 4,000 records for analysis.  In 2011, NPF will continue to roll out the QII program to NPF Centers of Excellence across the United States, Canada, and internationally. Data collection will be standardized through robust, web-interface system to facilitate data collection in place.

September 2011 Project Update:

Objectives:  Identify correlations between therapeutic choices and improved outcomes
Establish an evidence base as a foundation for future innovation
Improve the body of research to support evidence-based medicine decisions
Background:  With a goal of tracking a substantial and statistically-valid sample of the patients seen in the NPF Centers of Excellence (who provide care to approximately 50,000 patients), NPF’s QII already represents the largest international outcomes registry in Parkinson’s disease.  Our ultimate aim is to quantify and evaluate care as practiced at NPF’s Centers of Excellence in order to identify and share models of “best practices”, with the goal of providing every Parkinson’s patient the most effective treatment options available, whether they are seen by a movement disorder specialist, a general neurologist or their primary care physician.

Update/Results:
  At present, more than 4,000 unique patients at 17 NPF’s Centers of Excellence are tracked in the database.  Over 1,000 patients have had their one-year follow-up appointments.

Vanderbilt University and Georgetown University, two NPF Centers of Excellence, newly participating in the project, are beginning to recruit subjects.

Nine ancillary studies were generated in FY2011 as a result of the QII database. University of Florida and Struthers Parkinson’s Center investigators are currently using QII data to analyze motor and non motor issues associated with PD such as medication usage in DBS, excessive daytime sleepiness, falls prevention, diabetes and cognitive change, caregiver strain, mental health and DBS, and Timed Up and Go (TUG) performance.
A Data Analysis Working Group was created to revise and analyze data collected up to May of 2011. This group met at the Movement Disorder Society Meeting in Toronto and an action plan was drafted for the analysis of data and publication of data collected during the study period.

Dr. Eugene Nelson joined the QII steering committee. Dr. Nelson is the Director of Quality Administration for Dartmouth-Hitchcock Medical Center and is a national leader in health care improvement and the development and application of system performance, health outcomes, and costs of care.

Activities for 2011-2012: For FY2012 the 19 participating sites will continue to collect data from new and enrolled patients.  It is expected that, as the centers complete enrolling the 500 patients expected in each site, more follow up information will be collected. The Data Analysis Working Group will be comparing and analyzing data collected and is expected to submit several manuscripts to peer review journals for publication. An additional 10 to 20 ancillary studies will be conducted at participating QII centers.

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:

Project Title: Testing the neurotrophic and neuroprotective effects of SGK on the nigrostriatal dopaminergic system in vivo

Investigators: Xiqun Chen, PhD, Tatyana Kareva, Nikolai Kholodilov, MD, and Robert E. Burke, MD; Columbia University, New York, NY

Objective:  The goal of this study is to test whether the enzyme SGK can protect the neurons lost in animal models of Parkinson’s disease.

Project Description:  Neurons in a particular region of the brain called the substantia nigra die during Parkinson’s disease.  These neurons produce the important chemical dopamine.  As these neurons stop producing dopamine and die the symptoms of Parkinson’s begin to appear. The Burke lab is focused on elucidating the cellular mechanisms that cause these and other neurons to die, especially during Parkinson’s disease.  Our previous work found a cellular enzyme called AKT that was able to block cell death and restore neurons lost in animals models of Parkinson’s.  Recently, we have identified another enzyme closely related to AKT that is called SGK.  We found very high levels of SGK in the same dopamine neurons of the substantia nigra that are lost during Parkinson’s.  We propose that increasing the amounts of SGK in cells may be able to confer neuroprotection and help to induce the outgrowth of damaged neurons much like AKT.

Conclusion/Relevance to Parkinson’s Disease:  By identifying key cellular elements that might alter the neurodegeneration pathway in Parkinson’s, the work of the Burke lab will lead to a better understanding of how to develop new treatments for Parkinson’s disease.


September 2010 Project Update:

Results:  We increased the levels of SGK by using a form of the enzyme that was constitutively active.  Consistent with our hypothesis, high levels of SGK protect neurons from degradation in our model of Parkinson’s disease. A manuscript describing our results is currently in preparation.   

September 2011 Project Update:

Results: The project is now completed and the manuscript has been prepared.  It is planned to be submitted for peer-review in August, 2011.

