ETHANOL AND BCL-2 GENE INTERACTIONS IN DEVELOPING CNS

本研究将探讨发育性酒精神经毒性与Bcl2家族凋亡效应和抑制分子表达的关系。该家族成员可抑制或促进细胞凋亡(如Bax、Bad、Bclxs)。这些关系将在发育中的小脑中进行探索，小脑在出生后早期表现出对酒精的不同时间敏感性，在出生后45天有一个短暂的敏感期(P45)，导致浦肯野细胞和颗粒细胞的丧失，随后是该区域对酒精影响相对不敏感的时期(P7-8)。我们假设，Bcl-2相关分子表达的变化显著地导致了该人群对乙醇的相对时间易感性。通过人工饲养的方式将新生大鼠暴露在乙醇中，我们发现酒精在P4时会增加bax和bcl一xs的基因表达，但在P7时不会增加。在特定的实验中，我们将使用定量Western印迹程序来表征P4和P7乙醇暴露后Bcl2相关蛋白的表达动态。我们还将研究乙醇对某些Bcl2相关蛋白翻译后修饰的影响(例如，二聚化、磷酸化和分子完整性改变)。这样的过程会影响这些分子实施或抑制细胞死亡的能力。我们将使用免疫组织化学方法，在酒精暴露后的脆弱时间(P4)和“保护”时间(P7)，确定发育中的小脑内Bcl2相关蛋白的区域分布。最后，我们将确定Bcl2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基因bax的基因工程动物。纯合子、杂合子和野生型动物将在P45和Purkinje上暴露于乙醇，并随后进行颗粒细胞计数。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。这些研究将对首次确定细胞死亡效应分子和抑制分子在发育性酒精神经毒性中的作用具有重要意义，并将提供关于胎儿酒精综合征中破坏性中枢神经系统损害的关键机制的新信息。

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.