NEUROTOXICITY, NMDA RECEPTORS, AND FREE RADICALS

神经毒性、NMDA 受体和自由基

• 批准号: 6201026
• 负责人: ELIAS K MICHAELIS
• 金额: $ 13.26万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2001-04-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6201026
• 关键词: NMDA receptors; RNase protection assay; antisense nucleic acid; embryo /fetus tissue /cell culture; enzyme activity; free radical oxygen; glutamates; granule cell; laboratory rat; neural degeneration; neurons; neuroprotectants; neurotoxins; nitric oxide; nitric oxide synthase; nitroso compounds; northern blottings; oxidation; oxidative stress; protein sequence; protein structure function; receptor binding; receptor expression; superoxide dismutase; tissue /cell culture

Neuronal degeneration is a common phenomenon associated with aging in the central nervous system. It has been hypothesized that the causes for either the degenerative changes associated with normal aging or the exaggerated damage seen in disease states such as Alzheimer's disease are linked to hyperfunction of L-glutamate as an excitatory transmitter. Such hyperactivity of glutamate neurotransmission leads to excessive intracellular calcium (Ca2+) accumulation through increased activation of glutamate receptors, especially of the N-methyl-D-aspartate (NMDA) receptors. Three hypotheses are being tested in the proposed experimental work. First, that there is a correlation between the expression of specific NMDA receptor proteins in brain neurons and the susceptibility of the same or neighboring neurons to damage induced by ROS. Second, that NO and ROS modify NMDA receptor proteins at specific sites so that the activity of the receptors is down-regulated. Third, that neurons that express high levels of NOS yet are partially protected from NMDA-induced damage may use a variety of processes to diminish the effects of NO and ROS on cell viability. The specific aims for the proposed studies are: 1. To determine the importance of various forms of NMDA receptors in the activation of NOS and the formation of ROS in primary neuronal cultures. The ROS and NO generation in neurons will be measured following exposure to glutamate, NMDA, and various agonists and antagonists acting on NMDA receptors. The NMDA receptor subunits expressed and their role in cytotoxicity will be evaluated. 2. To characterize the specific amino acid modifications in either NMDAR1 or glutamate-binding proteins and of the rate of turnover of these proteins following generation of NO and oxygen free radicals in neurons. These studies will include the characterization of specific amino acid modifications in cloned and expressed proteins following exposure of the proteins in vitro to NO or oxoperoxynitrate (ONOO-) or superoxide anion or hydrogen peroxide. 3. To characterize the possible protective mechanisms operating in neurons which express high levels of NOS, mechanisms that may include alteration in calmodulin (CaM) activation of NOS due to oxidative modification of CaM, rapid Fe-nitrosation of a few proteins or high levels of expression of proteins such as BCL-2, BCL-X and superoxide dismutase (SOD). These studies will include the measurement of the levels of Fe-nitrosyl complex formation in neurons following activation of NMDA receptors and the determination of the comparative expression of the mRNA for MnSOD, CuZn- SOD, BCL-2, BCL-X and NOS. Two important outcomes of these studies will be a better understanding of the receptor macromolecules that are involved in the regulation of NO and ROS formation in neurons and of the mechanisms used by neurons to limit oxidative damage. If both goals are successfully accomplished, then we will have a better definition of potential targets for therapeutic intervention in neurodegenerative diseases associated with the aging process.

神经元变性是与衰老有关的常见现象 中枢神经系统。 已经假设 与正常衰老相关的退化变化或 在阿尔茨海默氏病等疾病状态下看到的夸大损害是 与L-谷氨酸作为兴奋性发射器的过度功能有关。 这样的 谷氨酸神经传递的多动症导致过度 细胞内钙（Ca2+）通过增加的激活而积累 谷氨酸受体，特别是N-甲基-D-天冬氨酸（NMDA）的受体 受体。 在提出的实验中检验了三个假设 工作。 首先，在表达之间存在相关性 脑神经元中的特定NMDA受体蛋白和 ROS引起的损伤相同或相邻的神经元。 第二，那没有 ROS在特定位点修改NMDA受体蛋白，以便 受体的活性下调。 第三，那个神经元 表达高水平的NOS，但部分保护了NMDA诱导的 损害可能会使用各种过程来减少NO和 ROS在细胞活力上。 拟议研究的具体目的是： 1。确定各种形式的NMDA受体在 NOS的激活和原代神经元培养物中ROS的形成。 暴露后，将测量ROS和无产生的神经元中的发电 致谷氨酸，NMDA以及作用于NMDA的各种激动剂和拮抗剂 受体。 NMDA受体亚基表达了 将评估细胞毒性。 2。表征两种NMDAR1中的特定氨基酸修饰 或谷氨酸结合蛋白以及这些周转率 神经元中NO和氧自由基的产生之后的蛋白质。 这些研究将包括特定氨基酸的表征 暴露后，克隆和表达的蛋白质修饰 体外蛋白质至NO或氧化二硝酸盐（ONOO-）或超氧化物阴离子或 过氧化氢。 3。表征在神经元中运行的可能的保护机制 表达高水平的NOS，可能包括改变的机制 在钙调蛋白（CAM）激活NOS中，由于氧化的修饰 CAM，几种蛋白质或高水平表达的快速Fe硝化 蛋白质（例如Bcl-2，Bcl-X和超氧化物歧化酶（SOD））的蛋白质。这些 研究将包括测量Fe-硝基复合物的水平 NMDA受体激活后神经元的形成和 测定mnsod mRNA的比较表达 SOD，BCL-2，BCL-X和NOS。 这些研究的两个重要结果将更好地理解 与NO调节有关的受体大分子和 神经元中的ROS形成以及神经元用于限制的机制 氧化损伤。 如果两个目标都成功完成，那么我们 将对治疗的潜在靶标有更好的定义 与衰老相关的神经退行性疾病的干预 过程。