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）和中风之间存在关联。HHcy的产生是由于s -腺苷-同型半胱氨酸水解酶（SAHH）对蛋氨酸的去甲基化增加，甲基四氢叶酸还原酶（MTHFR）和胱硫氨酸-y-裂解酶（CSE，一种负责Hcy代谢为H2S的酶，H2S是最有效的血管舒张剂、抗氧化剂和降压剂）导致线粒体功能障碍（线粒体自噬）和缺血性中风。细胞色素c传递电子并促进线粒体生物能量。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）。然而，它对线粒体自噬和中风的影响尚不清楚。本项目的长期目标是了解I/R损伤过程中线粒体自噬、线粒体修复和脑血管通透性的机制。我们的初步研究表明，在I/R期间，总Hcy水平升高，引起N-Hcy-cyt-C，增加线粒体基质金属蛋白酶-9 (mtMMP-9)， mt-基质（连接蛋白和紧密连接蛋白，TJP）的部分降解，导致线粒体自噬和脑血管通透性。有趣的是，四氢大麻酚能降低Hcy水平，减轻脑损伤。四氢姜黄素（THC）是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。该建议的中心假设是，HHcy通过N-Hcy-cyt-C部分地通过增加氧化应激、mtMMP-9降解连接蛋白43和TJP来促进线粒体自噬介导的脑损伤（图1）。四氢大麻酚、CSE基因和SAHH shRNA基因的转移使线粒体自噬和通透性减弱。我们将通过以下三个具体目标来验证这一假设：具体目标#1：确定Hcy是否有助于线粒体自噬，部分原因是通过诱导氧化应激，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。特异性目的#2：确定细胞色素c的同型半胱氨酸化是否激活了缺血再灌注中mt-MMP-9、胶原/弹性蛋白比、mtCxn43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。具体目的#3：确定Hcy是否改变缺血再灌注时线粒体（mt）生物能量学和脑血管重塑，以及THC、CSE和SAHH shRNA基因治疗是否缓解。这些研究将揭示脑血管重塑的新机制，并对缺血性脑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. PUBLIC HEALTH RELEVANCE: These studies will delineate the mechanisms of mitophagy and cerebrovascular remodeling in brain vasculature. The treatment with dietary herbal antioxidant tetrahydrocurcumin, naked CSE DNA and SAHH shRNA gene therapy mediated stabilization of mitochondrial membrane potential by inhibition of mitochondrial DNA methylation; oxidant and proteolysis stress via novel mechanistic pathways will be powerful therapeutic strategy for preventing ischemic stroke.