Nitrosylation Mechanisms for Protection Against Neurovascular Inflammatory Injury

描述(申请人提供)：脑创伤导致内皮和脑实质的炎症，统称为神经血管单位。虽然创伤性脑损伤(TBI)后神经元会因坏死而迅速死亡，但内皮细胞炎症的恶性循环会加剧损伤。在激活的内皮细胞中，过量的超氧化物与一氧化氮(NO)反应生成过氧亚硝酸根。脑外伤后高水平的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障碍。它激活AMP激酶(AMPK)，进而上调内皮型一氧化氮合酶(ENOS)的超氧化物生成活性，从而维持神经炎性继发性损伤的恶性循环。亚硝化试剂S-亚硝基谷胱甘肽(GSNO)能降低过氧亚硝酸根含量，抑制AMPK活性。它还可以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治疗是否能通过亚硝酸化改善脑损伤后神经血管单位的神经炎性损伤。我们推测GSNO可阻断脑损伤后AMPK/eNOS/过氧亚硝酸盐的恶性循环，从而减轻神经血管损伤，帮助功能恢复。在具体目标1中，将使用药物(过氧亚硝酸盐清除剂GSNO和AMPK选择性抑制剂)作为治疗工具，在受控皮质撞击小鼠模型中剖析AMPK的调节和对脑损伤的改善。在特定目标2中，将使用AMPKα1/2基因敲除小鼠、AMPKα过表达转基因小鼠和野生型小鼠进一步检验和验证所阐明的机制。在具体目标3中，我们将确定GSNO介导的AMPK或其上游激酶LKB1的亚硝化是否与抑制eNOS异常活性和减少过氧亚硝酸盐的形成有关。互补的药理学和遗传学方法将决定AMPK在脑损伤中的作用。与传统的NO供体不同，GSNO是一种无毒的内源性NO调节剂和亚硝化试剂。了解S-亚硝化机制和独特的AMPK/eNOS/过氧亚硝酸盐通路可能为神经炎性脑损伤的治疗提供新的策略，不仅针对其神经元后果，而且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.