Cell Specific Proteomics highlights Molecular signatures of Manganese neurotoxicity in Dopaminergic neurons in vivo - Project Summary/Abstract Parkinsonism is a progressive neurodegenerative disorder that is characterized by loss of dopaminergic neurons. Various environmental factors like exposure to Mn has been implicated in PD etiology. Although we have known that Mn exposure can induce neuronal death and TH neuron toxicity for decades, the functional relevance of overexposure of Mn is still not clearly understood. Mn has also been recently shown to act interact with various PD related genes, like α-synuclein and LRRK2 suggesting a complex gene-environment interactions. Further, various studies in rodent models of Mn toxicity have shown that Mn leads to molecular changes in the brain in a time-dependent manner. However, studying these molecular changes in a cell-specific manner at the protein level has been difficult due to the lack of a model system. Although single-cell RNA sequencing technology has provided some insight, understanding the proteome of the dopaminergic neurons at various stages of Manganese toxicity will provide key insight into early and late changes associated with these neurons. We propose to use the Cell type specific In vivo Biotinylation of Proteins (CIBOP) approach to study the proteome of TH neurons in vivo. The overarching hypothesis is using the CIBOP approach, we will successfully identify the proteomic signatures of TH neurons at different stages of Mn-induced neurotoxicity. Further, this model system will identify potential drug targets that can modify Mn-induced neurodegeneration and will also provide a tool for the field that can be used to study other cell types as well as the peripheral nervous system. In Aim 1, we will generate the TurboID/TH mice and will confirm the biotinylation of TH neurons in not only different regions of the central nervous system but also the peripheral nervous system. Further, we will expose these mice to Mn and perform proteomics to identify TH-specific changes in the molecular signatures at different stages of Mn toxicity. We will perform various bioinformatic analyses, including pathway analysis and MAGMA analysis, to further identify specific pathways altered in the TH neurons. Aim 2 will involve mechanistic validation of the top hits from our proteomic studies in our Drosophila model of Mn exposure that we developed. This model recapitulates various motor and non-motor symptoms of PD. We will use a variety of scalable tools like behavioral assays, HPLC, and seahorse as endpoint assays in flies. Further, to add human relevance, we will also use patient-derived iPS cells to validate our findings in Drosophila. Having a complementary team at Yale and the University of Rochester will help us complete this ambitious, exploratory proposal. While Dr. Rangaraju's group at Yale is an expert in CIBOP and mouse generation, we have expertise in flies, iPSC, and mechanistic studies in the field of Manganese neurotoxicity.
