White Matter Protection in Cerebral Ischemia

说明(申请人提供)：脑白质极易受脑缺血的影响13，脑白质损伤(WMI)是导致中风功能性残疾的主要原因，中风是美国第三大死亡原因，也是导致永久性残疾的主要原因。因此，针对WMI的干预措施可能会显著改善中风后的生活质量。WMI是一个研究相对较少和了解较少的领域。烟酰胺磷酸核糖基转移酶(NAMPT)主要定位于胞浆中，是烟酰胺腺嘌呤二核苷酸(NAD)生物合成的限速酶。NAMPT对脑缺血具有神经保护作用。在非中枢神经系统中，NAMPT存在于细胞外间隙(ENAMPT)，并在血液中系统循环。CNS是否也有eNAMPT以及这个eNAMPT的作用尚不清楚。在我们的前期研究中，我们首先检测到神经细胞将NAMPT分泌到细胞外空间，并且eNAMPT，而不是其细胞内对应物(INAMPT)，在缺血后被选择性地诱导，这意味着细胞外NAMPT可能在缺血中起作用。在神经元特异性转基因小鼠脑缺血后，细胞外间隙和白质中NAMPT水平升高。最重要的是，除了灰质保护外，我们发现NAMPT转基因过表达对缺血后的WMI具有保护作用。这些发现使我们推测细胞外NAMPT可能介导了缺血后的脑白质保护。与此相一致，我们发现重组NAMPT蛋白可保护少突胶质细胞免受氧糖剥夺(OGD)和AMPA毒性所致的缺血样损伤。更重要的是，我们在体内发现外源性给予NAMPT蛋白可以穿越血脑屏障，保护缺血性WMI。进一步研究发现，重组NAMPT蛋白对缺血诱导的少突胶质细胞及其祖细胞的细胞死亡具有保护作用，并能促进少突胶质细胞向成熟少突胶质细胞的分化。这些发现提示NAMPT可能通过抑制少突胶质细胞的死亡和促进OPC的成熟来直接保护WMI。我们发现NAMPT还能增强小胶质细胞的髓鞘吞噬功能。这一点很重要，因为髓鞘受损被认为会抑制OPC的成熟，并在许多病理情况下导致脱髓鞘。因此，NAMPT可能通过调节小胶质细胞的髓鞘吞噬作用间接促进少突胶质细胞的形成。本课题的目的是进一步验证NAMPT的脑白质保护作用及其机制，为脑缺血后脑白质保护提供一种新的、具有临床应用价值的治疗方法。需要检验的中心假设是，NAMPT通过增强少突胶质细胞保护/再生和小胶质细胞介导的髓鞘吞噬功能来预防WMI。通过大脑中动脉闭塞(MCAO)建立小鼠局灶性脑缺血模型，目的1：验证全身应用NAMPT蛋白对局灶性脑缺血所致WMI的保护作用。这项拟议的研究将测试NAMPT在治疗局灶性脑缺血所致的WMI方面的潜在翻译价值。将NAMPT重组蛋白应用于小鼠腹腔注射，定量评价NAMPT对缺血性脑损伤的作用。评估的终点包括梗死体积、WMI和神经学结果。目的：验证NAMPT通过抑制脑缺血后少突胶质细胞死亡和促进少突胶质细胞生成来预防WMI的假说。这项拟议的研究将测试全身应用NAMPT蛋白是否减少局灶性脑缺血后少突胶质细胞的死亡。我们还将测试NAMPT是否刺激缺血后少突胶质细胞的再生。目的：验证NAMPT通过增强小胶质细胞髓鞘吞噬功能来预防WMI的假说。将使用两种方法来提高NAMPT的水平：系统地给药NAMPT蛋白和通过小胶质细胞/巨噬细胞特异性F4/80启动子驱动的AAV9载体靶向NAMPT过表达。将评估NAMPT对小胶质细胞髓鞘吞噬、少突胶质细胞形成、WMI和缺血后功能结果的影响。PHS 398/2590(06/09版)页面续格式页面

DESCRIPTION (provided by applicant): Cerebral white matter is highly vulnerable to cerebral ischemia13 and white matter injury (WMI) is a major cause of functional disability in stroke, the nation's third leading cause of death and the leading cause of permanent disability. Thus, interventions targeted at WMI, an area that remains relatively understudied and poorly understood, may significantly improve post-stroke quality of life. Nicotinamide phosphoribosyltransferase (NAMPT) primarily localizes in the cytosol and functions as the rate-limiting enzyme for the biosynthesis of nicotinamide adenine dinucleotide (NAD)14. NAMPT is neuroprotective against cerebral ischemia15,16. In non-CNS systems, NAMPT can be found in the extracellular space (eNAMPT) and circulates systemically in the blood. Whether the CNS also has eNAMPT and the role of this eNAMPT remains unknown. In our pilot studies, we first detected that neural cells secrete NAMPT into the extracellular space and that eNAMPT, but not its intracellular counterpart (iNAMPT), is selectively induced after ischemia, implying that extracellular NAMPT may play a role in ischemia. Increased NAMPT levels are present in the extracellular space and in white matter after ischemia in neuron-specific transgenic mice. Most importantly, in addition to gray matter protection, we found that NAMPT transgenic overexpression protects against WMI after ischemia. These findings lead us to speculate that extracellular NAMPT may mediate white matter protection