Nudix hydrolases and mitochondrial dynamics in acute neurodegenerative disease

急性神经退行性疾病中的 Nudix 水解酶和线粒体动力学

• 批准号: 9249385
• 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: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9249385
• 关键词: Acute; Acute Brain Injuries; Address; Adenosine Diphosphate Ribose; Adverse effects; Affect; Age; Animal Model; Animals; Astrocytes; Bioenergetics; Brain; Brain Injuries; Brain region; Catabolism; Cause of Death; Cell Culture Techniques; Cell Death; Cell Survival; Cellular Stress; Chronic; Clinical; Consumption; Data; Disease; Enzymes; Evaluation; Failure; Fluorescence; Generations; Genes; Genotoxic Stress; Glucose; Goals; Grant; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Heart Arrest; High Prevalence; Histologic; Impairment; Injury; Inner mitochondrial membrane; Ischemia; Ischemic Brain Injury; Lead; Link; Long-Term Care; Membrane Fusion; Metabolic; Metabolism; Mitochondria; Morphology; Myocardial Infarction; NAD+ Nucleosidase; Nerve Degeneration; Neurodegenerative Disorders; Neurologic; Neurological outcome; Neurons; Organelles; Outcome; Oxidative Phosphorylation; Oxidative Stress; Oxygen; Pathologic; Play; Poly(ADP-ribose) Polymerases; Population; Process; Production; Prosencephalon; Protein Isoforms; Proteins; RNA Interference; Recovery; Research; Resistance; Respiratory physiology; Risk Factors; Role; Stroke; Study Subject; TBI treatment; Techniques; Testing; Therapeutic; Time; Transgenic Animals; Transgenic Mice; Traumatic Brain Injury; Veterans; Work; adenylate kinase; brain cell; brain tissue; cell type; clinical application; deprivation; disability; enzyme activity; improved; in vivo; inhibitor/antagonist; knock-down; mitochondrial dysfunction; mitochondrial metabolism; morphometry; mouse model; novel; novel therapeutic intervention; nucleotide metabolism; nudix hydrolase; overexpression; public health relevance; therapy development; translational approach

 DESCRIPTION (provided by applicant): 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 in neurons and astrocytes contributes to neurodegeneration is unknown, and the mechanisms leading to mitochondrial failure are elusive. Recently, it was suggested that an imbalance in mitochondrial dynamics could lead to neurodegeneration and brain damage. Furthermore, over-activation of NAD+ degrading poly-ADP-ribose polymerase (PARP1) causes excessive cellular and mitochondrial NAD+ depletion resulting in detrimental effects for cell survival. We hypothesize that the activity of ADP-ribose catabolizing enzyme, NUDIX hydrolase NUDT9, leads to downstream mitochondrial GTP consumption and consequently to inhibition of GTPases that control the organelle's fusion. Since ischemic insult triggers an extensive fission of mitochondria, the ability to refuse these organelles is essential for cell viability. This notio is strongly supported by our data showing irreversible fragmentation of mitochondria in ischemia vulnerable regions, re-fusion of mitochondria in ischemia resistant brain cells, and genotoxic stress induced depletion of mitochondrial NAD+ and GTP pools. Our preliminary data also show that over-expression of mitochondrial NUDT9 isoform is aggravating cell death; the mitochondria are more sensitive to ischemic insult and cell death maturation is accelerated in transgenic animals with increased levels of NUDT9. The primary goal of this study is to determine whether neuronal or astrocytic activity of NUDT9 is a major contributor to cell death mechanisms following ischemia. To address these questions we propose to: 1. Determine the specific role of NUDT9 in mitochondrial nucleotide metabolism, mitochondrial bioenergetics functions, dynamics and cell death. The role of NUDT9 in cell death of astrocytes and neurons will be examined by utilizing pure neuronal and astrocytic cell cultures prepared from brain tissue of our new transgenic animals that conditionally express mito-eYFP and NUDT9 either in neurons or in astrocytes. These cell cultures will be exposed to oxygen-glucose deprivation (OGD) and the effect of NUDT9 on bioenergetics metabolism, mitochondrial respiratory functions, mitochondrial dynamics and cell death will be determined. In addition, cellular and mitochondrial metabolism, mitochondrial respiratory functions, and mitochondrial fusion and fission will be analyzed. Small interference RNA (siRNA)-induced knockdown of the NUDT9 gene will be used to confirm the NUDT9 effect on impairment of mitochondrial dynamics and cell death mechanisms. 2. To study the specific role of NUDT9 in post-insult impairment of mitochondrial dynamics, we will use our transgenic animals that will be subjected to transient forebrain ischemia and at designated recovery periods the alterations in mitochondrial morphometry specifically in neurons or astrocytes in brain will be examined. Finally, we will assesse the effect of NUDT9 over-expression in neurons or astrocytes on the histological and neurological outcome after ischemic insult. Similarly, we will determine the role of NUDT9 enzyme activity in the mechanism of ischemic brain damage by assessing post-ischemic histological and neurological outcome of animals pre-treated with NUDT9 targeted siRNA. This work proposes both mechanistic and translational approaches to unravel the mechanisms of impairment in neuronal and astrocytic mitochondrial dynamics and determine its role in acute brain injury. Furthermore, the identification of a novel metabolic link between NAD+ catabolism and inhibition of mitochondrial fusion will offer new protective mechanisms that could significantly impact the clinical application of NUDT9 inhibitors as therapeutic compounds for acute brain injury such as global ischemia, stroke, TBI or chronic neurodegenerative disease.

