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Chromatin Modifications and Vulnerability to Glutamate Toxicity

染色质修饰和谷氨酸毒性脆弱性

基本信息

批准号：
7826976
负责人：
ALEXEI D KONDRATYEV
金额：
$ 23.03万
依托单位：
GEORGETOWN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-05-15 至 2012-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7826976
关键词：
Acute Address Affect Alzheimer&apos s Disease Amyotrophic Lateral Sclerosis Animals Apoptosis Ataxia Ataxia Telangiectasia Brain Cell Death Cells Cessation of life Chronic Clinical Commit Complement DNA DNA Damage DNA Double Strand Break DNA Repair DNA lesion Detection Disease Double Strand Break Repair Foundations Future Generations Glutamate Receptor Glutamates Goals Histones Huntington Disease Impairment Injury Internucleosomal DNA Fragmentation Ischemia Lead Lesion Measures Mediating Mediator of activation protein Modeling Mus N-Methylaspartate Nature Nerve Degeneration Neurodegenerative Disorders Neuroglia Neurologic Neuronal Injury Neurons Oxidative Stress Parkinson Disease Pathology Pathway interactions Phosphorylation Public Health Rattus Reactive Oxygen Species Receptor Activation Resistance Role Seizures Staging Stroke System Testing Therapeutic Intervention Time Toxic effect Transgenic Mice Variant Wild Type Mouse Work anticancer research base cell type chromatin modification endonuclease excitotoxicity histone modification human H2AX protein in vivo nervous system disorder neuron loss neuronal survival neurotoxicity novel public health relevance reconstitution repaired response restoration tumor

项目摘要

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.

描述（由申请人提供）：离子型谷氨酸受体的过度活化会增加氧化应激，这会导致神经损伤（如缺血和癫痫发作）后观察到的神经变性，并导致神经变性疾病（阿尔茨海默氏症、帕金森氏症等）中的神经元死亡。从临床的角度来看，这对大脑功能和生存是一个明显的威胁。据信，活性氧物质的产生和随后的氧化应激是谷氨酸毒性的主要贡献者。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（例如阿尔茨海默病、亨廷顿病和帕金森病、肌萎缩性侧索硬化症、共济失调毛细血管扩张症和许多其他神经系统疾病）下都可能导致神经元丢失和损伤。最致命的DNA损伤形式，双链断裂（DSB），以及细胞修复它们的能力尚未在谷氨酸受体过度刺激后直接证明。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中的重要性，但尚未在成熟神经元中进行系统的直接研究。我们已经开发了一个敏感的模型，开始解决DSB DNA损伤的作用，神经元的脆弱性，谷氨酸介导的侮辱使用磷酸化组蛋白变体H2A.X，这迅速发生后的DNA DSB。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损伤后的神经元存活。1）在离子型谷氨酸受体活化后，组蛋白H2 AX磷酸化的增加将导致DSB修复的增加;我们将通过测量大鼠皮层神经元培养物中的DSB修复活性来测试该假设; 2）由于DSB修复途径的破坏，H2 AX磷酸化的损害将导致谷氨酸毒性增加。为了测试这一点，我们将检查来自H2 AX-/-转基因小鼠的神经元对谷氨酸毒性的脆弱性，并评估其DSB修复能力。我们预计，H2 AX-/-神经元将更容易受到谷氨酸毒性，并表现出减少DSB修复相比，野生型细胞。此外，我们将使用慢病毒表达在H2 AX-/-神经元中重建功能性组蛋白H2 AX，并评估其对谷氨酸毒性的抗性的恢复。对这些假说的验证可能揭示多种神经退行性疾病中神经毒性的共同机制，为这些疾病的治疗寻找有吸引力的新靶点，并为将来的神经元DSB修复通路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，长时间癫痫发作、中风、TBI），而且在各种慢性神经退行性病症下，例如阿尔茨海默病、亨廷顿病、帕金森病、肌萎缩性侧索硬化、共济失调毛细血管扩张症以及目前没有有效治愈的其它神经病症。过度兴奋也有助于许多这些病理，并被认为是DNA损伤的主要原因。然而，对神经元中最致命类型的DNA损伤（双链断裂）的兴奋驱动形成的机制以及神经细胞承受这种损伤的能力知之甚少。该提案将检查这些机制，并将奠定基础，确定新的目标，治疗各种各样的神经系统疾病相关的兴奋性毒性。