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

烟酰胺单核苷酸依赖性线粒体活性氧生成在急性脑损伤中的作用

• 批准号: 9889770
• 负责人: TIBOR KRISTIAN
• 金额: --
• 依托单位: BALTIMORE VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9889770
• 关键词: Acetylation; Acute; Acute Brain Injuries; Address; Affect; Age; Animal Model; Animals; Astrocytes; Bioenergetics; Brain; Brain Injuries; Brain region; Catabolism; Cause of Death; Cell Death; Cell Survival; Cells; Chronic; Clinical; Clinical Trials; Complex; Consumption; Data; Deacetylase; Deacetylation; Death Rate; Disease; Dose; Drug Metabolic Detoxication; Enzymes; Failure; Female; Fluorescence; Generations; Glucose; Glutamate-ammonia-ligase adenylyltransferase; Goals; Health; Heart Arrest; High Prevalence; Histologic; Impairment; Injury; Ischemia; Ischemic Brain Injury; Lead; Link; Long-Term Care; Metabolic; Metabolism; Mitochondria; Mitochondrial Proteins; Modeling; Morphology; Myocardial Infarction; Nerve Degeneration; Neurodegenerative Disorders; Neurologic; Neurological outcome; Neurons; Nicotinamide Mononucleotide; Nicotinamide adenine dinucleotide; Oxygen; Pathologic; Pathway interactions; Pilot Projects; Play; Poly(ADP-ribose) Polymerases; Polymerase; Population; Process; Production; Prosencephalon; Protein Acetylation; Proteins; Reactive Oxygen Species; Regulation; Research; Respiration; Respiratory physiology; Ribose; Risk Factors; Role; SOD2 gene; Sirtuins; Stroke; Superoxides; TBI treatment; Testing; Therapeutic; Transgenic Animals; Transgenic Mice; Traumatic Brain Injury; Veterans; Work; aging population; brain cell; cell type; deprivation; disability; experimental study; improved; in vivo; insight; knockout animal; knockout gene; male; mitochondrial dysfunction; morphometry; mouse model; neuroprotection; novel; novel therapeutic intervention; nucleotide metabolism; overexpression; pre-clinical research; preservation; stroke outcome; stroke risk; stroke victims; therapy development; translational approach

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.

缺血性脑损伤常常涉及线粒体功能的损伤 与心脏骤停或中风有关然而，线粒体功能障碍在多大程度上有助于 神经退行性变的机制尚不清楚;导致线粒体功能衰竭的机制也不清楚 明白最近，有人提出，线粒体裂变/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活化 (NAD)+降解聚ADP-核糖聚合酶（PARP 1）导致过量的细胞和线粒体NAD+ 耗尽导致受损的细胞存活。我们假设烟酰胺单甘肽（NMN） 给药通过（a）逆转过度的线粒体膜电位， 通过刺激线粒体NAD+合成的裂变，（B）刺激线粒体NAD+的脱乙酰化 蛋白质，并导致（c）减少线粒体超氧化物的产生。 我们的初步数据显示，用NAD+前体NMN治疗动物具有显著的生物学效应。 神经保护作用，逆转过度的线粒体碎片化，增加大脑 线粒体NAD+水平。作为下游结果，NMN降低线粒体蛋白乙酰化， 抑制线粒体活性氧（ROS）的产生。本研究的主要目的是 确定NMN诱导的线粒体NAD+代谢、蛋白质 乙酰化、ROS生成和裂变抑制。为解决这些问题，我们建议： 1.确定sirtuin 3（SIRT 3）在线粒体活性氧（ROS）中的特定作用 生产，核苷酸代谢，线粒体生物能量功能和动力学。细胞将被 从我们的三种转基因动物模型制备：（1）表达线粒体靶向增强的动物 单独的黄色荧光蛋白（mito-eYFP），（2）表达mito-eYFP并过表达SIRT 3的动物 （3）表达线粒体-eYFP的SIRT 3敲除动物（线粒体-eYFP-SIRT 3 KO）。的 NMN诱导的线粒体蛋白乙酰化变化对线粒体ROS产生的作用， 将测定线粒体断裂和细胞死亡。细胞NAD+代谢，线粒体 呼吸功能，线粒体融合和分裂将进行分析，并在NMN中的作用 将测定神经保护和氧葡萄糖剥夺诱导的细胞死亡。 2.研究NMN治疗对线粒体缺血后调节的特异性作用， 我们将使用我们的转基因动物， 并检查神经元线粒体形态计量学的缺血后改变。此外，NMN- 引起NAD+代谢、线粒体蛋白乙酰化和线粒体ROS产生的变化 将被确定。此外，NMN诱导的NAD+代谢、线粒体蛋白 将测定乙酰化和线粒体ROS产生。最后，我们将评估NMN的效果 治疗对缺血后细胞和线粒体NAD+代谢和线粒体呼吸的影响。 这项工作的意义在于，它提出了机械和翻译的方法， 阐明NAD+依赖的线粒体ROS产生的机制， 动力学，并确定其在急性脑损伤中的作用。此外，一种新的代谢环节的鉴定 NAD+催化剂、线粒体蛋白的乙酰化/脱乙酰化、线粒体ROS生成之间 抑制线粒体分裂将确定新的神经保护机制， NMN作为急性脑损伤如全脑缺血、中风和TBI的治疗化合物的用途， 慢性神经退行性疾病，因此可能对退伍军人的健康产生重大影响。