TUMOR SUPPRESSOR P53 AND THE CNS

描述：这项提议的长期目标是界定 细胞周期调节异常导致选择性神经元的机制 退化。这将通过体内表达研究来进行研究， 翻译后修饰与细胞周期的生物学功能 神经细胞凋亡动物模型中的调控基因。具体目标1 将分析红藻氨酸诱导的癫痫发作对 P53转录的翻译后磷酸化和DNA结合 因子，细胞周期的主要调节因子，已被牵连到 神经细胞凋亡。将使用固相激酶分析来分离和 测定海马区裂解液中P53蛋白的活性。DNA结合活性 将通过凝胶迁移率变化分析进行分析。《特定目标2》将分析 肾上腺切除术中P53调控的细胞周期基因的激活 诱导海马区颗粒细胞凋亡。细胞周期基因是 研究包括细胞周期蛋白d1、p21WAF1和视网膜母细胞瘤的易感性 吉恩，RB。野生型小鼠和纯合子(p53-/-)或 P53缺失等位基因的杂合子(P53+/-)将被用来阐明 这些基因在P53介导的神经元死亡中的作用。至 识别结构性表达P53的神经元群，特定目的 3将确定p53基因表达的区域和细胞模式 正常的出生后、成年和老年中枢神经系统。基因表达 将通过原位杂交和免疫组织化学进行分析。这个 细胞损伤程度将通过组织学染色进行评估 技巧。这些研究的结果将大大增加我们的 对选择性神经元变性相关机制的认识 异常的细胞周期控制，并将为深入了解 神经退行性疾病的病理生理学。

DESCRIPTION: The long-term objective of this proposal is to define mechanisms of abnormal cell cycle regulation leading to selective neuronal degeneration. This will be investigated by in vivo studies of expression, post-translational modifications and biological function of cell cycle regulatory genes in animal models of neuronal apoptosis. Specific Aim 1 will analyze the effects of kainic acid-induced seizures on the post-translational phosphorylation and DNA binding of the p53 transcription factor, a major regulator of the cell cycle that has been implicated in neuronal apoptosis. A solid-phase kinase assay will be used to isolate and measure p53 kinase activity in hippocampal lysates. DNA binding activity will be analyzed by gel mobility shift assay. Specific Aim 2 will analyze the activation of p53-regulated cell cycle genes during adrenalectomy- induced apoptosis of hippocampal granule cells. The cell cycle genes to be studied include cyclin D1, p21 WAF1 and the retinoblastoma susceptibility gene, RB. Wild type mice and mice that are either homozygous (p53-/-) or heterozygous (p53+/-) for the p53 null allele, will be used to elucidate the roles of these genes as effectors of p53-mediated neuronal death. To identify neuronal populations that constitutively express p53, Specific Aim 3 will define regional and cellular patterns of p53 gene expression in the normal postnatal, adult and aged central nervous system. Gene expression will be analyzed by in situ hybridization and immunohistochemistry. The degree of cellular injury will be assessed by histological staining techniques. The results of these studies will add significantly to our knowledge of mechanisms of selective neuronal degeneration related to aberrant cell cycle control, and will provide insight into the pathophysiology of neurodegenerative diseases.