The goal of this research is to utilize novel research tools to investigate the molecular mechanisms that cause Parkinson’s disease (PD). The proposed work builds on previous funding from CIRM that directed the developed patient derived models of PD. The majority of PD patients suffer from sporadic disease with no clear etiology. However some PD patients harbor specific inherited mutations have been shown to cause PD. The most frequently observed form of genetic parkinsonism is caused by the LRRK2 G2019S mutation it the most common. This mutation accounts for approximately 1.5-2% of patients with apparently sporadic PD, increasing to 4-6% of patients with a family history of PD, and even higher in isolated populations. Importantly, LRRK2 induced PD is clinically and pathologically largely indistinguishable from sporadic PD.
This proposal focuses on studying the most frequent cause of familial PD and induces disease that is clinically and pathologically identical to sporadic PD cases. It is likely that LRRK2 regulates a pathway(s) that is important in the more common sporadic form of PD as well. Therefore by employing relevant models of PD, we hope to drive the biological understanding of LRRK2 in a direction that facilitates the development of disease therapeutics in the future. We ascertained patients harboring mutations in LRRK2 [heterozygous (+/G2019S) and homozygous (G2019S/G2019S)] as well as sporadic cases and age matched controls. We have successfully derived iPSCs from each genotype and differentiated these to DA neurons. We will use these as a model system to investigate these LRRK2 based models of PD.
We will adapt current biochemical assays of LRRK2, which are source material intensive, to the small culture volumes required for the differentiation of iPSCs to DA neurons. This is a crucial necessity for development for utilizing iPSC derived DA neurons as tractable models of LRRK2 based PD. We will then probe the roles of LRRK2 in neuronal cell differentiation and survival. We will also ask whether the mutant LRRK2 induces changes in autophagy, as this has been postulated as a mechanism of LRRK2 induced pathogenesis. By studying wild-type and disease mutant LRRK2, in DA models of PD we hope to provide crucial understanding of the role mutant LRRK2 has in disease.
It is estimated that by the year 2030, 75,000-120,000 Californians will be affected by Parkinson’s disease. Currently, there is no cure, early detection mechanism, preventative treatment, or effective way to slow disease progression. The increasing disability caused by the progression of disease burdens the patients, their caregivers as well as society in terms of healthcare costs. The majority of PD patients suffer from sporadic disease with no clear etiology, and a in a handful of these patients specific inherited mutations have been shown to cause PD. The most frequently mutated gene is called Leucine Rich Repeat Kinase 2 (LRRK2). Our goal is to study the mutated gene product in patient based models of Parkinson’s disease.
In previous CIRM funding, we have developed patient derived induced pluripotent stem cells (iPSCs) from patients harboring mutations in LRRK2. We have been successful in differentiating populations these iPSCs into the neurons that are depleted in PD. The next step is to utilize these cells as models of mutation induced PD ‘in a dish’. We will employ these pertinent disease models to answer basic biology questions that remain about the function of LRRK2.
This project brings together scientists previously funded by CIRM with scientists well versed in the study of LRRK2. This multidisciplinary approach to studying the causes of PD is a natural benefit to the State of California and its citizens. By bringing a better understanding of the role of LRRK2 in the cells that are lost in the progression of PD, we will bring more concrete knowledge of PD as a whole, bringing more hope for the development of a therapeutic for disease.
The goal of this project is to investigate the molecular mechanisms that cause Parkinson╒s disease (PD). The proposed work builds on previous work on the development of induced pluripotent stem cell (iPSC) lines derived from patients with a mutant familial form (leucine rich repeat kinase 2; LRRK2) of PD. LRRK2 is the most common cause of familial PD. The project encompasses three specific aims: Aim 1 will focus on the development and application of LRRK2 specific assays to iPSCs. Aim 2, will adapt the LRRK2 assays to differentiated iPS cells. This aim will apply the adapted biochemical approaches developed and employed in Aim 1 to iPSCs differentiated to neuronal stem cells and dopaminergic neurons. Finally, in Aim 3, the investigators will focus on studying LRRK2 activity in differentiated dopaminergic neurons.
Significance and Innovation:
- The proposal focuses on a major unsolved problem as the mechanisms that underlie neuronal cell death in PD are still not known. The development of assays designed to unravel the biological function of LRRK2 could be an important step forward toward the elucidation of those mechanisms.
- One reviewer described the main innovation of the proposal to be the combination of iPSC technology with innovative approaches to measure LRRK2 activity in cell based assays; in particular by monitoring specific phosphorylation sites that are regulated by LRRK2 downstream kinase.
- The proposed proximity ligation assay for detecting antibody binding at higher sensitivity represents an innovative approach to solve the detection problem.
- Concerns about the experimental design were raised as it made it difficult to determine the extent to which the experimental work as outlined would have a significant impact to the field and to what extent the project is truly innovative.
Feasibility and Experimental Design:
- Overall, the approach proposed is reasonably sound, however, reviewers agreed that the experimental design as laid out in the project is very descriptive and superficial in critical analysis. Controls for specific experiments are often ignored or trivialized, and quantitative analysis is lacking.
- A major concern shared by the reviewers was the preliminary data for the in vitro differentiation assays to generate authentic A9 dopaminergic (DA) neurons. No quantitative data regarding the efficiency and reproducibility of this procedure was presented. The reviewers felt that the PI should have attempted to address or at least mention the challenges associated with generating the appropriate neuron type.
- The applicants have developed new tools for the detection of LRRK2/LRRK2 activity, and have developed inhibitors of LRRK2. They plan to adapt those tools in LRRK2 iPSCs and in LRRK2 iPSC-derived dopamine neurons. Reviewers argued that some preliminary data using this assay in cell lines would have further strengthened the proposal.
- Alternative methodological approaches are only rarely mentioned should there be technical problems.
- Given that the investigators plan to test for impact of LRRK2 function on autophagy, reviewers were concerned with the lack of both preliminary data and publication on autophagy assays by the investigators.
- Research facilities and environment are adequate for the studies proposed.
Principal Investigator (PI) and Research Team:
- The PI is a biochemist with expertise in characterization of kinases that is relevant to the study of LRRK2.
- The co-investigator has a track record in PD and other movement disorders, and is part of the team that has generated and published on the iPSC derived from LRRK2 patients.
- The team has no specific expertise in the neural differentiation assays and in optimizing protocols for midbrain A9 DA neuron generation.
- Overall the research team is quite weak and of unknown composition and expertise.
Responsiveness to the RFA:
- The application is responsive to RFA by proposing the use of human patient specific iPSCs and by addressing mechanistic questions in the context of an important human disease.
- A motion was made to move this application into Tier 1, Recommended for Funding. Reviewers agreed that the application was responsive to the RFA as it does address mechanistic questions. The main point of criticism was whether reviewers were convinced that the applicants could make the right type of cells, which is a crucial point for success of this application. Motion carried.