Chromatin Modifications and Vulnerability to Glutamate Toxicity

描述(由申请人提供)：过度激活离子谷氨酸受体会增加氧化应激，这会导致在神经损伤后观察到的神经退化，如缺血和癫痫发作，以及导致神经退行性疾病(阿尔茨海默氏症、帕金森氏症等)的神经元死亡。从临床角度来看，它对大脑功能和生存是一个明显的威胁。据认为，活性氧的产生和随之而来的氧化应激是谷氨酸中毒的主要原因。同时，氧化应激是DNA损伤的主要原因，也是神经元损伤的常见组成部分。DNA损伤不仅在急性脑损伤后，而且在各种慢性神经退行性疾病，如阿尔茨海默病、亨廷顿病和帕金森病、肌萎缩侧索硬化症、共济失调毛细血管扩张症和许多其他神经疾病时，都可能导致神经元的丢失和损伤。最致命的DNA损伤形式是双链断裂(DSB)，细胞修复它们的能力在谷氨酸受体过度刺激后尚未被直接证明。虽然有限的证据表明DSB及其修复机制在谷氨酸诱导的损伤易感性中的重要性，但尚未对成熟神经元进行系统的直接研究。我们已经开发了一个敏感的模型，通过组蛋白变异体H2A.X的磷酸化开始研究DSB DNA损伤在谷氨酸介导的损伤中神经元易损性中的作用，组蛋白变异体在DNA DSB之后迅速发生。我们的一般工作假设是，终末分化神经元中未修复的DSB的后果是过度兴奋后神经元死亡的关键因素。相反，这些断裂的成功修复可能会增加谷氨酸驱动的侮辱后神经元的存活率。为了验证这一概念，我们将检验下列特定假说：1)组蛋白H2AX的磷酸化水平在离子型谷氨酸受体激活后会增加，从而导致DSB修复增加；我们将通过测量大鼠皮质神经元培养的DSB修复活性来验证这一假说；2)H2AX磷酸化受损将因DSB修复途径中断而导致谷氨酸毒性增加。为了测试这一点，我们将检查来自H2AX/-转基因小鼠的神经元对谷氨酸毒性的脆弱性，并评估它们的DSB修复能力。我们预计，与野生型细胞相比，H2AX-/-神经元将更容易受到谷氨酸毒性的影响，并表现出DSB修复的减弱。此外，我们将通过慢病毒表达来重建H_2AX-/-神经元中的功能性组蛋白H_2AX，并评估它们对谷氨酸毒性的抗性的恢复。验证这些假说可能会揭示一种导致多种神经退行性疾病神经毒性的新的共同机制，将有助于发现治疗这些疾病的有吸引力的新靶点，并将为未来针对神经元DSB修复途径的体内干预研究奠定基础。公共卫生相关性：DNA损伤是神经元损伤的常见组成部分。它可能不仅在急性脑损伤(例如，长时间癫痫、中风、脑外伤)后，而且在各种慢性神经退行性疾病下，如阿尔茨海默病、亨廷顿病、帕金森病、肌萎缩侧索硬化症、共济失调毛细血管扩张症等，也可能导致神经元丢失和损伤，以及其他目前无法有效治愈的神经疾病。过度兴奋也导致了许多这些病理现象，并被认为是DNA损伤的主要原因。然而，对于神经元中最致命的DNA损伤(双链断裂)在兴奋驱动下形成的机制以及神经细胞承受这种损伤的能力，人们知之甚少。这项建议将研究这些机制，并将为确定治疗与兴奋性毒性相关的各种神经系统疾病的新靶点奠定基础。

DESCRIPTION (provided by applicant): Excessive activation of ionotropic glutamate receptors increases oxidative stress, which contributes to the neurodegeneration observed following neurological insults such as ischemia and seizures, as well as contributes to neuronal death in neurodegenerative diseases (Alzheimer's, Parkinson's, etc.). From a clinical perspective, it is a clear threat to brain function and to survival. It is believed that generation of reactive oxygen species and ensuing oxidative stress is a major contributor to glutamate toxicity. At the same time, oxidative stress is a major cause of DNA damage, which is also a common component of neuronal injury. DNA damage may contribute to neuronal loss and injury not only after acute brain insults but also under various chronic neurodegenerative conditions, such as Alzheimer's, Huntington's, and Parkinson's diseases, amyotrophic lateral sclerosis, ataxia telangiectasia and many other neurological disorders. The most lethal form of DNA damage, the double strand breaks (DSBs), and the ability of cells to repair them has not yet been directly demonstrated following excessive stimulation of glutamate receptors. While limited evidence suggests the importance of DSBs and their repair machinery in vulnerability to glutamate-induced injury, no systematic direct studies have been done in mature neurons. We have developed a sensitive model to start addressing the role of DSB DNA damage in neuronal vulnerability to glutamate-mediated insults using phosphorylation of histone variant H2A.X, which occurs rapidly following DNA DSBs. Our general working hypothesis is that the consequences of unrepaired DSBs in terminally differentiated neurons are critical contributors to neuronal demise in the aftermath of excessive excitation. Conversely, successful repair of these breaks may increase neuronal survival following glutamate-driven insults. Specific Aims will test the following specific hypotheses aiming at proving this concept: 1) Increased phosphorylation of histone H2AX following activation of ionotropic glutamate receptors will result in increased DSB repair; this hypothesis we will tested by measuring DSB repair activity in rat cortical neuronal cultures; 2) Impairment of H2AX phosphorylation will result in increased glutamate toxicity due to the disruption of the DSB repair pathway. To test this, we will examine vulnerability of neurons from H2AX-/- transgenic mice to vulnerability to glutamate toxicity and evaluate their DSB repair capabilities. We expect that H2AX-/- neurons will be more vulnerable to glutamate toxicity and demonstrate diminished DSB repair as compared to wild-type cells. Moreover, we will reconstitute functional histone H2AX in H2AX-/- neurons using lentiviral expression and evaluate the restoration of their resistance to glutamate toxicity. Testing these hypotheses may reveal a novel common mechanism contributing to neurotoxicity in a variety of neurodegenerative disorders, will lead to identification of attractive new targets for therapy of these disorders, and will lay a foundation for future interventional studies in vivo targeting DSB repair pathway in neurons. PUBLIC HEALTH RELEVANCE: Damage to DNA is a common component of neuronal injury. It may contribute to neuronal loss and injury not only after acute brain insult (e.g., prolonged seizures, stroke, TBI) but also under various chronic neurodegenerative conditions, such as Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, ataxia telangiectasia, among other neurological disorders that currently have no effective cure. Excessive excitation also contributes to many of these pathologies and is believed to be the major cause of DNA damage. However, little is known about the mechanisms responsible for the excitation- driven formation of the most lethal type of DNA damage (double strand breaks) in neurons and the ability of nerve cells to withstand this damage. This proposal will examine these mechanisms and will lay the foundation for identification of new targets for therapy of a broad variety of neurological conditions relevant to excitotoxicity.