INVOLVEMENT OF CELL CYCLE REGULATORS IN NEURONAL DEATH

描述：细胞程序性死亡(PCD)是一种必要的发育过程 神经系统中普遍存在的现象。最近的证据 这表明PCD也发生在几种病理条件下。我们和 另一些人则假设剥夺神经营养因子会导致PCD 是由关键的细胞周期调节因子介导的，这些调节因子是由 促进生存的因素。最近，我们提供了第一个证据 对于神经元中特定基因细胞周期蛋白D1表达的增加 正在接受PCD。之前描述的细胞周期蛋白D1的唯一功能 它在细胞周期的G1期进程中所起的作用。我们 将研究细胞周期蛋白D1的具体作用假设 这是神经元死亡程序的必要部分。 为了实现这一目标，我们将直接评估细胞周期蛋白D1在 神经生长因子(NGF)的死亡剥夺了交感神经元。 利用细胞内微量注射，我们将表达(1)抑制 细胞周期蛋白D1的功能，(2)反义细胞周期蛋白D1序列，或(3) 中和细胞周期蛋白D1抗体检测细胞周期蛋白D1 NGF剥夺诱导的PCD需要表达。我们还将 检查细胞周期蛋白D1的异位过度表达是否足够 诱导在NGF存在下维持的神经元的PCD。生化 方法，包括蛋白激酶分析、免疫沉淀和 免疫印迹，将用于鉴定和表征分子 在PCD过程中与细胞周期蛋白D1相互作用。这些实验将解决 死亡神经元中细胞周期蛋白依赖的蛋白激酶是否被激活 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 一种细胞死亡的机制。最后，我们将使用反转 转录-聚合酶链式反应技术继续 细胞周期基因在死亡神经元中的表达目录。 这些研究将确定增加表达的意义 细胞周期蛋白D1在PCD中的表达，并将检验普遍假设 神经细胞死亡涉及细胞周期事件的激活。这 这项工作应该推动我们阐明分子的长期目标 神经元性PCD的机制及其调控手段的研究进展 从药理上来说。

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.