Cytoplasmic aggregation of TDP-43 has been reported in almost every age-dependent neurodegenerative disease, including in >40% of frontal temporal dementia (FTD), in the hippocampal neurons of Alzheimer's disease (AD) patients, in >90% of ALS, and in ~100% of a recently recognized AD-like dementia in the oldest of the elderly, an AD-like syndrome identified in 2019 and named Limbic-predominant Age-related TDP-43 Encephalopathy (LATE). We have demonstrated that TDP-43 phase separation and aggregation can drive neuronal death independent of RNA binding, stress granule formation, and TDP-43 association with stress granules. We have subsequently identified that acetylation of TDP-43 (which abolishes its RNA binding) drives its separation into liquid spherical annular bodies. These nuclear annuli have liquid annular shells enriched in TDP-43 and liquid centers highly enriched in HSP70 family molecular chaperones. Use of inhibitors of known deacetylases or the proteasome (to mimic the known age-dependent declines in deacetylase and proteasome activities) provokes cytoplasmic TDP-43 aggregation. We propose to determine the biological and pathological role(s) of acetylated TDP-43 and how HSP70 chaperone activity regulates nuclear TDP-43 function and its aggregation in the cytoplasm. We will determine the regulation and biological consequences of acetylated TDP-43 in neurons by identifying the key regulatory enzymes (acetyltranferases and deacetylases) of acetylated TDP-43 and alter TDP-43’s function in RNA splicing and its subcellular localization/aggregation. To understand how HSP70 family molecular chaperones regulates phase transition of TDP-43, we will use Hsc70 (encoded by the HSPA8 gene and the most abundant HSP70 in neurons) and determine how Hsc70 interacts with TDP-43. We will also determine if enhancing the activity of HSP70 (such as HSPA8, which is highly expressed in neurons) ameliorates TDP-43 pathology. We will also develop a potential therapeutic approach for TDP-43 proteinopathies in which rapid proteasome-mediated degradation of aggregated TDP-43 is achieved through an engineered E3 ubiquitin ligase linked to a synthetically evolved nanobody (a single chain antibody derived from an antibody heavy chain) recognizing either acetylated or phosphorylated TDP-43. Outcomes of these efforts will provide key insights for understanding basic aspects of TDP-43 biology and pathobiology in common dementia, and for developing a new concept of therapy that specifically targets TDP- 43 pathology that could potentially benefit aged patients with TDP-43-related dementia.