5. The Michael J. Fox Foundation for Parkinson's Research is using its distribution to fund the following:

PD Online Research  is an efficient Web-based platform for research professionals across the globe to engage daily on key research hurdles and breaking scientific findings.  This virtual workplace has a simple goal:  enable quicker knowledge turns and therefore faster progress toward transformative therapies.

Project Update:

Investigators:  MJFF staff, Tim Clark, Massachusetts General Hospital

Objective:  PDonline Research (PDOR) was launched in June 2009 as a Web-based community for PD researchers and funders to collaborate and converse across the globe.  MJFF’s goal in developing the site was to facilitate a space for sharing of data and research resources and therefore accelerate progress in developing transformative new therapies for people with Parkinson’s disease. The website extends productive and efficient interactions established at meetings via a scientific social/professional network by allowing constant interaction amongst PD researchers.  The site was conceptualized and built by MJFF in collaboration with Massachusetts General Hospital and the Initiative in Innovative Computing at Harvard University. 

Background/Methods/Design:
  PD Online Research content is offered as a free resource to the general public. But since MJFF’s primary focus is on building a technical hub for Parkinson’s research, only scientists, clinicians, allied healthcare professionals directly engaged in scientific research, and investment decision-makers interested in Parkinson’s disease in the public, nonprofit, and private sectors have the ability to post directly to the site.  In order to post on the site, one has to be a member, which will also allow them to comment on other posts.   PDOR provides information on recent scientific publications, Parkinson’s disease in the news, funding opportunities, available resources, and Parkinson’s disease related conferences.  MJFF has used PDOR to seed discussions on key Parkinson’s disease research roadblocks and provide opportunities for community input into MJFF funding priorities. 

Results:  Since 2009, PDOR has been successful in becoming a hub of news and information for Parkinson’s disease research.  One example of the site’s success involves the sharing and distribution of MJFF generated research antibodies targeting the PD associated gene, LRRK2.  In this example, MJFF offered these antibodies for free to the research community and in exchange researchers were asked to post data detailing their use of the antibodies onto PDOR.  Over 150 research laboratories participated in the community validation of these important research tools and the data is now available for all researchers to view and integrate into their future research studies.

Conclusion:  MJFF is in the process of optimizing PDOR to better serve the needs of the PD research community and improve information sharing and promote more real-time member discussion.  MJFF is excited about the changes set forth for PDOR and looks forward to rolling out these improvements in the coming year.

6. The Parkinson Alliance is using its distribution to fund the following:

Project Title: Creating dopaminergic neurons with authentic Parkinson's disease - A new path to drug discovery and understanding mechanisms of neurodegeneration

Investigator: Dr. J. William Langston, Founder, CEO, and Scientific Director of the Parkinson's Institute.

Objective: The goal of this study is to derive induced stem cell lines (known as human-induced pluripotent stem cells or hiPSCs) from skin biopsies or hair taken from adult patients with Parkinson's disease (PD).  More specifically, we wish to derive hiPSCs from patients with parkinsonism due to genetic causes, including the LRRK 2 G2019S and alpha-synuclein triplication mutations. We will then use established technologies to differentiate these cells into dopamine neurons, one of the main neuronal populations that degenerate in PD.  This work will also include collaborations with academic medical centers as well as industry.

Hypothesis to be tested:  The underlying hypothesis of this proposal is that these "re-born" dopaminergic neurons that started out as skin or hair cells from adult patients with PD will recapitulate one or more of key molecular aspects of neural degeneration associated with PD in a tissue culture dish (“PD in a dish”).  We believe that this is likely since these cells will have been obtained from patients with parkinsonism of a known cause and that cause will have been carried with the hiPSCs in the form a known mutation in their DNA. 

Significance:
These proposed studies have the potential to provide an entirely new tool for investigating disease mechanisms of PD.   Furthermore, these "parkinsonian cells" could provide a transformative way to screen large number of drugs to see if they can prevent or even reverse the disease process.  The power of this approach is that it could, for the first time, provide authentic Parkinson's disease cells that are not a "best guess" model, but neuronal populations with authentic disease.  This is important because the results would be directly relevant to humans with the disease.  It is worth noting that we already have a large number of potentially “neuroprotective” drugs ready for screening. 

