Nitrosylation Mechanisms for Protection Against Neurovascular Inflammatory Injury

描述（由申请方提供）：脑创伤诱导内皮和脑实质（统称为神经血管单位）的炎症。虽然创伤性脑损伤（TBI）后神经元因坏死而迅速死亡，但内皮细胞中炎症的恶性循环加剧了损伤。在活化的内皮细胞中，过量的超氧化物与一氧化氮（NO）反应形成过氧亚硝酸盐。在高水平TBI后，过氧亚硝酸盐参与血脑屏障（BBB）渗漏、酶功能改变和神经行为损伤。它激活AMP激酶（AMPK），这反过来又可能上调内皮型一氧化氮合酶（eNOS）的超氧化物生成活性，从而维持神经炎症继发性损伤的恶性循环。亚硝基化剂S-亚硝基谷胱甘肽（GSNO）能够降低过氧亚硝酸盐的水平并抑制AMPK的活性。它还恢复谷胱甘肽的水平，保护神经血管单位的完整性。 因此，本研究将探讨GSNO治疗是否通过亚硝基化改善TBI诱导的神经血管单位的神经炎症损伤。我们推测GSNO阻断恶性AMPK/eNOS/过氧亚硝酸盐循环，从而减少神经血管损伤并有助于TBI的功能恢复。 在具体目标1中，药理学试剂（GSNO，过氧亚硝酸盐清除剂和AMPK选择性抑制剂）将用作治疗工具，以在受控皮质撞击小鼠模型中剖析AMPK的调节和TBI的改善。在具体目标2中，将使用AMPK α 1/2敲除小鼠、AMPK α过表达转基因小鼠和野生型小鼠进一步检查和验证阐明的机制。在具体目标3中，我们将确定是否GSNO介导的亚硝基化的AMPK或其上游激酶LKB 1是负责异常eNOS活性的抑制和过氧亚硝酸盐的形成减少。互补的药理学和遗传学方法将确定AMPK在TBI中的作用。 与传统的NO供体不同，GSNO是一种无毒的内源性NO调节剂和亚硝基化剂。了解S-亚硝基化机制和独特的AMPK/eNOS/过氧亚硝酸盐途径可能会导致治疗神经炎性脑创伤的新策略，不仅针对其神经元后果，还针对其血管原因和恶化。

DESCRIPTION (provided by applicant): Brain trauma induces inflammation in both the endothelium and the brain parenchyma, collectively termed the neurovascular unit. While neurons die quickly by necrosis following traumatic brain injury (TBI), a vicious cycle of inflammation in endothelial cells exacerbates the injury. In activated endothelial cells, excessive superoxide reacts with nitric oxide (NO) to form peroxynitrite. At high levels following TBI, peroxynitrite is involved in blood brain barrier (BBB) leakage, altered enzymatic functions, and neurobehavior impairment. It activates AMP Kinase (AMPK), which in turn may up regulate the superoxide-producing activity of endothelial nitric oxide synthase (eNOS), and thus maintains a vicious cycle of neuroinflammatory secondary injury. The nitrosylating agent S-nitrosoglutathione (GSNO) is capable of reducing the levels of peroxynitrite and inhibiting the activity of AMPK. It also restores the levels of glutathione and protects the integrity of the neurovascular unit. Therefore, this study will investigate whether GSNO treatment ameliorates TBI-induced neuroinflammatory damage to the neurovascular unit via nitrosylation. We hypothesize that GSNO blocks the vicious AMPK/eNOS/peroxynitrite cycle, thus reducing the neurovascular injury and aiding functional recovery in TBI. In Specific Aim 1, pharmacological agents (GSNO, a peroxynitrite scavenger, and an AMPK selective inhibitor) will be used as therapeutic tools to dissect the regulation of AMPK and amelioration of TBI in a controlled cortical impact mouse model. In Specific Aim 2, the elucidated mechanisms will be further examined and validated using AMPK alpha 1/2 knockout mice, AMPK alpha over-expressing transgenic mice, and wild type mice. In Specific Aim 3, we will determine whether GSNO-mediated nitrosylation of either AMPK or its upstream kinase LKB1 is responsible for the inhibition of aberrant eNOS activity and the reduced formation of peroxynitrite. The complementary pharmacological and genetic approach will determine the role of AMPK in TBI. GSNO, unlike conventional NO donors, is a non-toxic endogenous NO modulator and nitrosylating agent. Understanding S-nitrosylation mechanism and the unique AMPK/eNOS/peroxynitrite pathway may lead to new strategies for the treatment of neuroinflammatory brain trauma that target not only its neuronal consequences but also its vascular causes and exacerbations.