EVALUATION OF PARS ACTIVATION IN NO NEUROTOXICITY

无神经毒性情况下 PARS 激活的评估

• 批准号: 2379702
• 负责人: VALINA L. DAWSON
• 金额: $ 10.58万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 2000-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2379702
• 关键词: ADP ribosylation; DNA damage; NMDA receptors; SDS polyacrylamide gel electrophoresis; adenosine triphosphate; cell death; electron microscopy; embryo /fetus; enzyme activity; enzyme inhibitors; glutamates; laboratory rat; neurotoxins; nicotinamide adenine dinucleotide; nitric oxide; pathologic process; polymerization; polymers; protease inhibitor; tissue /cell culture; western blottings

Many neurodegenerative diseases, including Alzheimer's disease, huntington's disease, stroke and epilepsy have been linked to derangements in glutamate neurotransmission. This neurotoxicity is mediated in part through the generation of nitric oxide (NO). While inhibition of NO formation can be neuroprotective, the mechanism of NO-induced neurotoxicity has yet to be elucidated. One potentially important pathway may involve activation of poly (ADP-ribose) synthetase (PARS) activity. To address the potential role of PARS in mediating glutamate-induced free radical neurotoxicity the following specific aims are proposed. Experiments will be designed and performed to evaluate the role of PARS in neurotoxicity induced by exposure of neurons to neurotoxic concentrations of glutamate or free radicals. The effect of PARS inhibitors on neurotoxicity elicited by glutamate and its analogs as well as by NO, superoxide anion and peroxynitrite will be examined. The time course of these effects and the specificity of PARS inhibitors will be evaluated to determine whether the neuroprotective effects of PARS inhibitors are due to pharmacologic inhibition of the enzyme or due to interference with other cellular functions. To address conflicting reports regarding DNA damage following NMDA or NO neurotoxicity, cultures will be examined for both single and double stranded DNA breaks, the nuclear morphology will be examined with bisbenzimide and by electron microscopy, and blots of agarose gels will be examined for laddering. The activation of PARS under similar conditions will be determined by Western blot analysis and polyacrylamide gel autoradiography. In thymocytes, PARS elicited cytotoxicity corresponds with cellular depletion of NAD and ATP. In in vitro nuclear preparations and in tumor cell lines, PARS is involved in apoptotic events following cleavage to its 85 kd form form the full length 120 kd form. These two events may be complimentary or may occur differentially in different cell types. To evaluate if either event contributes to the development of neurotoxicity, primary neuronal cortical cultures will be exposed to neurotoxic agents and the cellular levels of NAD and ATP will be determined and sister cultures will be examined for PARS cleavage. If cleavage occurs then inhibitors of the protease which cleaves PARS will be evaluated for potential neuroprotective effects.

许多神经退行性疾病，包括阿尔茨海默氏病， 亨廷顿氏病，中风和癫痫病与扰乱有关 在谷氨酸神经传递中。 这种神经毒性部分介导 通过一氧化氮的产生（NO）。 虽然抑制否 形成可以是神经保护作用，这是无诱导的神经毒性的机制 尚未阐明。 一个潜在重要的途径可能涉及 聚（ADP-核糖）合成酶（PARS）活性的激活。 解决 PAR在介导谷氨酸诱导的自由基中的潜在作用 神经毒性提出了以下特定目的。 实验会 设计和执行以评估PAR在神经毒性中的作用 神经元暴露于谷氨酸或神经毒性浓度或 自由基。 PAR抑制剂对谷氨酸和 它的类似物以及否，超氧化物阴离子和过氧亚硝酸盐将是 检查。 这些效果的时间过程和PAR的特异性 将评估抑制剂以确定是否神经保护 PAR抑制剂的作用是由于药物抑制作用 酶或由于干扰其他细胞功能。 解决有关NMDA或否之后DNA损伤的相互矛盾的报告 神经毒性，将检查单一和双重培养物 滞留的DNA断裂，将检查核形态 Bisbenzimide和电子显微镜以及琼脂糖凝胶的印迹将是 检查是否梯子。 在类似条件下par的激活 将通过蛋白质印迹分析和聚丙烯酰胺凝胶确定 放射自显影。 在胸腺细胞中，PARS引起的细胞毒性与细胞相对应 NAD和ATP的耗竭。 在体外核制剂和肿瘤中 细胞系，PARS参与裂解后的凋亡事件 85 kd形式的全长120 kd形式。 这两个事件可能是 免费或可能在不同的细胞类型中差异化。 到 评估这两个事件是否有助于神经毒性的发展， 原发性神经元皮质培养物将暴露于神经毒性剂和 NAD和ATP的细胞水平将确定和姊妹培养物 将检查是否有裂解。 如果发生裂解，则 裂解par的蛋白酶将被评估为电势 神经保护作用。