ETHANOL AND BCL-2 GENE INTERACTIONS IN DEVELOPING CNS

乙醇和 BCL-2 基因在中枢神经系统发育中的相互作用

• 批准号: 6509313
• 负责人: MARIETA B HEATON
• 金额: $ 29万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6509313
• 关键词: BCL2 gene /protein; apoptosis; cerebellum; developmental neurobiology; ethanol; gene expression; gene interaction; immunocytochemistry; immunoprecipitation; laboratory mouse; laboratory rat; neurogenetics; neurotoxicology; western blottings

The proposed research will investigate the relationship between developmental ethanol neurotoxicity and the expression of apoptosis effector and repressor molecules of the Bcl-2 family. Members of this family can inhibit apoptosis (e.g., Bcl-2, Bcl- xl) or promote it (e.g., Bax, Bad, Bcl-xs). These relationships will be explored in the developing cerebellum, which exhibits a differential temporal susceptibility to ethanol during the early postnatal period, with a brief period of sensitivity on postnatal days 45 (P45), which results in loss of Purkinje and granule cells, followed by a period during which this region is relatively refractory to ethanol effects (P7-8). We hypothesize that alterations in the expression of Bcl-2-related molecules contribute significantly to this population's relative temporal vulnerability to ethanol. Neonatal rats will be exposed to ethanol via artificial rearing, which we have found to produce increases in bax and bcl-xs mRNA on P4, but not on P7. In specific experiments we will use quantitative Western blot procedures to characterize the dynamics of expression of Bcl-2- related proteins following ethanol exposure at P4 and P7. We will also examine the influence of ethanol on certain post- translational modifications of Bcl-2-related proteins (e.g., dimerization, phosphorylation, and altered molecular integrity). Such processes affect the capacity of these molecules to implement or inhibit cell death. We will determine the regional distribution of Bcl-2-related proteins within the developing cerebellum following ethanol exposure at a vulnerable time (P4) and at a "protected" time (P7), using immunohistochemical procedures. Finally, we will establish whether a causal relationship exists between expression of Bcl-2-related proteins and ethanol-induced neurotoxicity. For this study we will use genetically engineered animals lacking the pro-apoptotic bax gene. Homozygous, heterozygous and wild-type animals will be exposed to ethanol on P45 and Purkinje and granule cell counts subsequently made. We hypothesize that loss of this apoptosis promoter will eliminate or significantly mitigate ethanol-induced cerebellar neuronal death. These studies will be important in producing the first characterization of the role of cell death effector and repressor molecules in developmental ethanol neurotoxicity, and will provide new information concerning a critical mechanism underlying the devastating central nervous system damage seen in the Fetal Alcohol Syndrome.

本研究将探讨发育性乙醇神经毒性与Bcl-2家族细胞凋亡效应分子和抑制分子表达的关系。该家族成员可抑制细胞凋亡（如Bcl-2、Bcl- xl）或促进细胞凋亡（如Bax、Bad、Bcl-xs）。这些关系将在发育中的小脑中进行探讨，小脑在出生后早期对乙醇表现出不同的时间敏感性，在出生后45天（P45）有一段短暂的敏感性，这导致浦肯野细胞和颗粒细胞的损失，随后一段时间该区域对乙醇的影响相对不耐（P7-8）。我们假设bcl -2相关分子表达的改变对该人群对乙醇的相对时间易感性有重要贡献。新生大鼠将通过人工饲养暴露于乙醇中，我们已经发现P4上的bax和bcl-xs mRNA增加，但P7上没有。在特定的实验中，我们将使用定量Western blot程序来表征乙醇暴露在P4和P7后Bcl-2相关蛋白的表达动态。我们还将研究乙醇对bcl -2相关蛋白的某些翻译后修饰的影响（例如，二聚化、磷酸化和分子完整性改变）。这些过程影响这些分子实现或抑制细胞死亡的能力。我们将使用免疫组织化学方法，在脆弱时间（P4）和“保护”时间（P7）暴露于乙醇后，确定bcl -2相关蛋白在发育中的小脑内的区域分布。最后，我们将确定bcl -2相关蛋白的表达与乙醇诱导的神经毒性之间是否存在因果关系。在这项研究中，我们将使用缺乏促凋亡bax基因的基因工程动物。纯合子、杂合子和野生型动物将暴露于乙醇中，然后进行P45和浦肯野颗粒细胞计数。我们假设这种凋亡启动子的缺失将消除或显著减轻乙醇诱导的小脑神经元死亡。这些研究对于首次描述细胞死亡效应和抑制分子在发育乙醇神经毒性中的作用具有重要意义，并将为胎儿酒精综合征中破坏性中枢神经系统损伤的关键机制提供新的信息。