PROJECT SUMMARY Sporadic amyotrophic lateral sclerosis (sALS) is the most common progressive motor-neuron disease, affecting 3 in 100,000 individuals 50 years or older worldwide. sALS is pathologically characterized by the presence of hyper-phosphorylated, insoluble aggregates of the RNA/DNA binding protein Transactive Response DNA Binding Protein (TDP-43). It is well established that aggregated TDP-43 causes dysfunction throughout the central nervous system (CNS), ultimately resulting in neuronal cell death and symptom onset. However, the exact molecular mechanism(s) by which aggregated TDP-43 imparts neurotoxicity has not yet been determined. Recent evidence implicates innate immune cells, particularly infiltrating peripheral macrophages and microglia, as a source of inflammation when stimulated by misfolded protein aggregates. This neuroinflammatory response is therefore expected to compromise neuron integrity and contribute to neurodegeneration. Furthermore, and adaptive immune cells, T- and B-lymphocytes, have been reported in post-mortem analyses of ALS brain and spinal cord, however their mechanistic role in pathology remains unclear. This implicates both innate and adaptive immune responses in sALS pathogenesis. Recent developments also suggest that microglia promote secondary T-cell responses when stimulated by protein aggregates. This T-cell activation may then result in direct neuronal death. Therefore, I hypothesize that aggregated TDP-43 causes a pro-inflammatory, neurodegenerative cascade mediated by innate and adaptive immune responses that drives neurotoxicity in sALS. In Aim 1, I will treat microglia with aggregated mammalian-derived TDP-43 and monitor modulation of phagolysosomal integrity and inflammatory activation. Preliminary data using models of microglia treated with TDP-43 aggregates demonstrate robust aggregate uptake and phagosome rupture. Additionally, mass spectrometry analysis of TDP-43-treated macrophage cultures revealed an increase in pro-inflammatory and immune activation factors, and markers of phagocytic dysfunction. In Aim 2, I will assess the ability of TDP-43- treated microglia to promote motor-neuron death by way of T-cell activation. I will determine if TDP-43- treated microglia engage with and promote CD4+ and CD8+ T-cell activation in culture. Finally, I will determine if activated cytotoxic CD8+ T-cells prompt motor-neuron death in vitro using sALS patient-derived T-cells and induced pluripotent stem cell (iPSC)-derived motor-neurons. In all, this work will use novel in vitro models to elucidate the unknown mechanism by which TDP-43 aggregation leads to motor-neuron death and sALS onset. This knowledge lends itself to biomarker and therapeutic development to improve diagnostic and treatment strategies for sALS patients.