NAD catabolism and mitochondrial dysfunction in acute neurodegenerative disease

描述(由申请人提供)： 线粒体功能受损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的线粒体功能障碍在多大程度上导致神经退行性变尚不清楚，导致线粒体功能障碍的机制也是难以捉摸的。线粒体损伤可能是由于通透性转换孔的激活或线粒体过度分裂导致基质吡啶核苷酸(NAD，NADP)的丢失，从而导致有害的NAD分解代谢。我们假设，细胞内主要的NAD调节酶CD38可以显著促进缺血性损伤后细胞内NAD的水解，抑制该酶将显著改善缺血性脑损伤。我们的初步数据有力地支持了这一观点，这些数据表明，烟酰胺单核苷酸(NMN)有希望预防缺血性脑损伤，NMN是一种天然存在的化合物，它可以抑制CD38 NAD糖水解酶，也可以进入NAD挽救途径。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变化是否先于脑组织NAD耗竭，以及CD38的神经元或星形胶质细胞活性是否是缺血后NAD水解的主要贡献因素。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，表达神经元或星形细胞线粒体特有的荧光标记蛋白。这些动物将被用来量化线粒体的形态变化，特别是在大脑中的神经元或星形胶质细胞。2.为了确定CD38在创伤后NAD分解代谢中的特定作用，我们利用CD38缺失的小鼠。CD38在星形胶质细胞和星形胶质细胞死亡中的作用将通过将纯神经元和星形胶质细胞暴露在缺氧/葡萄糖剥夺中以及通过使CD38缺乏的动物进行短暂性前脑缺血来研究。3.探讨NMN对缺血损伤的保护作用机制。我们将对缺血损伤后给予NMN进行剂量依赖和时间效应研究。在指定的恢复期后，将检查组织学和神经学结果。这项工作的意义在于，它提出了机械和翻译两种方法来解开神经元和星形细胞NAD分解代谢的机制，并确定其在急性脑损伤中的作用。此外，NMN保护机制的确定将显著影响NAD前体作为治疗化合物的临床应用，如中风、脑外伤或慢性神经退行性疾病等急性脑损伤。 公共卫生相关性： 随着包括年龄在内的中风危险因素在退伍军人中的高度流行，中风在这一人群中极其常见也就不足为奇了，每年约有40,000人中风。在美国，中风是第三大死亡原因和第一大残疾原因，这对退伍军人管理局提供残疾和长期护理提出了巨大的需求。尽管在卒中预防方面取得了进展，但仍迫切需要新的治疗方法来改善急性卒中幸存者和卒中后有身体和认知障碍的退伍军人的预后。通过重点了解导致线粒体并最终导致细胞生物能量衰竭的损伤机制，这项赠款的研究将促进具有改善中风患者长期临床结果的治疗方法的开发。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对脑外伤患者的治疗也具有重要意义。

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.