INVOLVEMENT OF CELL CYCLE REGULATORS IN NEURONAL DEATH

细胞周期调节因子参与神经元死亡

• 批准号: 2431283
• 负责人: ROBERT S FREEMAN
• 金额: $ 15.91万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-29 至 1999-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2431283
• 关键词: antisense nucleic acid; apoptosis; cyclins; immunoprecipitation; neurotrophic factors; neutralizing antibody; polymerase chain reaction; protein kinase; retinoblastoma protein; sympathetic nervous system; western blottings

DESCRIPTION: Programmed cell death (PCD) is a necessary developmental phenomenon that is widespread in the nervous systems. Recent evidence suggests that PCD also occur in several pathological conditions. We and other have hypothesized that neurotrophic factor deprivation induced PCD is mediated by key cell cycle regulators activated by the removal of survival promoting factors. Recently, we provided the first evidence for the increased expression of a specific gene, cyclin D1, in neurons undergoing PCD. The only previously described function for cyclin D1 is its role in progression through the G1-phase of the cell cycle. We shall investigate the specific hypothesis that cyclin D1 functions as a necessary part of the death program in neurons. Toward this objective, we shall assess directly the role of cyclin D1 in the death of nerve growth factor (NGF) deprived sympathetic neurons. Using intracellular microinjections, we shall express (1) inhibitors of cyclin D1 function, (2) antisense cyclin D1 sequences, or (3) neutralizing cyclin D1 antibodies to examine whether cyclin D1 expression is required for NGF deprivation-induced PCD. We shall also examine whether overexpression of cyclin D1 ectopically is sufficient to induce PCD in neurons maintained in the presence of NGF. Biochemical approaches, including protein kinase assays, immunoprecipitations, and immunoblotting, will be used to identify and characterize the molecules that interact with cyclin D1 during PCD. These experiments will address whether a cyclin-dependent protein kinase is activated in dying neurons or whether cyclin D1 interacts with the retinoblastoma protein as part of a mechanism for cell death. Lastly, we shall use reverse transcription-polymerase chain reaction technology to continue to catalog cell cycle gene expression in dying neurons. These studies will determine the significance of the increased expression of cyclin D1 during PCD and will test the general hypothesis that neuronal cell death involves the activation of cell cycle events. This work should further our long-term goals of elucidating the molecular mechanism of neuronal PCD and of developing the means to manipulate the process pharmacologically.

描述：程序性细胞死亡（PCD）是一种必要的发育过程， 这种现象在神经系统中普遍存在。最近的证据 表明PCD也发生在几种病理条件下。我们和 其他人则假设神经营养因子剥夺诱导PCD， 是由关键的细胞周期调节因子介导的， 生存促进因素。最近，我们提供了第一个证据 神经元中一种特殊基因细胞周期蛋白D1的表达增加 接受PCD。之前描述的细胞周期蛋白D1的唯一功能 是其在细胞周期G1期进展中的作用。我们 将研究细胞周期蛋白D1作为 是神经元死亡程序的必要组成部分 为了实现这一目标，我们将直接评估细胞周期蛋白D1的作用， 神经生长因子（NGF）剥夺交感神经元的死亡。 使用细胞内显微注射，我们将表达（1） 细胞周期蛋白D1功能，（2）反义细胞周期蛋白D1序列，或（3） 中和细胞周期蛋白D1抗体，以检查细胞周期蛋白D1是否 表达是NGF剥夺诱导的PCD所必需的。我们亦会 检查细胞周期蛋白D1异位过表达是否足够 在NGF存在下维持的神经元中诱导PCD。生化 方法，包括蛋白激酶测定，免疫沉淀， 免疫印迹，将用于识别和表征分子 与细胞周期蛋白D1相互作用。这些实验将解决 细胞周期蛋白依赖性蛋白激酶是否在垂死的神经元中被激活 或者细胞周期蛋白D1是否与视网膜母细胞瘤蛋白相互作用， 细胞死亡的机制。最后，我们将使用反向 转录-聚合酶链反应技术继续 细胞周期基因在垂死神经元中的表达。 这些研究将确定表达增加的意义， 的细胞周期蛋白D1在PCD，并将测试的一般假设， 神经元细胞死亡涉及细胞周期事件的激活。这 我们的工作应该进一步阐明我们的长期目标， 神经元PCD的机制和发展手段来操纵 工艺流程