Abstract: More than 200 million individuals are at risk for chronic traumatic encephalopathy (CTE) each year. CTE is strongly associated with exposure to repetitive head impacts (RHI) such as those received through contact sports and military activities. Neuropathologically, CTE is defined by progressive accumulation of hyperphosphorylated tau (p-tau) which is potentiated by microglial neuroinflammation. RHI exposure can induce microglial inflammation even before the onset of tau pathology, but the mechanisms connecting RHI, inflammation, and tau in CTE are unknown, representing a major knowledge gap. Proper lysosomal function in microglia is a key mechanism of p-tau clearance that, when impaired, contributes to p-tau deposition. Recently, a genetic risk factor for more severe tau pathology in CTE, TMEM106b, was found to influence microglial inflammation, tau pathology, and CTE severity. TMEM106b is a lysosome-associated protein and the risk allele produces larger, poorly acidifying lysosomes, pointing to a potential role for lysosomal processing in CTE pathogenesis. Furthermore, preliminary single nucleus RNA sequencing (snRNAseq) data from CTE tissue also suggest an alteration to lysosomal functions in microglia. These data led us to hypothesize that exposure to RHI triggers persistent microglial lysosomal dysfunction which is exacerbated by TMEM106b genetic risk allele and drives p-tau deposition in CTE. To address this hypothesis, we will utilize snRNAseq, spatial transcriptomics, and multiplex immunofluorescence to tease apart the genetic underpinnings of RHI-induced microglial lysosomal dysfunction and understand its role as a potential mechanism conferring TMEM106b genetic risk for more severe p-tau pathology in CTE. In Aim 1 we will assess cellularly and spatially resolved genomic changes to microglial transcripts in individuals with CTE. This will reveal the genomic underpinnings of lysosomal dysfunction following RHI and allow us to localize these changes to regions of p-tau deposition. In Aim 2 we will examine microglial lysosomal structure and content, markers of lysosomal function, stress, and protease expression. We will then analyze the relationship of these changes to TMEM106b allele status, years of RHI exposure, and presence and severity of CTE pathology. Previous work in the McKee and Cherry Labs (Sponsor and co-sponsor) and preliminary data have validated the proposed techniques. This work will be the first to study microglial lysosomal dysfunction as a mechanism of CTE pathology and genetic risk which will significantly advance or understanding of an early disease process and direct future discovery of novel biomarkers and therapeutically targetable mechanisms.