PD Therapeutics 2009: Poster Abstracts

Recognizing that increased knowledge of the clinicopathological correlates of Parkinson’s disease is critical to understanding the progression of the disease and developing novel treatment strategies, MJFF has supported a unique collaboration at the Arizona Parkinson’s Disease Consortium (APDC) through its Prescott Family Initiative. APDC comprises a consortium of Arizona researchers including investigators from Mayo Clinic Arizona and Sun Health Research Institute (SHRI) and leverages the resources of the SHRI Brain & Body Donation Program (BBDP). The APDC & BBDP perform standardized cognitive and motor assessments of a population of PD and elderly normal subjects who are then autopsied at death. The consortium has already uncovered exciting results that have revised the pathoanatomical staging of PD, including the mapping of alpha-synuclein pathology in the peripheral nervous system, and have initiated a process leading towards the development of clinical predictive algorithms for PD and dementia in PD.  MJFF seeks to facilitate collaborations between the APDC and the broader PD research community.

The BBDP is located in Sun City Arizona and has been in existence since 1987 [1]. It is a longitudinal, community-based clinicopathological study of cognition, motor function and the brain in normal elderly persons and in those with neurodegenerative diseases. More than 1175 donors have expired and been autopsied for research and tissue banking since the Program’s inception.  Brain tissue and cerebrospinal fluid is available from approximately 234 elderly controls, 131 with PD, 131 with Alzheimer’s disease with Lewy bodies (ADLB), 131 with dementia with Lewy bodies (DLB) and 37 with incidental Lewy body disease (ILDB). Of these, a subset have had antemortem standardized motor and cognitive assessments, including 72 with PD, 32 with ADLB, 23 with ILBD, 17 with DLB and 70 elderly controls.  In addition to brain tissue, fixed and frozen tissue is available from all major bodily organs and tissues for more than 130 subjects, including 13 with PD, 12 with DLB, 19 with ADLB and 4 with ILDB.  A unique feature of the BBDP is its consistently short postmortem interval (PMI), with a median of three hours for all cases. This makes the banked tissue useful for all forms of molecular biological studies, including those utilizing modern methods for genomics, gene expression (RNA) and proteomics.

Brain and body tissue as well as correlative clinical data is available in collaboration with the general PD research community, by inquiry to either Dr. Thomas G. Beach of SHRI (thomas.beach@bannerhealth.com) or Dr. Charles H. Adler of the Arizona Mayo Clinic (cadler@mayo.edu). 

Cell death caused by alpha-synuclein is thought to play a central role in the pathogenesis of Parkinson’s disease. We have previously shown that a class of compounds called Hsp90 inhibitors can lead to an increase in heat shock protein (Hsp) 70 and prevent alpha-synuclein aggregation and toxicity in cells in culture. Here, in collaboration with Pfizer, we have investigated if novel, small molecule, Hsp90 inhibitors can prevent neurodegeneration in a rat model of Parkinson’s disease.

We have used a rodent viral-vector model of Parkinson’s disease whereby the substantia nigra region of rats is injected with a viral vector (AAV) that expresses the protein alpha-synuclein. The targeted overexpression of alpha-synuclein in the rat brain has been shown to produce 30-50% cell death in the injected region after 6-8 weeks. Following viral vector injections, we treated the rats with novel Hsp90 inhibitors three times a week for six weeks. These inhibitors are orally available and can cross the blood brain barrier. After six weeks we examined the rodent brains to determine if the Hsp90 inhibitors were able to prevent the cell death that is normally caused by the overexpression of alpha-synuclein.  Preliminary data will be discussed herein.

A severe and unfortunate roadblock in Parkinson’s disease (PD) research is that to date no genetic mammalian models have recapitulated well the cardinal PD pathology of dopaminergic neuronal death in substantia nigra.  To overcome this prominent obstacle and leave no stones unturned, we propose to take advantage of the different biology between mouse and rat, and to establish bacterial artificial chromosome (BAC) transgenic rat models for α-synuclein (Park1) and LRRK2 (Park8), two disease genes for autosomal dominant Parkinson’s diseases.

