Parkinson's disease (PD) studies suggest that non-specific stressor combinations can cause cell type-specific damage and neuronal degeneration. The cardinal PD symptoms are caused by the selective death of midbrain dopamine (mDA) neurons, yet the majority of its environmental and genetic risk factors have the potential to affec many cell types. Induced pluripotent stem cells (iPSCs) reprogrammed from patient skin fibroblasts offer a potentially unlimited source of disease-relevant neurons for modeling the interaction of extrinsic stressors with cell type and genotype-specific factors. Further, the heat shock protein 90 (HSP90) co-chaperone complex has been shown to modulate disease progression in both PD-relevant genetic mutant and toxin-stressed cells. We hypothesize that analysis of co-chaperone dynamics and HSP90-related protein networks will identify cell and disease specific vulnerabilities in PD human patient iPSC-derived mDA neurons. Recent studies support that in genetic or toxic stress conditions, Hsp90 has the potential to facilitate neurodegenerative events. We hypothesize that the Hsp90 co-chaperone complex is uniquely altered in midbrain dopamine (mDA) neurons of PD patients. Analyzing this complex in patient specific iPSC-derived mDA neurons will allow us to identify and validate potential novel therapeutic targets. Toxin exposure can mimic PD-related phenotypes in vitro and in vivo. I will toxin-stress iPSC-derived mDA neurons of mutant PD patient lines (and isogenic controls) to pursue the following aims: First, determine chaperone dynamics in a patient-specific iPSC-derived mDA neuron model of PD. In my preliminary data, I have adapted co-sponsor's techniques to reveal disease-related phenotypes and Hsp90 activation in stressed iPSC-derived PD patient mDA neurons. I propose to validate findings with isogenic, gene-corrected iPSC lines and to optimize stress paradigms to reveal cell and disease specific phenotypes. Second, identify chaperone-mediated protein interactions in stressed mDA neurons using mass spectrometry analysis of PU-H71-bound Hsp90 co-chaperone complexes. In my preliminary data I have successfully adapted my Co-Sponsor's (G. Chiosis) proteomic assays. PU-H71 (Hsp90 inhibitor developed by Co- Sponsor's lab) selectively recognizes the stress form of the Hsp90 complex in cancer cells. I propose to use PU-H71-based mass spec to establish the first global profile of proteins that interact with the Hsp90 co- chaperone stress complex in neurons, and to observe this complex in PD-iPSC-derived mDA neurons. Third, perform functional studies to test the role of candidate pathways differentially regulated in stressed PD- versus control-iPSC derived mDA neurons. Through collaborations and analysis similar to the one described in Aim 2, my Co-Sponsor's laboratory has identified potential cancer drug targets to pair with PU-H71 treatment. I will generate candidate pathways from my preliminary data and Aim 2 data. will perform loss and gain-of- function and pharmacological studies to explore their contribution in cell and disease specific PD phenotypes.
Our collaboration will merge novel technologies pioneered by our laboratories in the induced pluripotent stem cell and medicinal chemistry fields to advance drug discovery for Parkinson's disease. To our knowledge, the cell stress response has not yet been examined in any functional, disease-relevant human neurons. Further, our system could help advance the study of druggable targets in other neurodegenerative diseases such as Alzheimer's disease.
