Mechanisms of Mitochondrial Dysfunction in Brain Vasculature

描述（由申请人提供）：临床数据表明Hcy水平升高（也称为高同型半胱氨酸血症（HHcy））与卒中之间存在相关性。由于S-腺苷-高半胱氨酸水解酶（SAHH）引起的甲硫氨酸去甲基化增加以及甲基四氢叶酸还原酶（MTHFR）和胱硫醚-γ-裂解酶（CSE，一种负责将Hcy代谢为H2S的酶，一种最有效的血管扩张剂、抗氧化剂和抗高血压剂）的减少而产生HHcy，导致线粒体功能障碍（线粒体自噬）和缺血性卒中。细胞色素C传输电子并促进线粒体生物能量学。有趣的是，在HHcy期间，细胞色素C变成同型半胱氨酸化（N-Hcy-cyt-c）。但其对线粒体自噬和脑卒中的影响尚不清楚。本项目的长期目标是了解I/R损伤时脑血管线粒体自噬、线粒体修复和通透性的机制。我们的初步研究表明，I/R期间，总Hcy水平升高，导致N-Hcy-cyt-C，线粒体基质金属蛋白酶-9（mtMMP-9）增加，线粒体基质（连接蛋白和紧密连接蛋白，TJP）部分降解，导致脑血管的线粒体自噬和通透性。有趣的是，THC降低了Hcy水平，减轻了脑损伤。四氢姜黄素（THC），一种主要的草药抗氧化剂和抗炎剂，已被证明可以保护大脑免受I/R损伤。该提议的中心假设是，HHcy通过增加氧化应激、mtMMP-9、降解连接蛋白-43和TJP，部分地通过N-Hcy-cyt-C促进线粒体自噬介导的脑损伤（图1）。用THC、CSE基因和SAHH shRNA基因转移的处理减弱了线粒体自噬和通透性。我们将通过以下三个具体目标来测试这一假设：具体目标#1：确定Hcy是否有助于线粒体自噬，部分通过诱导氧化应激，加剧缺血再灌注中细胞色素-c的同型半胱氨酸化，以及THC，CSE和SAHH shRNA基因治疗是否减轻这些变化。具体目标#2：确定细胞色素c的同型半胱氨酸化是否激活缺血再灌注中的mt-MMP-9、胶原/弹性蛋白比率、mtCxn 43和mt-紧密连接蛋白的破坏，以及THC、CSE和SAHH shRNA基因治疗是否改善。具体目标#3：确定Hcy是否改变缺血再灌注时线粒体（mt）生物能量学和血管重塑，以及THC、CSE和SAHH shRNA基因治疗是否缓解。这些研究将证明脑血管重塑的新机制，并对脑缺血性卒中的线粒体修复具有治疗意义。

DESCRIPTION (provided by applicant): Clinical data suggest an association between elevated levels of Hcy, also known as hyperhomocysteine-mia (HHcy) and stroke. HHcy is generated due to increase in de-methylation of methionine by S-adenosine- homocysteine hydrolase (SAHH) and a decrease in methyltetrahydrofolate reductase (MTHFR) and cystathionine-y-lyase (CSE, an enzyme responsible for Hcy metabolism to H2S, a most potent vasodilator, antioxidant and anti-hypertensive agent) contribute to mitochondria dysfunction (mitophagy) and ischemic stroke. Cytochrome-C transports electrons and facilitates mitochondrial bioenergetics. Interestingly, during HHcy, cytochrome-C becomes homocysteinylated (N-Hcy-cyt-c). However, it's consequence to mitophagy and stroke is unclear. The long-term goal of this project is to understand the mechanism of mitophagy, mitochondrial repair and permeability in brain vasculature during I/R injury. Our preliminary studies suggest that during I/R ,total Hcy levels increases, causes N-Hcy-cyt-C , increases mitochondrial matrix metalloproteinase-9 (mtMMP-9), in-part degradation of mt-matrix (connexin and tight junction protein, TJP) which led to mitophagy and permeability in brain vasculature. Interestingly, THC decreases Hcy level and mitigates brain damage. Tetra hydro-curcumin (THC), a major herbal antioxidant and anti-inflammatory agent, has shown to protect brain against I/R injury. The central hypothesis of this proposal is that HHcy contributes to mitophagy mediated brain damage through N-Hcy-cyt-C in part, by increasing oxidative stress, mtMMP-9, degrades connexin-43 and TJP (Figure 1). The treatment with THC, CSE gene and SAHH shRNA gene transfer attenuates mitophagy and permeability. We will test this hypothesis by following three specific aims: Specific Aim #1: To determine whether the Hcy contributes to mitophagy, in part by inducing oxidative stress, exacerbating homocysteinylation of cytochrome-c in ischemia reperfusion and if THC, CSE and SAHH shRNA gene therapy mitigates these changes. Specific Aim #2: To determine whether the homocysteinylation of cytochrome-c activates mt-MMP-9, disruption of collagen/elastin ratio, mtCxn43 and mt-tight junction proteins in ischemia reperfusion and if THC, CSE and SAHH shRNA gene therapy ameliorate. Specific Aim #3: To determine whether Hcy alters mitochondrial (mt) bioenergetics and cerebro-vascular remodeling in ischemia reperfusion and if THC, CSE and SAHH shRNA gene therapy alleviate. These studies will demonstrate the novel mechanism of cerebrovascular remodeling and have therapeutic ramifications for mitochondrial repair in cerebral ischemic stroke.