Deliverables/Milestones: 
The specific milestones for this project, which we estimate will take one year to 18 months, are as follows:

1.  Obtain appropriate human subjects approvals, including IRB approval for skin biopsies, and SCRO approval for derivation of iPS cells.
2.  Recruitment of 15 PD patients and controls for biopsy and establishment of fibroblast cultures.
3.  Reprogramming of fibroblasts for induced pluripotent cells (iPSCs).
4.  Characterization of ~150 iPSC clones for pluripotency and karyotype.
5.  Directed differentiation of iPSCs into dopaminergic neurons.

While this is a very ambitious set of deliverables, but we believe that they are achievable within the timeframe proposed for this research project.

September 2010 Project Update:

Progress in specific areas: Over the last nine months we have made a exciting progress towards the goal of this study that is modeling pathology of Parkinson’s disease in a culture dish.

  • Isolation of human fibroblasts: Human skin cells were isolated from biopsies of 25 patients with both sporadic and genetic forms PD and controls individuals from the Parkinson’s Institute and we have established several collaborations with other clinical centers to ascertain skin cells from different mutation carriers.
  • iPS cell derivation: Our first focus has been on fibroblasts derived from a patient with an SNCA triplication and an unaffected sibling. Primary skin cells were reprogrammed through infection with four viruses that expressed specific proteins for reprogramming named Oct3/4, Sox2, Klf4, and c-Myc. Approximately 20-25 days after infection, potential iPS cell colonies were mechanically picked based upon morphological similarity to human embryonic stem cell (hESC) colonies and were expanded on feeder plates. These clones survived in culture for more than 30 passages and were similar to undifferentiated hESCs in morphology and show all characteristics of hESCs.
  • Derivation of midbrain dopaminergic neurons: To determine the ability of our genetically defined SNCA triplication hiPSC line and the sibling control, we differentiated them into midbrain DA cells using mechanical isolation and temporal application of growth and signaling factors. To confirm the enrichment of DA neurons, mRNA and protein markers were assessed that were indicative of immature neurons and midbrain DA neurons.
  • Phenotypic differences in hiPSC-derived dopaminergic cells: To determine specific protein overexpression in derived neurons, cells were analyzed by rt-PCR and immunocytochemistry, and Western blot analysis. We consistently observed an overexpression/accumulation of specific proteins known to be involved in the pathology of PD.
  • New Grant: We are very excited to announce that this research can now be greatly expanded and accelerated with a recently awarded $3.7 million grant for the California Institute for Regenerative Medicine.  Thank you Parkinson’s Alliance for playing such an important role in helping this research get started!


Conclusion: We have demonstrated, for the first time, the ability to generate DA neurons from a PD patient with genetically defined PD through cellular reprogramming and directed differentiation. These iPS cell lines are similar in morphology and molecular signatures to hESCs. Importantly, the SNCA triplication line shows early signs of a cellular PD phenotype with an increase of specific protein levels compared to control cell lines.  These critical first steps provide the foundation for further refining a pathological phenotype that can be study disease mechanisms and be applied to the discovery of drugs aimed at disease modification.

 

Project Title:  Novel, small-molecule inhibitors of  a-synuclein assembly and toxicity for disease-modifying therapy of Parkinson's disease.

Investigator:  Dr. Gal Bitan

Project Description:  The generous gift of The Parkinson Alliance and Team Parkinson will support exciting research in the Bitan Laboratory geared towards development of disease-modifying therapy for Parkinson's disease (PD). Current treatment of PD focuses on compensation for dopamine deficiency, predominantly by using L-dopa. Though this kind of treatment is successful in alleviating major symptoms, such as tremor, it does not address “non-dopaminergic” symptoms, including falling and freezing, which affect 80-90% of patients with PD and 100% of patients with early-onset PD, or dementia which afflicts ~30% of patients with PD. These non-dopaminergic symptoms are major causes of mortality and morbidity in PD. In addition, treatment with L-dopa causes complications such as “off-time” and dyskinesia (involuntary movement). To address these unmet needs, disease-modifying, rather than symptomatic, therapy is needed. We have discovered a new experimental drug that disarms what most researchers believe to be real culprit causing PD - toxic aggregates of a protein called alpha-synuclein. These toxic aggregates are the main component of Lewy bodies, the hallmark pathologic lesion in the brain of patients with PD. The new drug inhibits the formation of these toxic aggregates and thereby prevents the initial processes that lead to development of PD. Through collaboration with Dr. Bronstein's group, the new drug was found to block the toxic effect of alpha-synuclein aggregates in cultured cells and zebra fish. The fish, which without the drug are severely deformed and die within a few days, appear healthy and normal by simple addition of the drug to the water in which they swim. Related experiments in mice show that the drug can get into the brain and remove toxic protein aggregates.

