Neurorestorative Therapy for Stroke Injury

衰老是人类中风的主要不可改变的危险因素，缺血性中风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 与中风的老年动物模型一样，老年中风患者的功能恢复程度慢于 在年轻的时候。在老年脑中，兴奋性毒性诱导的持续神经元过氧亚硝酸盐产生更多 突出，导致持续的钙蛋白酶激活，从而导致神经变性。我们观察到， 过氧化亚硝酸根的有害活性可以被S-亚硝基谷胱甘肽（GSNO）阻断，S-亚硝基谷胱甘肽是NO 和谷胱甘肽。GSNO抑制小鼠神经元型一氧化氮合酶（nNOS）诱导的过氧亚硝基阴离子形成 中风模型因此，使用老年小鼠模型，我们建议研究 过氧亚硝酸盐与GSNO在卒中功能恢复中的比较 使用动物源性细胞培养模型的机制性中风研究尚未在人类中得到验证， 阻碍了基于机制的神经保护性人类中风治疗的发展。所以我们 建议使用诱导多能干细胞（iPSC）衍生的人类神经元来研究 过氧亚硝酸盐与GSNO介导的机制在中风条件下兴奋性毒性诱导的毒性。 对nNOS/calpain系统调节的研究表明， nNOS/钙蛋白酶的特异性抑制剂不仅限制了细胞损伤，而且限制了重要的生理功能。在 相反，所提出的GSNO介导的nNOS和钙蛋白酶的可逆抑制提供了一种新的方法， 我们假设其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/钙蛋白酶系统的有害活性得以维持， 在老年人中比在年轻中风的大脑中更持久。高水平的过氧亚硝酸盐和上调 钙蛋白酶的活性导致严重的神经退行性变，而GSNO通过S- 亚硝基化，可逆地抑制nNOS和钙蛋白酶的活性，以减少过氧亚硝基诱导的 神经变性和加速功能恢复。假设将被检验 通过两个具体目标。具体目标1阐明了GSNO通过其抑制 中风条件下iPSC衍生的人神经元中有害的nNOS/过氧亚硝酸盐/钙蛋白酶系统 文化模式具体目标2研究GSNO是否通过以下方式帮助和加速功能恢复： 阻断老年小鼠中风模型中有害的nNOS/过氧亚硝酸盐/钙蛋白酶系统。 预期的结果包括阐明GSNO介导的抑制细胞凋亡的机制。 nNOS/过氧亚硝基阴离子/钙蛋白酶系统与老年动物功能恢复iPSC衍生的人类神经元将 提供了一个新的工具来了解人类中风损伤的机制，导致发展一个 对中风幸存者（包括老年人和退伍军人）具有转化价值的治疗。GSNO是一个 组成部分的人类大脑和身体，其疗效已被记录在几个动物模型， 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.