Neurorestorative Therapy for Stroke Injury

衰老是人类主要的不可改变的卒中危险因素，而缺血性卒中在老年人中非常普遍 退伍军人和其他美国人，经济负担超过700亿美元/年。此外，利率和 老年中风患者的功能恢复程度，就像老年中风动物模型一样，慢于 在年轻人身上。在老年脑中，兴奋性毒性诱导的持续性神经元过氧亚硝酸盐的产生更多 突出，导致持续的钙蛋白酶激活，从而导致神经退化。我们观察到这一点 NO的反应产物S亚硝基谷胱甘肽可阻断过氧亚硝酸根的毒害作用 和谷胱甘肽。GSNO抑制小鼠神经元型一氧化氮合酶(NNOS)源性过氧亚硝酸盐的形成 中风的模型。因此，使用衰老的小鼠模型，我们建议研究 过氧亚硝酸盐与GSNO在卒中后功能恢复中的比较。 使用动物来源的细胞培养模型进行的机械性中风研究尚未在人类身上得到验证， 阻碍了以机制为基础的人类中风神经保护性疗法的发展。因此，我们 建议使用诱导多能干细胞(IPSC)来源的人神经元来研究 卒中条件下兴奋性毒性诱导毒性中过氧亚硝酸盐与GSNO介导的机制。 对nNOS/calain系统调节的研究表明，使用 NNOS/calain的特异性抑制剂不仅限制了细胞损伤，还限制了重要的生理功能。在……里面 相反，GSNO介导的对nNOS和calain的可逆抑制提供了一种新的途径 我们假设它将通过维持生理功能和抑制病理活动而有效。 我们假设nNOS/过氧亚硝酸盐/钙蛋白系统的有害活动保持不变。 在老年人中比年轻的中风脑更顽强。高水平的过氧亚硝酸盐和上调 钙蛋白酶的活性可引起严重的神经变性，而GSNO则通过S的作用机制 亚硝酸化可逆地抑制nNOS和钙蛋白酶的活性以减少过氧亚硝酸盐诱导的 促进老年卒中小鼠神经退变和功能恢复。这一假设将得到检验。 通过两个具体目标。具体目标1阐明了GSNO通过其抑制 卒中条件下IPSC来源的人神经细胞中有害的nNOS/过氧亚硝酸盐/钙蛋白系统 文化模式。特定目的2研究GSNO是否通过以下方式帮助和加速功能恢复 阻断老年小鼠卒中模型中有害的nNOS/过氧亚硝酸盐/钙蛋白系统。 预期结果包括阐明GSNO介导的抑制 NNOS/过氧亚硝酸盐/钙蛋白系统与老年动物功能恢复IPSC来源的人类神经元将 为了解人类中风损伤的机制提供了新的工具，导致了 治疗中风幸存者的翻译价值，包括老年人和退伍军人。GSNO是一个 是人类大脑和身体的组成部分，其疗效已在几个动物模型中得到证实。 神经血管、心血管和血管疾病。

Aging is the major non-modifiable stroke risk factor in humans, and ischemic stroke is highly prevalent in aged Veterans and other Americans, with an economic burden exceeding $70 billion/year. Furthermore, the rate and the degree of functional recovery in aged stroke patients, as in aged animal models of stroke, are slower than in the young. In the aged brain, excitotoxicity-induced sustained neuronal peroxynitrite production is more prominent, causing sustained calpain activation and thus neurodegeneration. We observed that this deleterious activity of peroxynitrite can be blocked by S-nitrosoglutathione (GSNO), a reaction product of NO and glutathione. GSNO inhibits neuronal nitric oxide synthase (nNOS)-derived peroxynitrite formation in mouse models of stroke. Therefore, using aged mouse models, we propose to investigate the critical roles of peroxynitrite versus GSNO in functional recovery from stroke. Mechanistic stroke studies using animal-derived cell culture models have not been validated in humans, frustrating the development of mechanism-based neuroprotective human stroke therapy. Therefore, we propose using induced pluripotent stem cell (iPSC)-derived human neurons to investigate the effect of peroxynitrite versus GSNO-mediated mechanisms in excitotoxicity-induced toxicity under stroke conditions. Studies on the regulation of nNOS/calpain system indicate that a complete irreversible inhibition using specific inhibitors of nNOS/calpain limits not only the cellular insult but also vital physiologic functions. In contrast, the proposed GSNO-mediated reversible inhibition of nNOS and calpain offers a novel approach which we hypothesize will be effective by maintaining physiologic function and inhibiting pathological activities. We hypothesize that deleterious activities of the nNOS/peroxynitrite/calpain system are maintained more persistently in the aged than the young stroke brain. High levels of peroxynitrite and up regulated activity of calpains cause profound neurodegeneration, whereas, GSNO, via the mechanism of S- nitrosylation, reversibly inhibits the activities of nNOS and calpain to reduce peroxynitrite-induced neurodegeneration and accelerate functional recovery in aged stroke mice. The hypothesis will be tested via two specific aims. Specific aim 1 elucidates the regulatory mechanisms through which GSNO inhibits the deleterious nNOS/peroxynitrite/calpain system under stroke conditions in iPSC-derived human neuronal culture model. Specific Aim 2 investigates whether GSNO aids and accelerates functional recovery by blocking the deleterious nNOS/peroxynitrite/calpain system in aged mouse stroke models. Expected outcomes include elucidation of GSNO-mediated mechanisms for inhibition of the nNOS/peroxynitrite/calpain system and functional recovery in aged animals. iPSC-derived human neurons will provide a new tool to understand the mechanisms of human stroke injury, leading to the development of a therapy of translational value for stroke survivors including senior citizens and Veterans. GSNO is a component of the human brain and body, and its efficacy has been documented in several animal models of neurovascular, cardiovascular and vascular diseases.