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

项目摘要 本研究旨在探讨内溶酶体损伤在缺血性脑损伤中的作用。最近， 在了解内溶酶体系统方面已经取得了重大进展， 溶酶体系统这种内溶酶体系统现在包括三种基本结构：（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 (e.g.,管腔内组织蛋白酶和结构蛋白）和来自高尔基体的废物。 内吞和自噬途径。虽然LE含有组织蛋白酶和废物，但LE的 由于LE的酸性较低（6.0），组织蛋白酶不能有效地降解这些废物。LE必须熔断 与酸性更强的溶酶体（L）（pH 4.0-4.5）结合，形成杂合内溶酶体（EL）， 这些废物。该限速LE-至-L融合步骤由N-乙基马来酰亚胺敏感性蛋白介导。 因子ATP酶（NSF）依赖性机制。我们最近的研究表明，脑缺血导致NSF 注定会死亡的神经元的缺陷。我们产生了一个神经元特异性NSF缺陷小鼠系，以了解 NSF缺乏在全脑和局灶性脑缺血后观察到的内溶酶体损伤中的作用。在 NSF缺陷小鼠（无缺血），存在异常“多聚集”的显著积聚， 内溶酶体结构，随后是自主神经元死亡。这种表型复制相同的 在全脑和局灶性脑缺血后，在野生型（WT）同窝仔中观察到的神经病理学特征。 神经元组织蛋白酶B（CTSB）的释放是在我们的NSF- 没有缺血的缺陷型小鼠和在全脑和局灶性脑缺血后的WT同窝小鼠。大多数组织蛋白酶 在中性pH下具有低活性或没有活性，但CTSB独特地在中性pH下表现出内肽酶活性。 神经元中最主要的组织蛋白酶。我们最近的研究进一步表明，传统的（所有组织）CTSB 敲除（KO）在小鼠中显著保护小鼠局灶性脑缺血模型中的脑。基于这些新 结果，我们假设缺血后NSF缺乏导致内溶酶体损伤，并引起大的 其内容物的量释放，例如，CTSB，进入细胞质和细胞外空间。这么大的数量 CTSB的释放导致缺血性脑损伤。我们将通过调查来测试这个新的假设：（一）如何后- 缺血性NSF缺乏导致内溶酶体损伤，使用无脑的神经元特异性NSF缺乏小鼠 缺血和它们的同窝出生仔经受脑缺血（Aim 1）;（ii）如果转基因（tg）中NSF过表达， 小鼠可以减少内溶酶体损伤和脑缺血性损伤（Aim 2）;（iii）是否以及如何神经元 使用神经元特异性CTSB KO小鼠，CTSB释放导致缺血性脑损伤（Aim 3）;和（iv）CTSB的作用。 使用小胶质细胞和巨噬细胞CTSB在长时间缺血后炎症中的作用 特异性CTSB KO小鼠（目的4）。这些研究将有助于开发新的治疗方法， 内溶酶体损伤和潜在地恢复脑缺血后的NSF活性。

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.