# The Role of Lysosomal Membrane Permeabilization and Cathepsin B Release in Stroke Brain Injury

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA, SAN DIEGO · 2023 · $525,917

## Abstract

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...

## Key facts

- **NIH application ID:** 10736263
- **Project number:** 1R01NS129553-01A1
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN DIEGO
- **Principal Investigator:** Bingren Hu
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $525,917
- **Award type:** 1
- **Project period:** 2023-07-01 → 2028-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10736263

## Citation

> US National Institutes of Health, RePORTER application 10736263, The Role of Lysosomal Membrane Permeabilization and Cathepsin B Release in Stroke Brain Injury (1R01NS129553-01A1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10736263. Licensed CC0.

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