PROJECT SUMMARY Glial pathology is increasingly recognized as a key feature in neurodegenerative diseases. However, the exact mechanisms that promotes glial pathology remain unclear. Several studies implicate the autophagy machinery, a highly conserved lysosomal degradation pathway, in maintaining protein homeostasis in neurons during brain aging and neurodegeneration. In addition, autophagy regulates the check-and-balance of microglia-mediated inflammatory response to misfolded proteins, including β-amyloid peptides, hyperphosphorylated Tau, and TDP- 43, which accumulate during neurodegeneration. Collectively, these results underscore the critical and cell type- specific role of the autophagy machinery in maintaining neuron-glia homeostasis by regulating the trafficking of intracellular organelles and cargoes, such as misfolded proteins. In the previous funding period, we uncovered new autophagy-dependent secretory processes in non-neuronal and glial cells, including most notably, LC3- dependent extracellular vesicle loading and secretion (LDELS). LDELS targets include RNA binding proteins and extracellular vesicle (EV) cargoes that have been implicated in the pathogenesis of frontotemporal dementia (FTD). In addition, our results showed that loss of function in FTD gene Progranulin (Grn) leads to age-dependent endolysosomal defects and hypersecretory phenotypes in microglia that promotes neuronal loss and abnormal accumulation of the RNA binding protein TDP-43 in Grn-/- mouse brain. Together, these results underscore the important roles of autophagy and endolysosomal trafficking in microglial activation in FTD. In follow up studies, we have further expanded the scope of our work and identified a highly regulated mechanism, Secretory Autophagy during Lysosome Inhibition (SALI), which is critically dependent on multiple ATGs for autophagosome formation and on Rab27a for vesicle exocytosis. Using TMT-based quantitative proteomics, we uncovered that loss of Progranulin leads to a marked increase in unconventional myosin Myo5a, a known Rab27a interacting partner. Given the well-documented role of Rab27a and Myo5a in the transport of intracellular organelles, these results broach the hypothesis that blockage of endolysosomal trafficking disrupts microglial homeostasis and promotes secretory autophagy and neurodegeneration in Grn-/- mice. To test this hypothesis, we will: 1) Delineate how FTD-associated endolysosomal defects influence secretory autophagy and dissect the effects of SALI on intracellular homeostasis; 2) Scrutinize how secretory autophagy and endolysosomal defects in microglia impact lipid metabolism and microglial toxicity; and 3) Determine whether genetic autophagy loss mitigates microglial toxicity and neurodegeneration in Progranulin-deficient mice. Together, the studies proposed in this renewal harness the unique expertise in Drs. Huang and Debnath’s laboratories and provide critical insights on how SALI controls the delicate balanc...