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.

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 与神经退行性疾病的关系尚不清楚；导致线粒体衰竭的机制尚不清楚 明白了。最近，有人提出线粒体裂变/融合动力学的不平衡可能导致 神经退行性变和脑损伤。此外，烟酰胺腺嘌呤二核苷酸的过度激活 (NAD)+ 降解聚 ADP-核糖聚合酶 (PARP1) 导致细胞和线粒体 NAD+ 过多 耗竭导致细胞存活受损。我们假设烟酰胺单核苷酸（NMN） 给药通过以下方式抑制缺血后神经变性：(a) 逆转过多的线粒体 通过刺激线粒体 NAD+ 合成进行裂变，(b) 刺激线粒体脱乙酰化 蛋白质并导致 (c) 线粒体超氧化物产生减少。 我们的初步数据表明，用 NAD+ 前体 NMN 治疗动物具有显着效果。 神经保护作用，逆转线粒体过度破碎，增加大脑 线粒体 NAD+ 水平。 NMN 的下游结果是减少线粒体蛋白乙酰化， 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 乙酰化、ROS 产生和裂变抑制。为了解决这些问题，我们建议： 1.确定sirtuin 3（SIRT3）在线粒体活性氧（ROS）中的具体作用 生产、核苷酸代谢、线粒体生物能功能和动力学。细胞将是 从我们的三种转基因动物模型中制备：（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 SIRT3 的动物 （mito-eYFP-SIRT3OE），或（3）mito-eYFP表达SIRT3敲除动物（mito-eYFP-SIRT3KO）。这 NMN 诱导的线粒体蛋白乙酰化变化对线粒体 ROS 产生的作用， 将确定线粒体碎片和细胞死亡。细胞 NAD+ 代谢，线粒体 将分析呼吸功能、线粒体融合和裂变及其在 NMN 中的作用 将确定神经保护和缺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具体作用 在大脑动力学方面，我们将使用将遭受短暂前脑缺血的转基因动物 并将检查神经元线粒体形态测量的缺血后变化。此外，NMN- 诱导 NAD+ 代谢、线粒体蛋白乙酰化和线粒体 ROS 生成的变化 将被确定。此外，NMN 诱导 NAD+ 代谢、线粒体蛋白的变化 将确定乙酰化和线粒体 ROS 生成。最后我们来评估一下NMN的效果 对缺血后细胞和线粒体 NAD+ 代谢和线粒体呼吸的治疗。 这项工作的意义在于它提出了机械和转化方法 揭示 NAD+ 依赖的线粒体 ROS 产生、线粒体损伤的机制 动力学并确定其在急性脑损伤中的作用。此外，新的代谢联系的鉴定 NAD+ 分解代谢、线粒体蛋白乙酰化/脱乙酰化、线粒体 ROS 生成之间 线粒体裂变的抑制将确定神经保护的新机制，这可能导致 使用 NMN 作为治疗急性脑损伤（如全身缺血、中风和 TBI）的化合物，或 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。