With the help of The Parkinson Alliance and Team Parkinson, we will develop this promising research in pre-clinical studies in transgenic mice in collaboration with Dr. Chesselet. Specifically, we will use mice engineered to overexpress human alpha-synuclein. The mice develop alpha-synuclein aggregates in the brain and show motor deficits already at 2 months of age. We will conduct experiments to optimize dose and treatment duration examining the effect of the drug on clearance of alpha-synuclein aggregates in the brain and on the motor abilities of the mice. In addition, we will conduct pharmacological studies to determine the optimal route of administration of the drug. We expect that these pre-clinical experiments will lead to FDA approval of Investigational New Drug (IND) status for our lead compound, facilitate initiation of clinical trials, and hopefully result in effective treatment and cure for PD.

September 2010 Project Update:

Background:
Several lines of evidence implicate self-assembly of a-synuclein as a key causative event in PD. In particular, formation of neurotoxic a-synuclein oligomers is believed to be a major cause for the neurodegeneration observed in multiple brain regions. a-Synuclein is a ubiquitous, naturally unstructured protein whose function is not well understood. Fibrillar a-synuclein is the main component of Lewy Bodies and Lewy neurites, the pathological hallmarks of PD. Mutations in, or multiplication of, the a-synuclein encoding gene cause familial PD. Recent genome wide association studies have repeatedly found a-synuclein as a major risk factor for PD. CLR01 is a novel compound we have discovered using a rational approach. The mechanism of action of CLR01 is novel and unique. The compound binds potently to Lys residues in proteins (Kd ~20 uM), with 10-times lower affinity to Arg, and with little, if any, affinity to most other cationic biomolecules. By binding to Lys residues, CLR01 inhibits a combination of hydrophobic and electrostatic interactions that are key to nucleation and aggregation by most amyloidogenic proteins. During the nucleation events, intermolecular contacts among protein monomers are fairly weak and therefore the moderate binding affinity of CLR01 to Lys is sufficient to interrupt them. At the same time, the uM binding of CLR01 to Lys does not affect the structure or function of “normal,” natively folded proteins unless substantially higher MT concentrations are present.

Methods/Design: 
We tested:
1. The capability of CLR01 to inhibit a-synuclein aggregation in vitro using thioflavin T fluorescence and electron microscopy.
2. Inhibition of a-synuclein-induced toxicity in cell culture.
3. Rescue of a-synuclein-induced toxicity leading to deformation and mortality in a zebrafish model.
4. Rescue of a-synuclein-induced motor deficits in a transgenic mouse model expressing human, wild-type a-synuclein under the control of the Thy1 promoter.
These experiments included extensive collaboration with Drs. Jeff Bronstein and Marie-Françoise Chesselet, Department of Neurology, UCLA.

Results:
1. CLR01 inhibited a-synuclein aggregation completely at a 1:1 concentration ratio and partially at a 1:10 concentration ratio, respectively.
2. CLR01 inhibited the toxicity of both exogenous a-synuclein oligomers and endogenously expressed in cell culture.
3. CLR01 showed significant rescue of the phenotype and survival of zebrafish embryos expressing human, wild-type a-synuclein. The mechanism of rescue was shown to be maintaining a-synuclein in a soluble form, preventing formation of toxic aggregates, and enabling degradation of a-synuclein via the proteasome.
4. CLR01 was found to significantly ameliorate motor deficits in transgenic mice expressing human, wild-type a-synuclein in the brain when administered subcutaneously for 28 days at a low dose (0.4 mg/kg/day). The mechanism is still under study but initial results suggest that it involves reduction in the levels of soluble, likely oligomeric, a-synuclein.
5. Importantly, in cell culture and zebrafish experiments, the dose of CLR01 showing toxicity was substantially higher than the dose needed for the beneficial effects. In the transgenic mice, no side effects were observed.

