Parkinson’s disease (PD) is the second most common neurodegenerative disorder and is poised to increase in prevalence as the population ages. Hallmark pathologies of the disease include aggregation of alpha-synuclein (α-Syn) into Lewy bodies accompanied by the loss of dopamine neurons in the nigrostriatal system. A potential third pathological hallmark of PD is the presence of reactive microglia within the substantia nigra (SN), surrounding inclusion-bearing neurons. The contribution of early inclusion-associated microglial activation to nigrostriatal degeneration is not well understood. The rat α-Syn preformed fibril (PFF) model of synucleinopathy demonstrates two distinct phases of pathology progression in the SN. The first phase is the aggregation phase, characterized by the accumulation of α-Syn into phosphorylated alpha-synuclein (pSyn) inclusions, followed by the degeneration phase, characterized by the loss of tyrosine hydroxylase (TH) phenotype and overt degeneration of nigral neurons. By leveraging the distinct aggregation phase of the PFF model, we are able to specifically investigate the transcriptomic response within the SN to pSyn inclusion formation prior to neurodegeneration. In the present dissertation we used a laser capture microdissection (LCM) technique and RNA sequencing to investigate differential gene expression within the inclusion-bearing nigra at one- and two-months post PFF injection, prior to phenotype loss and subsequent neurodegeneration. Our results suggest that genes and pathways associated with inflammation and the immune response are increased at both timepoints, and that genes and pathways associated with synaptic transmission and synaptic signaling are decreased at both timepoints. In addition, we found that upregulation of genes and pathways implicating defense responses to virus are uniquely upregulated at the two-month time point. Furthermore, we identified a family of proteases, called cathepsins, that are upregulated in the inclusion-bearing SN at both timepoints. Subsequent follow up analysis using droplet digital PCR (ddPCR), fluorescent in situ hybridization (FISH), and immunofluorescence (IF) revealed increases in multiple cathepsins within the inclusion-bearing SN. Specifically, cathepsin H (Ctsh), S (Ctss), and X (Ctsx) were all found to be enriched within microglia in the inclusion-bearing SN at either one, two, or both timepoints following PFF injection. Collectively, these results suggest that the impact of microglia to pSyn inclusions in the SNpc is temporally dynamic. These results suggest a role for reactive microglia in early alpha-synuclein pathology and may provide therapeutic targets for disease intervention.
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