Brain ischemia-reperfusion (IR) injury due to stroke and cardiac arrest is a leading cause of death and long-term disability, affecting thousands of Veterans every year. However, the molecular mechanisms underlying brain IR injury are still not completely understood. The objective of this proposal is to study a novel hypothesis that brain IR leads to a cascade of events resulting in inactivation of N-ethylmaleimide sensitive factor (NSF), massive buildup of damaged late endosomes (LEs), fatal release of cathepsin B (CTSB), induction of mitochondrial outer membrane permeabilization (MOMP), and delayed neuronal death. NSF is the sole ATPase controlling membrane trafficking from the Golgi apparatus to the endosome- lysosome system. Our recent studies show that NSF ATPase is progressively trapped as inactive aggregates within hippocampal CA1 neurons destined to undergo delayed neuronal death after brain IR. Our electron microscopic (EM) studies further show massive accumulation of damaged Golgi, transport vesicles (Vs), and late endosomes (LEs) in the CA1 neurons. Consequently, CTSB is extensively released from damaged Golgi/Vs/LEs structures, followed by delayed neuronal death after brain IR. We therefore generated a neuron-specific NSF activity-deficient transgenic (tg) mouse line (replacement of NSF 329 glutamate with glutamine, i.e., E329Q). The most prominent pathological phenotypes of this E329Q tg mouse line are massive accumulation of damaged Golgi/Vs/LEs, and release of CTSB, followed by delayed neuronal death. These phenotypes are virtually identical to those observed in hippocampal CA1 neurons destined to undergo delayed neuronal death after brain IR. Based on these new discoveries, we propose to test a novel hypothesis strongly supported by preliminary studies, i.e., brain IR leads to a cascade of events of NSF inactivation, massive buildup of Golgi/Vs/LEs, release of CTSB, induction of MOMP, and delayed neuronal death. We will use the E329Q tg mouse line, CTSB knockout (KO) mice, and cutting-edge technologies to study these molecular events after brain IR. Aim 1 will test the novel hypothesis that NSF inactivation results in fatal release of CTSB and delayed neuronal death after brain IR. We will use the E329Q tg mice without brain IR and wildtype (wt) littermates subjected to brain IR to test this novel hypothesis. Aim 2 will test the novel hypothesis that fatal release of CTSB leads to induction of MOMP after brain IR. We will use CTSB KO mice to test this novel hypothesis. This Aim is based on the finding that cytosolic release of CTSB induces MOMP, resulting in delayed neuronal death after brain IR. Aim 3 will test the hypothesis that the NSF inactivation-induced cascade of events is a common pathway responsible for delayed neuronal death after brain IR. The rationale for Aim 3 is that studies in Aims 1 and 2 focus on the role of NSF inactivation in the hippocampal CA1 neurons after brain IR. A broader and perhaps even more importa...