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 i s 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 important question is: is NSF inactivation commonly responsible for delayed neuronal death in all populations of hippocampal and neocortical neurons after brain IR? We will test this hypothesis by: (i) studying the damaging effects on the NSF-CTSB-MOMP pathway in ?ischemic resistant? CA3, DG and neocortical neurons after prolonged brain IR; (ii) examining whether CTSB KO can prevent delayed neuronal death in ?ischemic resistant? neurons after prolonged brain IR; and (iii) investigating whether the increased vulnerability in ?ischemic resistant? neurons in E329Q tg mice is due to the weakening of the NSF active store, resulting in CTSB release and induction of MOMP after brain IR. These studies will provide insights into the novel mechanism underlying brain IR injury and identify new therapeutic targets for the treatment of brain IR injury.

Public Health Relevance

Ischemia-reperfusion injury after cardiac arrest or stroke is a devastating neurological disease that is particularly prevalent in elderly Veterans. This proposal will study the role of the membrane trafficking activity in ischemia-reperfusion injury.! The proposed studies will form the basis for translational studies that will improve the treatment of acute brain damage due to cardiac arrest and stroke in Veterans; and potentially include treatment of ischemic and traumatic brain injury.

Agency
National Institute of Health (NIH)
Institute
Veterans Affairs (VA)
Type
Non-HHS Research Projects (I01)
Project #
1I01BX003926-01A1
Application #
9452325
Study Section
Neurobiology C (NURC)
Project Start
2018-01-01
Project End
2021-12-31
Budget Start
2018-01-01
Budget End
2018-12-31
Support Year
1
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Baltimore VA Medical Center
Department
Type
DUNS #
796532609
City
Baltimore
State
MD
Country
United States
Zip Code
21201
Yuan, Dong; Liu, Chunli; Wu, Jiang et al. (2018) Nest-building activity as a reproducible and long-term stroke deficit test in a mouse model of stroke. Brain Behav 8:e00993
Yuan, Dong; Liu, Chunli; Wu, Jiang et al. (2018) Inactivation of NSF ATPase Leads to Cathepsin B Release After Transient Cerebral Ischemia. Transl Stroke Res 9:201-213
Yuan, Dong; Liu, Chunli; Hu, Bingren (2018) Dysfunction of Membrane Trafficking Leads to Ischemia-Reperfusion Injury After Transient Cerebral Ischemia. Transl Stroke Res 9:215-222