NAD catabolism and mitochondrial dysfunction in acute neurodegenerative disease

描述（由申请人提供）： 线粒体功能的损伤经常与中风或心脏骤停相关的缺血性脑损伤有关。然而，神经元和神经胶质细胞中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。线粒体损伤可由渗透性转换孔的活化或过度线粒体分裂导致基质吡啶核苷酸（NAD+、NADP+）的损失和随后的有害NAD+催化剂引起。我们推测，主要的细胞NAD调节酶CD 38可以显着有助于细胞内NAD+水解缺血性损伤后，抑制这种酶将显着改善缺血性脑损伤。这一观点得到了我们初步数据的有力支持，这些数据表明烟酰胺单甘肽（NMN）有希望保护缺血性脑损伤，NMN是一种天然存在的化合物，可抑制CD 38 NAD+糖水解酶，也可进入NAD+补救途径。本研究的主要目的是确定神经元或星形胶质细胞线粒体的病理形态学变化是否先于脑组织NAD+耗竭，以及CD 38的神经元或星形胶质细胞活性是否是缺血后NAD+水解的主要贡献者。为了解决这些问题，我们建议：1。利用我们独特的转基因动物，表达特异性荧光标记蛋白，无论是神经元或星形胶质细胞线粒体。这些动物将用于定量脑中神经元或星形胶质细胞的线粒体形态学改变。2.为了确定CD 38在损伤后NAD+ catalysts中的具体作用，我们将利用CD 38-null小鼠。将通过将纯神经元和星形胶质细胞培养物暴露于氧/葡萄糖剥夺和通过使CD 38缺陷动物经历短暂前脑缺血来检查CD 38在星形胶质细胞和神经元的细胞死亡中的作用。3.研究NMN对缺血性损伤的保护机制。我们将进行剂量依赖性和时间效应研究与NMN管理后缺血性损伤。在指定的恢复期后，将检查组织学和神经学结局。这项工作的意义在于，它提出了机制和翻译方法来解开神经元和星形胶质细胞NAD+ catalysts的机制，并确定其在急性脑损伤中的作用。此外，NMN保护机制的鉴定将显著影响NAD+前体作为治疗性化合物用于急性脑损伤如中风和TBI或慢性神经退行性疾病的临床应用。 公共卫生相关性： 由于退伍军人中中风风险因素的高患病率，包括年龄，中风在这一人群中非常常见并不奇怪，每年约有40，000例中风。中风是美国第三大死亡原因和残疾的主要原因，对VA提供残疾和长期护理的需求很大。虽然在预防中风方面取得了进展，但仍然非常需要新的治疗方法来改善急性中风幸存者和中风后有身体和认知障碍的退伍军人的结局。通过专注于了解导致线粒体和最终细胞生物能量衰竭的损伤机制，该研究将促进治疗方法的发展，从而改善中风患者的长期临床结果。由于中风引发的细胞死亡机制与创伤性脑损伤引起的脑损伤非常相似，因此本项目研究的新治疗方法也将对TBI患者的治疗产生重大影响。

DESCRIPTION (provided by applicant): Impairments in mitochondrial functions have been frequently implicated in ischemic brain injury associated with stroke or cardiac arrest. However, the extent to which mitochondrial dysfunction in neurons and glia contribute to neurodegeneration is unknown and the mechanisms leading to mitochondrial failure are elusive. Mitochondrial impairment can result from activation of the permeability transition pore or excessive mitochondrial fission leading to loss of matrix pyridine nucleotides (NAD+, NADP+) and consequent detrimental NAD+ catabolism. We hypothesize that the major cellular NAD-regulating enzyme CD38 can significantly contributes to intracellular NAD+ hydrolysis following an ischemic insult and that inhibition of this enzyme will dramatically ameliorate the ischemic brain injury. This notion is strongly supported by our preliminary data that suggest promising protection against ischemic brain damage by nicotinamide mononucleotide (NMN), a naturally occurring compound that inhibits CD38 NAD+ glycohydrolase and also feeds into the NAD+ salvage pathway. The primary goal of this study is to determine whether pathologic morphological changes of neuronal or astrocytic mitochondria precedes brain tissue NAD+ depletion and, whether neuronal or astrocytic activity of CD38 is a major contributor to NAD+ hydrolysis following ischemia. To address these questions we propose to: 1. Utilize our unique transgenic animals that express fluorescent marker proteins specific either to neuronal or to astrocytic mitochondria. These animals will be used to quantify mitochondrial morphometric alterations specifically in neurons or astrocytes in brain. 2. To determine the specific role of CD38 in post-insult NAD+ catabolism we will utilize a CD38-null mice. The role of CD38 in cell death of astrocytes and neurons will be examined by exposing the pure neuronal and astrocytic cell culture to oxygen/glucose deprivation and by subjecting CD38 deficient animals to transient forebrain ischemia. 3. Examine the mechanisms of NMN protection against ischemic damage. We will perform both dose-dependent and time-effect studies with NMN administration following ischemic insult. After the designated recovery period, the histological and neurological outcome will be examined. The significance of this work is that it proposes both mechanistic and translational approaches to unravel the mechanisms of neuronal and astrocytic NAD+ catabolism and determine its role in acute brain injury. Furthermore, the identification of NMN protective mechanisms will significantly impact the clinical application of NAD+ precursors as therapeutic compounds for acute brain injury as stroke and TBI or chronic neurodegenerative disease. PUBLIC HEALTH RELEVANCE: With the high prevalence of stroke risk factors among veterans, including age, it is not surprising that stroke is extremely common in this population, with approximately 40,000 strokes per year. Stroke is the 3rd leading cause of death and the leading cause of disability in the US, placing a great demand on VA to provide disability and long-term care. Although there have been advances in stroke prevention, there is still a great need for new therapies to improve the outcomes of both acute stroke survivors and veterans with physical and cognitive disabilities after stroke. By focusing on understanding injury mechanisms that leads to mitochondrial and ultimately cellular bioenergetic failure, the research in this grant will promote the development of treatments with the ability to improve the long-term clinical outcome for stroke victims. Since cell death mechanisms triggered by stroke are very similar to those causing brain damage due to traumatic brain injury, the new therapeutic approaches studied in this project will also have significant implication for treatment of TBI victims.