Project Summary The proposed research aims to investigate the role of endolysosomal damage in ischemic brain injury. Recently, significant progress has been made in understanding the endolysosomal system, previously known as the lysosomal system. This endolysosomal system now includes three basic structures: (i) late endosome (LE), (ii) lysosome (L), and (iii) endolysosome/autolysosome (EL/AL). The LE receives incoming endolysosomal proteins (e.g., intraluminal cathepsins and structural proteins) from the Golgi apparatus and waste cargos from the endocytic and autophagic pathways. Although the LE contains both cathepsins and waste cargos, the LE’s cathepsins cannot efficiently degrade these waste cargos due to LE’s less acidic pH (6.0). The LE must fuse with the more acidic lysosome (L) (pH 4.0-4.5) to become a hybrid endolysosome (EL) to efficiently degrade these waste cargos. This rate-limiting LE-to-L fusion step is mediated by the N-ethylmaleimide sensitive factor ATPase (NSF)-dependent machinery. Our recent studies show that brain ischemia leads to NSF deficiency in neurons destined to die. We generated a neuron-specific NSF deficient mouse line to understand the role of NSF deficiency in the endolysosomal damage observed after both global and focal brain ischemia. In NSF-deficient mice (absence of ischemia), there is a prominent buildup of abnormal “multi-aggregated” endolysosomal structures, followed by autonomous neuronal death. This phenotype replicates the same neuropathological features observed in the wildtype (wt) littermates after both global and focal brain ischemia. Neuronal cathepsin B (CTSB) release is another key neuropathological feature observed in both our NSF- deficient mice without ischemia and the wt littermates after both global and focal brain ischemia. Most cathepsins have low or no activity at neutral pH, but CTSB uniquely exhibits endopeptidase activity at neutral pH. CTSB is the most dominant cathepsin in neurons. Our recent studies further show that conventional (all tissue) CTSB knockout (KO) in mice significantly protects the brain in a mouse focal brain ischemia model. Based on these new results, we hypothesize that post-ischemic NSF deficiency leads to endolysosomal damage and causes a large quantity release of its contents, e.g., CTSB, into the cytoplasm and extracellular space. This large quantity release of CTSB leads to ischemic brain injury. We will test this novel hypothesis by investigating: (i) how post- ischemic NSF deficiency leads to endolysosomal damage using neuron-specific NSF deficient mice without brain ischemia and their littermates subjected to brain ischemia (Aim 1); (ii) if NSF overexpression in transgenic (tg) mice can reduce both endolysosomal damage and brain ischemic injury (Aim 2); (iii) whether and how neuronal CTSB release leads to ischemic brain injury using neuron-specific CTSB KO mice (Aim 3); and (iv) the role of microglial and macrophage CTSB in prolonged post-ischemic inflammati...