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

• 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
• 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

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.

项目摘要 拟议的研究旨在研究内溶性损伤在缺血性脑损伤中的作用。最近， 在理解内溶血系统（以前称为内溶血系统）方面取得了重大进展 溶酶体系统。现在，此内溶血系统包括三个基本结构：（i）晚期内体（LE），（ii） 溶酶体（L）和（iii）内溶液体/自溶性（EL/AL）。 LE接收传入的内溶性蛋白 （例如，腔内组织蛋白酶和结构蛋白）来自高尔基体和废物肉 内吞和自噬途径。尽管LE包含calter蛋白和垃圾蜡，但LE的 由于LE的酸性pH值较少（6.0），组织蛋白酶无法有效地降解这些废物兑。 LE必须融合 随着更酸性的溶酶体（L）（pH 4.0-4.5）成为杂交内溶性（EL）以有效降解 这些垃圾货物。限制速率的LE-TO-L融合步骤是由N-乙基马来酰亚胺敏感的 因子ATPase（NSF）依赖性机械。我们最近的研究表明，脑缺血导致NSF 注定要死的神经元缺乏。我们生成了一个神经元特异性的NSF缺陷鼠标线以了解 NSF缺乏在全球和局灶性脑缺血后观察到的内溶性损害中的作用。在 NSF缺乏小鼠（缺血），有一个明显的“多聚集” 内溶性结构，其次是自主神经元死亡。此表型复制相同 在全球和局灶性脑部缺血之后，在野生型（WT）窝窝中观察到的神经病理学特征。 神经元组织蛋白酶B（CTSB）释放是在我们的NSF-中都观察到的另一个关键神经病理学特征 在全球和局灶性脑缺血之后，没有缺血的小鼠和无元窝窝。大多数组织蛋白酶 中性pH值低或没有活性，但是CTSB在中性pH值下唯一表现出内肽酶活性。 CTSB是 神经元中最优势的组织蛋白酶。我们最近的研究进一步表明，常规（所有组织）CTSB 小鼠中的敲除（KO）可显着保护小鼠局灶性脑缺血模型中的大脑。基于这些新的 结果，我们假设缺血后的NSF缺乏会导致内溶性损害，并导致大量 其内容的数量释放，例如CTSB，进入细胞质和细胞外空间。大量数量 CTSB的释放导致缺血性脑损伤。我们将通过调查以下方法来检验这一新假设：（i） 缺血性NSF缺乏会导致使用神经特异性NSF缺乏小鼠无脑的内溶性损害 缺血及其同窝室受到脑缺血的影响（AIM 1）； （ii）如果转基因（TG）中的NSF过表达 小鼠可以减少内溶性损伤和脑缺血性损伤（AIM 2）； （iii）是否以及如何神经元 CTSB释放使用神经特异性CTSB KO小鼠导致缺血性脑损伤（AIM 3）； （iv） 小胶质细胞和巨噬细胞CTSB在长时间的缺血后炎症中使用小胶质细胞和巨噬细胞 - 特定的CTSB KO小鼠（AIM 4）。这些研究将促进靶向新的疗法的发展 内溶性损伤并有可能恢复脑缺血后的NSF活性。