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米亚(HHcy)和中风之间的联系。HHcy的产生是由于S-腺苷-同型半胱氨酸水解酶增加蛋氨酸的去甲基化，而甲基四氢叶酸还原酶和胱硫醚-y-裂解酶(CSE)的减少导致线粒体功能障碍(有丝分裂)和缺血性卒中。CSE是一种负责Hcy代谢成H_2S的酶，是最有效的血管扩张剂、抗氧化剂和抗高血压药。细胞色素C运输电子，促进线粒体生物能量学。有趣的是，在HHcy过程中，细胞色素-C变成同型半胱氨酸化(N-Hcy-Cyt-c)。然而，它对有丝分裂和中风的后果尚不清楚。该项目的长期目标是了解I/R损伤时脑血管系统中有丝分裂、线粒体修复和通透性的机制。我们的初步研究表明，在I/R期间，总Hcy水平升高，产生N-Hcy-Cyt-C，增加线粒体基质金属蛋白酶-9(MtMMP9)，部分降解mt-基质(连接蛋白和紧密连接蛋白，TJP)，从而导致脑血管的有丝分裂和通透性。有趣的是，THC可降低同型半胱氨酸水平，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。这一建议的中心假设是，HHcy通过N-Hcy-Cyt-C部分地通过增加氧化应激，mtMMP9，降解连接蛋白-43和TJP，促进有丝分裂介导的脑损伤(图1)。THC、CSE基因和SAHH shRNA基因转移处理可降低有丝分裂吞噬和通透性。我们将通过以下三个特定目标来验证这一假说：特定目标1：确定Hcy是否有助于有丝分裂，部分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基因治疗是否缓解了这些变化。特异性目的#2：探讨细胞色素-c同型半胱氨酸化是否在缺血再灌注时激活mt-MMP9、胶原/弹性蛋白比值、mtCxn43和mt紧密连接蛋白以及THC、CSE和SAHH shRNA基因治疗是否有改善作用。具体目的#3：确定同型半胱氨酸是否改变缺血再灌注时线粒体生物能量学和脑血管重构，以及THC、CSE和SAHH shRNA基因治疗是否减轻。这些研究将揭示脑血管重塑的新机制，并对脑缺血卒中的线粒体修复具有治疗意义。 公共卫生相关性：这些研究将描述脑血管系统中有丝分裂和脑血管重塑的机制。膳食中草药抗氧化剂四氢姜黄素、裸CSE DNA和SAHH shRNA基因治疗通过抑制线粒体DNA甲基化介导线粒体膜电位的稳定；氧化应激和蛋白水解性应激通过新的机制途径将成为预防缺血性卒中的有效治疗策略。

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.