Inactivation of NSF ATPase Leads to Cathepsin B Release After Transient Cerebral Ischemia

Original Article
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Abstract

Neurons have extraordinary large cell membrane surface area, thus requiring extremely high levels of intracellular membrane-trafficking activities. Consequently, defects in the membrane-trafficking activities preferentially affect neurons. A critical molecule for controlling the membrane-trafficking activities is the N-ethylmaleimide-sensitive factor (NSF) ATPase. This study is to investigate the cascade of events of NSF ATPase inactivation, resulting in a massive buildup of late endosomes (LEs) and fatal release of cathepsin B (CTSB) after transient cerebral ischemia using the 2-vessel occlusion with hypotension (2VO+Hypotension) global brain ischemia model. Rats were subjected to 20 min of transient cerebral ischemia followed by 0.5, 4, 24, and 72 h of reperfusion. Neuronal histopathology and ultrastructure were examined by the light and electron microscopy, respectively. Western blotting and confocal microscopy were utilized for analyzing the levels, redistribution, and co-localization of Golgi apparatus and endosome or lysosome markers. Transient cerebral ischemia leads to delayed neuronal death that occurs at 48–72 h of reperfusion mainly in hippocampal CA1 and neocortical (Cx) layers 3 and 5 pyramidal neurons. During the delayed period, NSF ATPase is irreversibly trapped into inactive protein aggregates selectively in post-ischemic neurons destined to die. NSF inactivation leads to a massive buildup of Golgi fragments, transport vesicles (TVs) and late endosomes (LEs), and release of the 33 kDa LE type of CTSB, which is followed by delayed neuronal death after transient cerebral ischemia. The results support a novel hypothesis that transient cerebral ischemia leads to NSF inactivation, resulting in a cascade of events of fatal release of CTSB and delayed neuronal death after transient cerebral ischemia.

Keywords

N-ethylmaleimide sensitive factor ATPase (NSF) SNAREs Brain ischemia-reperfusion injury Membrane trafficking  Cathepsin B (CTSB) Golgi fragments Transport vesicle Late endosome Lysosome. 

Abbreviations

NSF

N-ethylmaleimide sensitive factor ATPase

SNAREs

Soluble NSF attachment protein receptors

SNAP

Soluble NSF attachment protein

CTSB

Cathepsin B

TVs

Transport vesicles

LE

Late endosome

EL

Endolysosome

L

Lysosome

MOMP

Mitochondrial outer membrane permeabilization

IRI

Ischemia-reperfusion injury

DG

Dentate gyrus

EM

Electron microscopy

Vti1b

Vesicle transport through interaction with t-SNAREs homolog 1B

TGN38

Trans-Golgi network membrane protein 38 kDa

Notes

Compliance with Ethical Standards

Conflict of Interest

Dong Yuan, Chunli Liu, and Bingren Hu declare no conflict of interest.

Ethical Approval

This article does not contain any studies with human subjects. All the experimental procedures involving using animals were approved by the Animal Use and Care Committee in the University of Maryland School of Medicine.

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Copyright information

© Springer Science+Business Media, LLC (Outside the USA) 2017

Authors and Affiliations

  1. 1.Department of Neurology and Anesthesiology, Shock Trauma and Anesthesiology Research CenterUniversity of Maryland School of MedicineBaltimoreUSA
  2. 2.Department of Neurology, The First Teaching HospitalJilin UniversityChangchunChina
  3. 3.Veterans Affairs Maryland Health Center SystemBaltimoreUSA

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