Conclusion/Relevance to Parkinson’s disease:
The data accumulated so far suggest that CLR01 is a novel and promising therapeutic candidate for Parkinson’s disease and support its further development for human clinical trials.

September 2011 Project Update:

These experiments included extensive collaboration with Drs. Jeff Bronstein and Marie-Françoise Chesselet, Department of Neurology, UCLA.

Results:

1. CLR01 inhibited ?-synuclein aggregation completely at a 1:1 concentration ratio and partially at a 1:10 concentration ratio, respectively.
2. CLR01 inhibited the toxicity of both exogenous a-synuclein oligomers and endogenously expressed in cell culture.
3. CLR01 showed significant rescue of the phenotype and survival of zebrafish embryos expressing human, wild-type a-synuclein. The mechanism of rescue was shown to be maintaining a-synuclein in a soluble form, preventing formation of toxic aggregates, and enabling degradation of a-synuclein via the proteasome.
4. CLR01 was found to significantly ameliorate motor deficits in transgenic mice expressing human, wild-type a-synuclein in the brain when administered subcutaneously for 28 days at a low dose (0.4 mg/kg/day). The mechanism is still under study but initial results suggest that it involves reduction in the levels of soluble, likely oligomeric, a-synuclein.
5. Upon ICV administration for 28 days, the effect of CLR01 was particularly significant in the challenge beam test, where is was consistent in all 5 trials and also increased by 2.2-fold relative to subcutaneous administration. The data demonstrate the efficacy of CLR01 and suggest that blood–brain barrier penetration likely needs to be optimized in future medicinal chemistry and/or formulation studies.

Conclusion/Relevance to Parkinson’s disease:  The data accumulated so far suggest that CLR01 is a novel and promising therapeutic candidate for Parkinson’s disease and support its further development for human clinical trials.
A manuscript describing the data summarized in points 1–3 above has been submitted for publication and is now being revised to address reviewers’ comments. The mouse experiments are still being analyzed and will be published in the near future.

 

Project Title:  Is there abnormal network activity in the motor cortex of 6-OHDA lesioned mice.

Investigator:  Dr. Carlos Portera, M.D., PhD

Project Description:  The current treatments for Parkinson disease (PD) offer some symptomatic relief, but often at the cost of serious side effects, including dyskinesias. The introduction of deep brain stimulation (DBS) in the treatment of PD two decades ago has arguably been the most effective treatment strategy since the discovery of levodopa. Understanding the exact mechanisms of how DBS helps PD patients will help improve this therapeutic strategy. A recent study using the 6-hydroxydopamine (6-OHDA) rodent model of PD (Gradinaru et al., 2009) suggests that DBS may help by reducing the activity of the subthalamic nucleus (STN) through its effects on the firing of neurons in motor cortex, which is the part of the brain that controls movement. This raises the possibility that neurons in the motor cortex of PD patients fire less than normal, leading to an overactive STN that produces some of the symptoms of PD. I propose to test the hypothesis that neurons are hypoactive in the motor cortex of mice that had been rendered parkinsonian by injection of the neurotoxin 6-OHDA into the substantia nigra. Specifically, I intend to examine the spontaneous activity of neurons in the motor cortex of mice before and after administering 6-OHDA. We will use the cutting-edge technique of two-photon calcium imaging to record the activity of large numbers of cortical neurons non-invasively.

These experiments will shed light into the mechanisms of circuit dysfunction in PD and may lead to improved treatments for this devastating disorder.

September 2010 Project Update:

We originally proposed to test the hypothesis that neurons in the motor cortex of Parkinsonian mice are hypoactive. In a way, this is expected in patients with Parkinson disease (PD), because the low levels of dopamine lead to reduce activity in a part of the brain called the thalamus, which normally stimulates the motor cortex to initiate movement. Specifically, we proposed to render mice Parkinsonian by injecting the neurotoxin 6-hydroxy-dopamine (6-OHDA) into the substantia nigra. Next, we planned to examine the spontaneous activity of neurons in the motor cortex of mice before and after the 6-OHDA injection. However, after discussions with UCLA investigators and PD research experts Marie-Francoise Chesselet and Michael Levine, we decided instead to examine neuronal activity in motor cortex of the Alphasynuclein over-expressing transgenic mice (the so-called ASO mice). Drs. Chesselet and Levine considered the ASO mice to be a better model of PD.

