Roles of NAMPT and NAD+ in hypoxic conditioning-induced neurovascular protection in subarachnoid hemorrhage

项目摘要/摘要 动脉瘤性蛛网膜下腔出血(SAH)是一种发病率很高的疾病，很大程度上是由于继发性脑出血。 早期脑损伤(EBI)和迟发性脑缺血(DCI)造成的损伤。EBI发生在发病后1-3天， 以血脑屏障破裂、神经炎症和神经细胞死亡为特征。DCI发生4-12个月 发病后数天，大动脉血管痉挛和微循环障碍共同作用的结果。vt.给出 EBI和DCI是由广泛的神经血管缺陷引起的，我们相信有效的SAH治疗将 需要多种保护作用才能最大限度地提高疗效。因此，我们应用了一个强大的 多效性效应已知的保护策略--条件性治疗--对动物实验模型的保护 萨哈。条件反射是一个概念，大脑固有的对损伤的抵抗力可以通过暴露来增强 对无害的压力刺激。先前，我们发现在SAH(低氧)之前就开始了低氧适应。 预适应)以eNOS依赖的方式提供针对DCI的强大保护。最近，我们延长了 在这些结果的基础上有三个重要的方面：1)我们发现SAH后3h开始出现低氧条件反射 (低氧后处理；HPostC)也产生强大的神经血管保护；2)我们证明了 依赖NAD的脱乙酰酶Sirtuin1(SIRT1)是这种保护的关键媒介；3)我们发现 初步认为，烟酰胺磷酸核糖基转移酶(NAMPT)可能是一个关键的上游分子，推动 HPostC提供的神经血管保护。NAMPT是NAD挽救途径中的限速酶 它将烟酰胺(NAM)转化为烟酰胺单核苷酸(NMN)，从而能够生物合成NAD，这 是SIRT1激活的重要辅助因子。 在目前的拨款中，我们将检验我们的中心假设，即NAMPT驱动的NAD生产起到了因果关系 HPostC在SAH的神经血管保护中的作用，这种保护是部分或 完全由SIRT1介导。具体目标是(1)检验NAMPT对于 HPostC在SAH中提供的EBI和DCI保护；(2)检验治疗策略设计的假设 要增强NAMPT活动或提高NAD水平，请模仿HPostC在 SAH；如果是，确定这种保护是部分还是完全由SIRT1介导的；以及(3)确定 以NAMPT和NAD为靶点的治疗策略的翻译潜力 蛛网膜下腔出血后的术语认知障碍。使用的方法包括：(A)两种互补的SAH小鼠模型；(B) 评估NAMPT、NAD和SIRT1水平；(C)评估神经炎症、神经细胞死亡、 血管痉挛、微循环障碍以及短期和长期神经行为缺陷；(D)药理学和 NAMPT和SIRT1的遗传抑制；以及(E)NAMPT或NAD的药理和遗传增强。 总体而言，目前拨款中提议的工作有可能确定一种全新的治疗方法 脑动脉瘤破裂患者的治疗-NAMPT激活或NAD增强。如果成功， 这些研究将有助于更好地理解HPostC的广度、机制和可持续性。 SAH诱导的神经血管保护作用及NAMPT和NAD的可译性 治疗学。

Project Summary/Abstract Aneurysmal subarachnoid hemorrhage (SAH) is a highly morbid condition, in large part due to secondary brain injury from Early Brain Injury (EBI) and Delayed Cerebral Ischemia (DCI). EBI occurs 1-3 days after ictus and is characterized by blood brain barrier breakdown, neuroinflammation, and neuronal cell death. DCI occurs 4-12 days after ictus and results from a combination of large artery vasospasm and microcirculatory deficits. Given that EBI and DCI are caused by wide-ranging neurovascular deficits, we believe that effective SAH therapy will require a multiplicity of protective effects to maximize the chance of efficacy. We therefore applied a powerful protection strategy with known pleiotropic effects – Conditioning-based therapy – to experimental models of SAH. Conditioning is a concept whereby the brain's inherent resistance to injury can be enhanced by exposure to non-harmful stress stimuli. Previously, we showed that hypoxic conditioning initiated before SAH (Hypoxic Preconditioning) provides robust protection against DCI in an eNOS-dependent manner. Recently, we extended upon these results in three important ways: 1) We showed that hypoxic conditioning initiated 3h after SAH (Hypoxic Post-Conditioning; HPostC) also produces robust neurovascular protection; 2) We showed that the NAD+-dependent deacetylase, Sirtuin 1 (SIRT1), is a key mediator of this protection; and 3) We showed preliminarily that Nicotinamide phosphoribosyltransferase (NAMPT) is likely a key upstream molecule driving the neurovascular protection afforded by HPostC. NAMPT is the rate-limiting enzyme in the NAD+ salvage pathway that converts nicotinamide (NAM) to nicotinamide mononucleotide (NMN) enabling biosynthesis of NAD+, which is an essential co-factor of SIRT1 leading to its activation. In the present grant, we will test our central hypothesis is that NAMPT-driven NAD+ production plays a causal role in the neurovascular protection afforded by HPostC in SAH, and that this protection is either partially or completely SIRT1-mediated. The Specific Aims are (1) Test the hypothesis that NAMPT is necessary for the EBI and DCI protection afforded by HPostC in SAH; (2) Test the hypothesis that therapeutic strategies designed to augment NAMPT activity or increase NAD+ levels mimic the EBI and DCI protection afforded by HPostC in SAH; and if so, determine if this protection is partially or completely SIRT1-mediated; and (3) Determine the translational potential of therapeutic strategies targeting NAMPT and NAD+ by assessing their impact on long- term cognitive deficits after SAH. Methods used include: (a) Two complementary mouse models of SAH; (b) Assessment of NAMPT, NAD+, and SIRT1 levels; (c) Assessment of neuroinflammation, neuronal cell death, vasospasm, microcirculatory deficits, and short- and long-term neurobehavioral deficits; (d) Pharmacologic and genetic inhibition of NAMPT and SIRT1; and (e) Pharmacologic and genetic augmentation of NAMPT or NAD+. Overall, the work proposed in the present grant has the potential to identify an entirely new therapies for the treatment of patients with ruptured brain aneurysms – NAMPT activation or NAD+ augmentation. If successful, these studies will result in an improved understanding of the breadth, mechanism, and sustainability of HPostC- induced neurovascular protection in SAH and determine the translatability of NAMPT- and NAD+-directed therapeutics.