Nuclear Bcl-2 Promoted Apoptosis: Relevance to Stroke

核 Bcl-2 促进细胞凋亡：与中风的相关性

• 批准号: 6936729
• 负责人: BRYCE P PORTIER
• 金额: $ 2.65万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-14 至 2007-02-13
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6936729
• 关键词: BCL2 gene /protein; PC12 cells; annexins; apoptosis; binding proteins; calcium flux; calcium indicator; chelating agents; confocal scanning microscopy; flow cytometry; fluorescent dye /probe; immunoprecipitation; mitochondria; molecular chaperones; neurons; neuroprotectants; nuclear membrane; predoctoral investigator; protein binding; protein localization; protein protein interaction; small interfering RNA; stroke; terminal nick end labeling; transfection; western blottings

DESCRIPTION (provided by applicant): Ischemic stroke is an especially grievous disease due to the functional deficits inflicted on victims following initial insult. One proposed treatment regimen is Bcl-2 overexpression following initial ischemia. While Bcl-2 is a known antiapoptotic protein when stably expressed at the mitochondria, its function at extra-mitochondrial sites (nuclear membrane and ER) is uncharacterized. Therefore, generalized Bcl-2 overexpression following initial ischemia could result in Bcl-2 at multiple cellular locations (mitochondria, ER and nuclear membrane) and even increase rather than prevent cell death. This project will investigate Bcl-2's localization following differential expression and determine localization specific changes in apoptosis regulation in the well characterized, neuron-like, PC12 cell line. The techniques of subcellular fractionation, western blot, flow cytometry (nuclei and whole cells) and confocal calcium imaging will be used to test the following hypotheses: 1.) Short term, induced Bcl-2 expression promotes nuclear Bcl-2 localization an event that promotes apoptosis. 2.) Bcl-2 localized at the nuclear membrane can be removed following elevation in FKBP38 (chaperone protein) expression. 3) The mechanism nuclear Bcl-2 utilizes to induce apoptosis is acted through elevating intra-nuclear calcium concentrations. These results will provide important new information on the cellular localization and function of Bcl-2 following rapid overexpression (endogenous or gene-therapy). They will also provide valuable insight into potential therapeutic interventions that would invoke proper Bcl-2 localization which in turn would induce anti-apoptotic Bcl-2 function, ultimately saving neurons and decreasing functional loss following ischemic stroke.

描述（由申请人提供）： 缺血性中风是一种特别严重的疾病，因为初次损伤后受害者会出现功能缺陷。一种提议的治疗方案是初始缺血后 Bcl-2 过度表达。虽然 Bcl-2 在线粒体稳定表达时是一种已知的抗凋亡蛋白，但其在线粒体外位点（核膜和内质网）的功能尚不清楚。因此，初始缺血后普遍的 Bcl-2 过度表达可能导致 Bcl-2 在多个细胞位置（线粒体、内质网和核膜）出现，甚至增加而不是阻止细胞死亡。该项目将研究差异表达后 Bcl-2 的定位，并确定在特征明确的神经元样 PC12 细胞系中细胞凋亡调节的定位特异性变化。亚细胞分级分离、蛋白质印迹、流式细胞术（细胞核和全细胞）和共聚焦钙成像技术将用于测试以下假设： 1.) 短期内，诱导的 Bcl-2 表达促进核 Bcl-2 定位，这是一个事件促进细胞凋亡。 2.) FKBP38（伴侣蛋白）表达升高后，可以去除位于核膜的 Bcl-2。 3) 核Bcl-2诱导细胞凋亡的机制是通过提高核内钙离子浓度来发挥作用。这些结果将为 Bcl-2 快速过表达（内源性或基因治疗）后的细胞定位和功能提供重要的新信息。他们还将为潜在的治疗干预措施提供有价值的见解，这些干预措施将调用适当的 Bcl-2 定位，进而诱导 Bcl-2 抗凋亡功能，最终拯救神经元并减少缺血性中风后的功能丧失。