To date, we have succeeded in establishing the BAD transgenic technology in rat, and generating five genotypes of transgenic rats for α-synuclein and LRRK2.  Multiple founder lines were obtained for each genotype (16 for E46K α-synuclein, 9 for A53T α-synuclein, 9 for Wild type α-synuclein, 4 for R1441G LRRK2, and 13 for G2019S LRRK2).  Interestingly, the old animals started to show deficits in movements, and the deficit was rescued by L-dopa.  Currently, we are collaborating with Dr. Tim Greenamyre’s group to fully characterize these rat models.

Parkinson’s disease (PD) interventional trials rely predominantly on clinical outcomes that often vary within subjects and are not consistent across subjects. Reliable and well-validated biomarkers to monitor PD progression would dramatically improve patient care and accelerate research into both PD etiology and therapeutics.  During the past two decades much progress has been made in identifying and assessing PD biomarkers, but as yet, no fully validated biomarker for PD is currently available. Given the recent advances in molecular genetics, neurobiology, imaging technology and radiochemistry that have provided new tools that may be useful for PD biomarkers, and the recognition that the lack of PD progression biomarkers has created a roadblock for further studies of disease modifying therapies, there is increasing consensus that a major initiative to develop PD progression biomarkers is both necessary and feasible. MJFF is planning a large clinical study to enroll 400 PD subjects and 200 healthy controls with the objective to collect clinical, imaging, and biologic data to develop biomarkers of Parkinson’s disease progression. The data will be collected over 3-5 years and will include motor and non-motor clinical data, dopamine scans, and biologic fluid (blood, urine, cerebral spinal fluid) collection. The data will be made available to the PD research community. This initiative will create a consortium of academic centers, government agencies and PD foundations, and pharmaceutical and biotech companies to collectively design, implement and fund a comprehensive program to develop markers of PD progression.  MJFF is currently in the pre-enrollment stage of this study with the goal to launch the study in spring of 2010.

The most widely prescribed therapy for Parkinson’s disease is levodopa (L-DOPA), which allows patients to regain motor control. However, many patients develop adverse reactions to L-DOPA, including abnormal involuntary movements termed dyskinesias. We seek to understand the basis of these abnormal movements by studying genes that we discovered in animal models of dyskinesias. We will test whether manipulation of these genes can alleviate L-DOPA-induced abnormal movements in animals, thereby laying the groundwork for novel therapeutic targets.

Two of the genes that we discovered to be abnormally regulated in animal models of dyskinesia, called CalDAG-GEF1 and CalDAG-GEF2, are well situated to play a central role in driving L-DOPA-induced movements. These genes are expressed in the striatum, which is the main dopamine-modulated, motor-control center of the brain that suffers loss of dopamine in Parkinson’s disease. These genes can activate ERK proteins, which mediate abnormal motor responses to L-DOPA. We will test whether manipulation of the CalDAG-GEFs can alleviate L-DOPA-induced dyskinesias by using two strategies: First, we will test whether mice that we have engineered to lack CalDAG-GEF1 or 2 are resistant to developing L-DOPA-induced motor complications. Second, we will use viruses to either repress or activate the CalDAG-GEFs in parkinsonian rodents, to test for mitigation of motoric side-effects at various stages of L-DOPA therapy.

Co-author: Jill Crittenden

PD Online Research (PDOR) is a collaborative community for technical discussion and problem-solving in Parkinson’s disease science, powered by he Michael J. Fox Foundation for Parkinson’s Research in collaboration with the Initiative in Innovative Computing at Harvard University. Our goal is to speed timely information-sharing and foster productive debate that accelerates PD therapeutic development and enables smart, informed decision-making by scientists and funders alike. PDOR is an experiment that aims to extend to the web the informal but highly productive and efficient interactions that happen in scientific conferences and lab hallways every day.  Our professional members move their work forward by posting commentary and questions instantly – unmoderated and  uncensored.