We have been using the cutting-edge technique of two-photon microscopy to record the activity of large numbers of cortical neurons non-invasively, with calcium imaging. So far, we find that the average firing rate of neurons in the motor cortex is lower in ASO mice than in normal controls, meaning that the motor cortex of mutant mice is less active. Although the data are very preliminary because we have only imaged 2 ASO mice and 4 control mice, the result is what we expected. Next, we intend to image more mice to confirm these results.  Eventually, we will attempt to stimulate neurons in motor cortex of ASO mice with light or with electrodes to restore their activity levels back to normal. The hope is that this will have a therapeutic benefit in the Parkinsonian mice. Our goal is to shed light into the mechanisms of circuit dysfunction in PD that may lead to improved treatments for this devastating disorder. The funds from the Parkinson Alliance will hopefully allow us to generate sufficient preliminary data for other grants, including Federal funding.

September 2011 Project Update:

In addition to acute two-photon calcium imaging experiments using the synthetic calcium indicator dye Oregon Green BAPTA-1, we also intend to pursue chronic recordings of neuronal activity in the neocortex of ASO mice with the genetically encoded calcium indicator GCaMP3. Using viral mediated transfections we will be able to record the firing of cortical neurons over periods of several weeks.

 

7. The Parkinson’s Institute and Clinical Center is using its distribution to fund the following:

Project Title:  Epidemiologic Studies of PD Etiology

Investigator:  Tanner CM, Goldman SM, Langston JW

Objective:  The goal of this study is to further investigate the hypothesis that Parkinson’s disease is a complex disorder with both genetic and environmental determinants

Background:  The cause of Parkinson’s disease is not known.  Our work indicates that no single factor causes Parkinson’s disease.  Instead, combinations of risk factors and genetic makeup determine whether an individual develops Parkinson’s disease or not. Our recent work found that specific environmental factors only increase Parkinson’s disease risk in those carrying certain genetic variants.  This effect was very large for some factors, with up to an 11-fold increased risk of Parkinson’s disease.  The goal of this research project is to expand on this ongoing work, focusing on the interplay between inherited (genetic) and acquired (environmental) factors to identify the causes of Parkinson’s disease.

September 2010 Project Update:

Methods/Design:  We conducted analyses on the effects of genes and environment, taking advantage of extensive work in several large epidemiologic studies totaling nearly 2000 individuals.  Detailed lifelong information on environmental risk factors was collected, and DNA tested for over 1500 variations in 120 genes thought to play a role in PD.  We investigated the following specific hypotheses: 1) Does exposure to occupational solvents increase the risk of PD?  2) Is the effect of head injury on PD dependent on genetic makeup?

Results:  (1) Studying 100 pairs of twins in which one twin had PD and one did not (i.e., twins discordant for PD), we found that occupational exposure to the common solvent trichloroethylene (TCE) was associated with a more than 6-fold increased risk of PD.  Those exposed to either TCE or tetrachloroethylene (PERC) were at more than 10-fold increased risk.  (2) We investigated the effect of head injury with loss of consciousness or concussion in two large studies.  Overall, the effect of head injury was modest.  However, in those who had both a head injury and a common variant of the alpha-synuclein gene, the risk of PD was increased more than 6-fold.

 

2011 Project Update:

Conclusion/Relevance to Parkinson’s disease:  (1) This is the first population-based study to find an association of TCE with PD.  Solvent exposures are very common at work and at home.  TCE is present in numerous household products and is the most frequently reported organic contaminant in groundwater.  If our results are confirmed in future research, then public health measures will need to be implemented to reduce TCE exposures in the population.  (2) We found a significant and strong interplay between head injury and a common variant in a gene known to be important in PD.  The independent effect of either of these raised risk only slightly, but the combination raised risk dramatically.  More than 3 million head injuries occur every year in the U.S.  In our study, the injuries preceded PD by an average of 35 years, providing hope that protective medications and measures could help prevent future PD in susceptible individuals. Thanks to Unity Walk funding, manuscripts reporting each of these findings have now been accepted for publication in Annals of Neurology, and are in press.