 描述（由申请人提供）： 线粒体功能损伤经常与心脏骤停或中风相关的缺血性脑损伤有关。然而，神经元和星形胶质细胞中的线粒体功能障碍在多大程度上导致神经变性尚不清楚，导致线粒体功能衰竭的机制也难以理解。最近，有人提出，线粒体动力学的不平衡可能导致神经退行性变和脑损伤。此外，NAD+降解聚ADP核糖聚合酶（PARP 1）的过度激活会导致细胞和线粒体NAD+过度消耗，从而对细胞存活产生不利影响。我们假设ADP-核糖分解代谢酶NUDIX水解酶NUDT 9的活性导致下游线粒体GTP消耗，从而抑制控制细胞器融合的GTP酶。由于缺血性损伤引发线粒体的广泛分裂，因此拒绝这些细胞器的能力对于细胞活力至关重要。这一观点得到了我们的数据的有力支持，这些数据显示了缺血易受损伤区域中线粒体的不可逆碎片化、抗缺血脑细胞中线粒体的再融合以及遗传毒性应激诱导的线粒体NAD+和GTP池的耗竭。我们的初步数据还表明，线粒体NUDT 9同种型的过表达加剧了细胞死亡;线粒体对缺血性损伤更敏感，并且在NUDT 9水平增加的转基因动物中细胞死亡成熟加速。本研究的主要目的是确定NUDT 9的神经元或星形胶质细胞活性是否是缺血后细胞死亡机制的主要贡献者。为了解决这些问题，我们建议：1。确定NUDT 9在线粒体核苷酸代谢、线粒体生物能量学功能、动力学和细胞死亡中的特定作用。NUDT 9在星形胶质细胞和神经元的细胞死亡中的作用将通过利用从我们的新转基因动物的脑组织制备的纯神经元和星形胶质细胞细胞培养物来检查，所述新转基因动物在神经元或星形胶质细胞中有条件地表达mito-eYFP和NUDT 9。将这些细胞培养物暴露于氧-葡萄糖剥夺（OGD），并测定NUDT 9对生物能量代谢、线粒体呼吸功能、线粒体动力学和细胞死亡的影响。此外，还将分析细胞和线粒体代谢、线粒体呼吸功能以及线粒体融合和分裂。小干扰RNA（siRNA）诱导的NUDT 9基因敲低将用于证实NUDT 9对线粒体动力学和细胞死亡机制的损伤的作用。 2.为了研究NUDT 9在线粒体动力学损伤后损伤中的具体作用，我们将使用我们的转基因动物，其将经受短暂前脑缺血，并且在指定的恢复期，将检查脑中特别是神经元或星形胶质细胞中线粒体形态计量学的改变。最后，我们将阿塞塞神经元或星形胶质细胞中NUDT 9过表达对缺血损伤后组织学和神经学结果的影响。类似地，我们将通过评估用NUDT 9靶向siRNA预处理的动物的缺血后组织学和神经学结果来确定NUDT 9酶活性在缺血性脑损伤机制中的作用。 这项工作提出了机械和翻译的方法来解开神经元和星形胶质细胞线粒体动力学损伤的机制，并确定其在急性脑损伤中的作用。此外，NAD+催化剂和线粒体融合抑制之间的新型代谢联系的鉴定将提供新的保护机制，其可以显著影响NUDT 9抑制剂作为治疗急性脑损伤如全脑缺血、中风、TBI或慢性神经退行性疾病的治疗性化合物的临床应用。