Project Summary Cellular inclusions of proteins are primary hallmarks of a great majority of neurodegenerative diseases. In common forms of amyotrophic lateral sclerosis (ALS), Alzheimer’s disease related dementias (frontotemporal dementia; limbic-predominant age-related TDP-43 encephalopathy (LATE)) as well as some forms of Alzheimer’s disease, the essential human TAR DNA binding protein of 43 kDa (TDP-43) forms intraneuronal aggregates. In the parent grant, we examine in detail the structural biology of TDP-43 to yield new insights into the mechanism of disease and potential new therapies. Importantly, dozens of missense mutations in an aggregation-prone domain of TDP-43 have been found in familial and sporadic cases of ALS and frontotemporal dementia. In the parent award, we focus on two hotspots, regions of TDP-43 clustering disease mutations. In this supplement proposal, we will use the tools available in the laboratory to focus on a third new hotspot where mutations associated with and causative for ALS are found but currently no proposed biophysical / biochemical mechanism has been probed. The new proposed experiments fit within the scope of the parent award and the overall hypothesis that mutations in TDP-43 alter the molecular structure and interactions, leading to TDP-43 dysfunction. As rationale, 11 mutations associated with ALS and frontotemporal dementia are found in residues 378-390, including four in familial ALS, suggesting causation. Furthermore, our previous data indicate a potential role for this region in making contacts stabilizing functional interactions. We will test the hypothesis that residues 378-390 participate in biological self-interactions of TDP- 43 that are disrupted by mutations in this hot spot and that post translational modifications in the hot spot modulate this important interaction. In supplement Aim 1, we hypothesize that this region forms hydrophobic contacts mediating dynamic interactions, and that these interactions are disrupted by disease-associated mutations in this region leading to aggregation. Using our established NMR spectroscopy techniques paired with in vitro phase separation, we will determine the structural details of contacts at this site and evaluate the mechanistic role of disease mutations. In Aim 2, we will test the impact of known TDP-43 post-translational modifications in this region on the interactions and the resulting impact on TDP-43 aggregation, as well as the impact of mutations on the post-translational modification and vice versa (i.e. cross-talk). These studies provide future input to the design of strategies to prevent toxic disruption of TDP-43 in Alzheimer’s disease related dementias and ALS. These studies also form an important part of the PhD thesis plan for a URM graduate student. A comprehensive training and career development plan is presented here to prepare the student for his career goal to make sustained and impactful contributions in the molecular understanding an...