after ischemia. In line with this, we found that recombinant NAMPT protein protects oligodendrocytes against ischemia-like injury induced by oxygen glucose deprivation (OGD) and AMPA toxicity. More importantly, we found that exogenously administered NAMPT protein can cross the blood brain barrier and protect against ischemic WMI in vivo. Further studies found that recombinant NAMPT protein protects against cell death of oligodendrocytes and its progenitor cells (OPCs) induced by ischemia, and enhances the differentiation of OPCs into mature oligodendrocytes. These findings suggest that NAMPT may directly protect against WMI via inhibiting oligodendrocyte death and enhancing the maturation of OPCs. We found that NAMPT also enhances microglial myelin phagocytosis. This is important because damaged myelin is believed to suppress the maturation of OPCs and cause demyelination in many pathological conditions. Thus NAMPT may indirectly enhance oligodendrogenesis via modulating microglial myelin phagocytosis. The objective of this project is to further test the white matter protective effect of NAMPT and its underlying mechanisms, and to develop a novel and clinically relevant therapeutic approach for white matter protection after cerebral ischemia. The central hypothesis to be tested is that NAMPT protects against WMI via enhancing oligodendrocytic protection/ regeneration and microglia-mediated myelin phagocytosis. Focal ischemia will be induced in mice by middle cerebral artery occlusion (MCAO) The following Specific Aims are proposed: Aim 1: Test the hypothesis that systemic administration of NAMPT protein protects against WMI induced by focal ischemia. The proposed study will test the potential translational value of NAMPT in treating WMI induced by focal cerebral ischemia. NAMPT recombinant protein will be administrated intraperitoneally to mice and the effect of NAMPT on ischemic brain injury will be quantitatively evaluated. The endpoints for assessment include infarct volume, WMI, and neurological outcomes. Aim 2: Test the hypothesis that NAMPT protects against WMI by inhibiting oligodendrocytic death and enhancing oligodendrogenesis after cerebral ischemia. The proposed study will test whether systemic administration of NAMPT protein reduces oligodendrocyte death after focal ischemia. We will also test whether NAMPT stimulates the regeneration of oligodendrocytes after ischemia. Aim 3: Test the hypothesis that NAMPT protects against WMI by enhancing microglial myelin phagocytosis. Two approaches will be used to elevate NAMPT levels: systemic administration of NAMPT protein and microglia-targeted NAMPT overexpression via an AAV9 vector driven by microglia/macrophage specific F4/80 promoter. The effect of NAMPT on microglial myelin phagocytosis, oligodendrogenesis, WMI, and functional outcomes after ischemia will be evaluated. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page