Project Summary Frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are two progressive neurodegenerative disorders on a spectrum of disease related to the RNA/DNA binding protein TAR DNA- binding Protein of 43 kDa (TDP-43). Many patients demonstrate an intermediate phenotype of dementia with motor neuron disease (here called FTLD-ALS). The vast majority of pure FTLD and ALS cases are sporadic (sFTLD, sALS), with no family history or known genetic mutation. More than 50% of all FTLD and 90% of all ALS cases manifest a characteristic pathology in affected neurons: hyperphosphorylated, ubiquitinated inclusions of TDP-43. TDP-43 pathology is often observed in other neurodegenerative disorders, including Alzheimer’s and Parkinson’s Diseases, suggesting a common pathogenic mechanism linking TDP-43 dysfunction and neurodegeneration. TDP-43 aggregates are normally degraded by autophagy, but in FTLD-ALS this machinery fails, contributing to disease progression. In fact, some familial FTLD-ALS cases are caused by mutations in autophagy-related proteins. The mechanisms behind TDP-43 aggregation and the neurotoxicity it imparts remain poorly understood, particularly in sporadic disease. Most animal models rely on overexpression of disease- associated genetic variants; however, these may be limited in generalizability to sporadic disease. Our lab identified TDP-43 acetylated at a key lysine residue (Ac-K145) as a driver of TDP-43 pathology. Ac-K145 TDP- 43 is detected in the pathologic inclusions in sALS spinal cord. With the goal of better modeling sporadic illness, we used CRISPR/Cas9 technology to insert a K145Q acetylation-mimic mutation in the endogenous mouse Tardbp locus (TDP-43K145Q) to generate a novel model of TDP-43 proteinopathy in sporadic FTLD-ALS. TDP- 43K145Q mice show hallmark pathologies, such as age-dependent cognitive impairment and accumulation of insoluble TDP-43 in the cortex and spinal cord. This project aims to determine the role of acetylation-mimic TDP- 43 in neurodegeneration and autophagy impairment. Aim 1 will test the hypothesis that aging exacerbates the neurodegenerative phenotype in TDP-43K145Q mice, using behavioral assays of cognitive and motor function, neuropathologic assessment of cortical tissue, and electrodiagnostic studies of motor unit function. Aim 2 will test the hypothesis that autophagic flux is impaired in primary cortical neurons of TDP-43K145Q sFTLD-ALS mice, using an in vitro aging paradigm alongside pharmacologic manipulation of autophagy and biochemical and live- cell imaging techniques. The long-term goal of this project is to better understand the mechanisms behind TDP- 43-related neurodegeneration and reveal opportunities for therapeutic intervention. This work will provide me with comprehensive training in both translational and basic science research methods, and I will complement my research with mentored clinical activities caring for neurodegenerative disease patients....