Project Summary/Abstract Regardless of family history, site of onset, and sequence of symptoms and progression, all Amyotrophic Lateral Sclerosis (ALS) patients lose muscle mass as the motor neurons that connect to muscle in limbs to control movement stop working and eventually die. However, there appears to be an extensive delay between initial neuronal dysfunction and physical degeneration. The complex nervous system is maintained, in part, due to cell resiliency to stress in order to maintain function and resist activating cell death processes. Experiments in this proposal will investigate a novel hypothesis: neurons in ALS enter cellular senescence as part of a complicated stress response to avoid active degeneration and cell death. Nonetheless, while this protective mechanism may preserve the physical presence of the cell, senescent cell dysfunction and potentially toxic secretome promotes chronic tissue degeneration over time that facilitates disease progression. Senescence is activated by protein accumulation in other neurodegenerative diseases. Here we will determine whether TDP43 deposition differentially affects the cellular senescence stress response by evaluating postmortem human brain and spinal cord from veteran donors, accessed through the VA ALS Biorepository Brain Bank, with either TDP43 positive or negative neuropathology. To comprehensively investigate neurons and their environment we will utilize GeoMx digital spatial profiling technology. This high resolution, high-content non-destructive analytical method enables the investigation of 100s of proteins at the single cell level while maintaining spatial resolution. This affords the ability to map the data back to the tissue to correlate protein expression differences with cell morphology, location in the tissue, extracellular environment and proximity to vascular and / or other pathologies. The data generated by our research proposal will provided an unprecedented rigor for depth of information on cellular stress in ALS spinal cord and brain. The rich dataset generated through these experiments will open many novel avenues of investigation in ALS and potentially unveil cellular senescence as a new therapeutic target. This is an area desperately in need of development as the majority of ALS patients die, on average, 2-4 years after diagnosis.