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

溶酶体膜透化和组织蛋白酶 B 释放在中风脑损伤中的作用

基本信息

批准号：
10736263
负责人：
Bingren Hu
金额：
$ 52.59万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-07-01 至 2028-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10736263
关键词：
ATP phosphohydrolase Abbreviations Affect American Animal Model Autophagosome Brain Brain Injuries Brain Ischemia Cathepsins Cathepsins B Cell Death Cell Membrane Permeability Cells Cytoplasm Cytosol Development Disease Early Endosome Encephalitis Endopeptidases Enzymes Ethylmaleimide Event Exhibits Extracellular Space Family member Golgi Apparatus Hybrids I Kappa B-Alpha Inflammasome Inflammation Inflammation Mediators Inflammatory Inflammatory Response Injury Ischemia Ischemic Brain Injury Ischemic Stroke Knock-out Knockout Mice Lysosomes Macrophage Mediating Microglia Modeling Mus N-ethylmaleimide-sensitive protein NF-kappa B Neurons Outer Mitochondrial Membrane Pathologic Pathway interactions Pattern Phenotype Process Protein Deficiency Proteins Replacement Therapy Research Role Rupture Site Stroke Structural Protein Structure Suicide System Testing Therapeutic Tissues Transgenic Mice cytokine late endosome migration mouse model nervous system disorder neuron loss neuropathology new therapeutic target novel overexpression wasting

项目摘要

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 inflammation using microglia and macrophage- specific CTSB KO mice (Aim 4). These studies will facilitate the development of novel therapeutics that target endolysosomal damage and potentially restores NSF activity after brain ischemia.

项目概要拟议的研究旨在研究内溶酶体损伤在缺血性脑损伤中的作用。在理解内溶酶体系统（以前称为内溶酶体系统）方面取得了重大进展该溶酶体系统现在包括三个基本结构：（i）晚期内体（LE），（ii）溶酶体 (L) 和 (iii) 内溶酶体/自溶酶体 (EL/AL) LE 接收传入的内溶酶体蛋白。来自高尔基体的（例如，腔内组织蛋白酶和结构蛋白）和来自虽然 LE 含有组织蛋白酶和废物，但 LE 的内吞和自噬途径。由于 LE 的 pH 值较低 (6.0)，组织蛋白酶无法有效降解这些废物。与酸性更强的溶酶体 (L) (pH 4.0-4.5) 形成杂合内溶酶体 (EL) 以有效降解这些废物货物。限速的 LE 到 L 融合步骤是由 N-乙基马来酰亚胺敏感介导的。我们最近的研究表明，脑缺血会导致 NSF。我们构建了一个神经元特异性 NSF 缺陷小鼠品系来了解。 NSF 缺乏在全脑缺血和局灶性脑缺血后观察到的内溶酶体损伤中的作用。 NSF 缺陷小鼠（无缺血），存在明显的异常“多聚集”积聚内溶酶体结构，随后是自主神经死亡，这种表型复制相同。在全脑和局灶性脑缺血后，在野生型（wt）同窝小鼠中观察到神经病理学特征。神经元组织蛋白酶 B (CTSB) 释放是在我们的 NSF- 没有缺血的缺陷小鼠和大多数组织蛋白酶在全脑和局灶性脑缺血后的野生型同窝小鼠。在中性 pH 下具有低活性或无活性，但 CTSB 在中性 pH 下具有独特的内肽酶活性。我们最近的研究进一步表明，传统（所有组织）CTSB 是神经元中最主要的组织蛋白酶。基于这些新的研究，在小鼠局灶性脑缺血模型中，基因敲除（KO）可以显着保护小鼠的大脑。结果，我们发现缺血后 NSF 缺乏会导致内溶酶体损伤并导致大量其内容物（例如 CTSB）大量释放到细胞质和细胞外空间。 CTSB 的释放会导致缺血性脑损伤，我们将通过研究以下内容来检验这一新假设：(i) 后如何发生。使用神经元特异性 NSF 缺陷无脑小鼠，缺血性 NSF 缺陷导致内溶酶体损伤缺血及其同窝小鼠脑缺血（目标 1）；（ii）如果转基因（tg）中 NSF 过度表达小鼠可以减少内溶酶体损伤和脑缺血损伤（目标 2）；（iii）是否以及如何减轻神经损伤使用神经元特异性 CTSB KO 小鼠释放 CTSB 会导致缺血性脑损伤（目标 3）；以及 (iv) 小胶质细胞和巨噬细胞 CTSB 使用小胶质细胞和巨噬细胞治疗长期缺血后炎症特定的 CTSB KO 小鼠（目标 4）。内溶酶体损伤并可能在脑缺血后恢复 NSF 活性。