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.

项目总结