The role of nicotinamide mononucleotide dependent mitochondrial reactive oxygen species generation in acute brain injury

线粒体功能受损经常与缺血性脑损伤有关 与心脏骤停或中风有关。然而，线粒体功能障碍的程度 神经退行性变的原因尚不清楚，导致线粒体功能衰竭的机制也不清楚。 明白了。最近，有人提出线粒体分裂/融合动力学的不平衡可能导致 神经退化和脑损伤。此外，烟酰胺腺嘌呤二核苷酸的过度激活 (NAD)降解多聚ADP核糖聚合酶(PARP1)导致细胞和线粒体NAD过多 耗尽导致细胞存活受损。我们假设烟酰胺单核苷酸(NMN) 给药通过(A)逆转过量的线粒体来抑制缺血后的神经变性 通过刺激线粒体NAD合成而发生的裂变：(B)刺激线粒体脱乙酰化 蛋白质并导致(C)线粒体超氧化物生成减少。 我们的初步数据显示，NAD前体NMN对动物的治疗具有戏剧性 神经保护作用，逆转线粒体过度碎裂，增加大脑 线粒体NAD水平。作为下游结果，NMN正在减少线粒体蛋白质的乙酰化和 抑制线粒体活性氧(ROS)的产生。这项研究的主要目标是 确定NMN诱导的线粒体NAD代谢、蛋白质变化之间的机制联系(S) 乙酰化、ROS的产生和抑制分裂。为解决这些问题，我们建议： 1.确定sirtuin 3(SIRT3)在线粒体活性氧物种(ROS)中的具体作用 生产，核苷酸代谢，线粒体生物能量功能，和动力学。单元格将是 从我们的三个转基因动物模型制备：(1)表达线粒体的动物靶向增强 单独的黄色荧光蛋白(mito-EYFP)，(2)表达mito-EYFP和过表达SIRT3的动物 (3)表达SIRT3基因敲除动物(mito-EYFP-SIRT3KO)。这个 NMN诱导的线粒体蛋白乙酰化改变在线粒体ROS产生中的作用 线粒体碎裂和细胞死亡将被确定。细胞NAD代谢，线粒体 将分析呼吸功能、线粒体融合和裂变以及它们在NMN中的作用 神经保护和缺氧性葡萄糖剥夺导致的细胞死亡将被确定。 2.研究NMN治疗对线粒体缺血后调制的特异性影响 大脑的动力学，我们将使用我们的转基因动物，这些动物将受到短暂性前脑缺血的影响 并将检查缺血后神经元线粒体形态计量学的变化。此外，NMN- NAD代谢、线粒体蛋白乙酰化和线粒体ROS生成的变化 将会被确定。此外，NMN还引起NAD代谢、线粒体蛋白的变化 乙酰化和线粒体ROS的产生将被确定。最后，我们将评估NMN的效果 对缺血后细胞和线粒体NAD代谢及线粒体呼吸的治疗。 这项工作的意义在于，它同时提出了机械式和转换式的方法 解开NAD依赖线粒体ROS产生、线粒体损伤的机制 并确定其在急性脑损伤中的作用。此外，一个新的代谢链的鉴定 NAD分解代谢、线粒体蛋白质乙酰化/去乙酰化、线粒体ROS生成 抑制线粒体分裂将确定新的神经保护机制，这可能导致 将NMN作为治疗化合物用于治疗急性脑损伤，如全脑缺血、中风和脑外伤或 慢性神经退行性疾病，因此对退伍军人的健康有潜在的重大影响。

Impairments in mitochondrial functions have been frequently implicated in ischemic brain injury associated with cardiac arrest or stroke. However, the extent to which mitochondrial dysfunction contributes to neurodegeneration is unknown; and the mechanisms leading to mitochondrial failure are not well understood. Recently, it was suggested that an imbalance in mitochondrial fission/fusion dynamics can lead to neurodegeneration and brain damage. Furthermore, overactivation of nicotinamide adenine dinucleotide (NAD)+ degrading poly-ADP-ribose polymerase (PARP1) causes excessive cellular and mitochondrial NAD+ depletion resulting in impaired cell survival. We hypothesize that the nicotinamide mononucleotide (NMN) administration is inhibiting the post-ischemic neurodegeneration by (a) reversing excessive mitochondrial fission via stimulation of mitochondrial NAD+ synthesis that (b) stimulates deacetylation of mitochondrial proteins and leads to (c) reduction of mitochondrial superoxide production. Our preliminary data show that treatment of animals with NAD+ precursor NMN has dramatic neuroprotection effect, reverses the excessive mitochondrial fragmentation and increases the brain mitochondria NAD+ levels. As a downstream result NMN is decreasing mitochondrial proteins acetylation and inhibits mitochondrial reactive oxygen species (ROS) production. The primary goal of this study is to determine the mechanistic link(s) between NMN induced changes in mitochondrial NAD+ metabolism, protein acetylation, ROS generation and inhibition of fission. To address these questions, we propose to: 1. Determine the specific role of sirtuin 3 (SIRT3) in mitochondrial reactive oxygen species (ROS) production, nucleotide metabolism, mitochondrial bioenergetic functions, and dynamics. Cells will be prepared from our three transgenic animal models: (1) animals expressing mitochondria targeted enhanced yellow fluorescence protein (mito-eYFP) alone, (2) animals expressing mito-eYFP and overexpressing SIRT3 (mito-eYFP-SIRT3OE), or (3) mito-eYFP expressing SIRT3 knockout animals (mito-eYFP-SIRT3KO). The role of NMN-induced changes in mitochondrial protein acetylation on mitochondria ROS production, mitochondrial fragmentation and cell death will be determined. Cellular NAD+ metabolism, mitochondrial respiratory function, and mitochondrial fusion and fission will be analyzed and their role in NMN neuroprotection and oxygen glucose deprivation induced cell death will be determined. 2. To study the specific effect of NMN treatment on post-ischemic modulation of mitochondrial dynamics in brain, we will use our transgenic animals that will be subjected to transient forebrain ischemia and the post-ischemic alterations in neuronal mitochondrial morphometry will be examined. In addition, NMN- induced changes in NAD+ metabolism, mitochondrial protein acetylation and mitochondrial ROS generation will be determined. Additionally, NMN-induced changes in NAD+ metabolism, mitochondrial protein acetylation and mitochondrial ROS generation will be determined. Finally, we will assess the effect of NMN treatment on post-ischemic cellular and mitochondrial NAD+ metabolism and mitochondrial respiration. The significance of this work is that it proposes both mechanistic and translational approaches to unravel the mechanisms of NAD+ dependent mitochondrial ROS production, impairment in mitochondrial dynamics and determine its role in acute brain injury. Furthermore, the identification of a novel metabolic link between NAD+ catabolism, acetylation/deacetylation of mitochondrial proteins, mitochondrial ROS generation and inhibition of mitochondrial fission will identify new mechanisms for neuroprotection that could lead to the use of NMN as a therapeutic compound for acute brain injury such as global ischemia, stroke and TBI or chronic neurodegenerative disease, thus potentially have significant impact on the health of Veterans.