ETHANOL AND BCL-2 GENE INTERACTIONS IN DEVELOPING CNS

这项研究将探讨发育中乙醇神经毒性与Bcl-2家族凋亡效应分子和抑制分子表达之间的关系。该家族的成员可以抑制细胞凋亡（例如，Bcl-2、Bcl- xl）或促进它（例如，Bax、Bad、Bcl-xs）。 这些关系将在发育中的小脑中进行探索，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）出现短暂的敏感性，导致浦肯野细胞和颗粒细胞的丧失，随后是该区域对乙醇作用相对难治的时期（P7-8）。 我们假设Bcl-2相关分子表达的改变对这一人群对乙醇的相对时间脆弱性有显著影响。 将新生大鼠暴露于乙醇通过人工饲养，我们已经发现，P4的bax和bcl-xs mRNA的增加，但不是在P7。 在具体的实验中，我们将使用定量蛋白质印迹法来表征在P4和P7乙醇暴露后Bcl-2相关蛋白表达的动态。 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、磷酸化和改变的分子完整性）。这些过程影响这些分子实现或抑制细胞死亡的能力。 我们将确定区域分布的Bcl-2相关的蛋白在发展中的小脑乙醇暴露后，在脆弱的时间（P4）和“保护”的时间（P7），使用免疫组织化学程序。 最后，我们将确定Bcl-2相关蛋白的表达和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在P45和Purkinje暴露于乙醇，随后进行颗粒细胞计数。 我们假设，这种凋亡促进剂的损失将消除或显着减轻乙醇诱导的小脑神经元死亡。这些研究将是重要的，在生产的细胞死亡效应和抑制分子在发育乙醇神经毒性的作用的第一个特征，并将提供新的信息有关的关键机制的破坏性中枢神经系统损伤的胎儿酒精综合征。

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.