Launched on June 1 2009, PDOR has over 1200 members from the academic, industry, medical, government, non-profit and investment sectors.  PDOR members from the most senior professional levels to astute grad students have already posted hundreds of highly informative and provocative comments on the hottest topics in PD research. All content is  easily accessible through the PD Guide.

Members also benefit from early coverage of the hottest papers in PD science as they hit the press – with pre-embargo access to major scientific journals, PDOR is able to initiate relevant and timely scientific discussion of the newest research papers on the day of publication.

In addition to providing a platform for communication, PDOR also features a number of custom resources including a continuously updated bibliography of PD-relevant papers, a Clinical Trials Database, and a PD Target Database. PDOR members actively annotate and discuss the contents of these databases, providing unique on-the-record resources for the PD research community.

Since our launch, PDOR has hosted discussion by scientists from all over the globe, covering a broad range of PD science. We depend on the entire community to engage with this new platform and to share our goal of hastening research toward better treatments and ultimately a cure for PD.

Co-author: Sudeshna Das

Mutations in Leucine Rich repeat Kinase 2 (LRRK2) lead to a form of autosomal dominant Parkinson’s Disease (PD) with symptoms and pathological hallmarks that are similar to sporadic forms of the disorder.  A number of these mutations result in increased kinase activity of the protein, and it is hypothesized that inhibition of LRRK2’s kinase activity may slow or halt progression of this disease.  Hence, our objective is to identify LRRK2 kinase inhibitors for development as disease modifying therapies for PD patients. Furthermore, these studies will further elucidate the role played by LRRK2 in cells, and highlight intracellular pathways altered during the progression of PD.

To characterize the kinase activity of LRRK2, understand its enzymological properties, and develop assays suitable for compound screening, LRRK2 protein was purified using baculovirus expression systems.  LRRK2 was found to robustly phosphorylate a surrogate peptide substrate, LRRKtide, and also leads to a slow autophosphorylation of LRRK2.  One of the most prevalent human LRRK2 mutations, G2019S, was found to significantly increase the rate of both autophosphorylation and LRRKtide phosphorylation compared to wild-type. Furthermore, G2019S LRRK2 demonstrates a 2-fold lower binding affinity for ATP compared to wild-type LRRK2.  

In preparation for HTS we developed a Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET)-based assay (LanthaScreen^TM).  An analysis of five clinically observed LRRK2 mutations using either radiometric or Lanthascreen^TM assays revealed that the G2019S mutation results in the greatest enhancement of kinase activity using LRRKtide as substrate.  To develop secondary assays allowing the prioritization of hits obtained using in vitro assays, we have generated inducible stable cell lines expressing wild-type and mutant LRRK2, and can demonstrate that LRRK2 immunoprecipitated from these cells retains kinase activity. 

These cell-free and cell-based assays and reagents are also being used to identify relevant LRRK2 substrates and pathways in combination with peptide positional motif scanning and Protoarray^TM assays.  Initial results are highlighting preferred phosphorylation motifs and hence identifying putative cellular substrates.  Together, these studies facilitate the identification of substrates phosphorylated by human mutations of LRRK2, the identification of pathways modulated by this kinase, and the identification of selective LRRK2 kinase inhibitors for development as disease modifying therapies for the treatment of PD.

Steven P. Braithwaite¹, Michael Monaghan¹, Robert Martone¹, Vasanti S. Anand¹, Andres Hurtado-Lorenzo¹, Pooja P. Pungaliya², Brian Bates², Warren D. Hirst¹, Peter H. Reinhart¹

¹Wyeth Research, Princeton, NJ 08543  ²Wyeth Research, Cambridge, MA 02140