Nuclear clearance and cytoplasmic aggregation of TDP-43 have been reported in almost every age-dependent neurodegenerative disease, including as the defining feature of a recently recognized dementia in the oldest of the elderly, an AD-like syndrome named Limbic-predominant Age-related TDP-43 Encephalopathy (LATE), a proportion of the hippocampal neurons in Alzheimer's disease (AD), >40% of frontal temporal dementia (FTD), and >90% of instances of ALS. Our prior efforts (for which we now seek renewed support) have established that transient stress can induce Liquid-Liquid Phase Separation (LLPS) of cytoplasmic TDP-43 into liquid droplets that then transition to a solid state, slowly deplete nuclear TDP-43, and provoke cell death over a timescale of weeks. We also determined that partial or complete proteasome inhibition (to mimic the established decline in proteosome activity during normal aging) provokes TDP-43 mislocalization/accumulation within the cytoplasm. Quantitative mass spectrometry (with proximity-labeling and isobaric-tagging) has identified the small heat shock protein HSPB1 to be a regulator of cytoplasmic TDP-43 phase separation and subsequent aggregation. HSPB1 partitions into TDP-43 droplets, inhibits TDP-43 assembly into fibrils, and mediates disassembly of stress-induced, TDP-43 droplets. Building on our prior and continuing work, we now propose to determine 1) how the age-dependent decrease in proteasome activity drives TDP-43 loss of function, cytoplasmic mislocalization, phase separation, and aggregation and 2) how protein chaperone HSPB1, in conjunction with HSP70, affects cytoplasmic TDP-43 phase separation, inhibits TDP-43 assembly into fibrils, and mediates disassembly of TDP-43 droplets. We have also initiated development of an approach to generate new/replacement neurons in the aged adult mouse brain by transiently suppressing the RNA binding protein Polypyrimidine Tract Binding Protein-1 (PTB) using an antisense oligonucleotide (ASO) delivered by a single injection into cerebral spinal fluid (CSF). Radial glial-like cells (and possibly other GFAP-expressing cells) convert into new neurons over a two month period, acquire mature neuronal character, and functionally integrate into endogenous circuits that modify mouse behavior. Here we will systematically identify the functionality, localization, cell origin, timing, and molecular pathways of cells undergoing identity conversion, with a primary assay the development and utilization of single cell RNA signatures obtained with spatial transcriptomics (Multiplexed Error-Robust Fluorescence In Situ Hybridization [MERFISH]).