Ethanol and Bcl-2 Gene Interactions in the Developing CNS

描述（由申请人提供）：拟议研究的目的是确定神经系统发育期间暴露于乙醇所产生的破坏性后果的关键细胞机制。这种暴露可能导致胎儿酒精综合征（FAS）或酒精相关的出生缺陷（ARBD）。对这些机制的进一步理解将使最终设计出预防或减轻乙醇神经毒性的治疗策略成为可能。在这些研究中特别感兴趣的是乙醇对Bcl-2存活调节基因家族蛋白质的影响。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、Bid）。拟议的实验将集中在Bax蛋白，一种与乙醇诱导的细胞死亡密切相关的凋亡激动剂。这些关系正在探讨在发展中的小脑，这是非常敏感的乙醇在出生后早期。该区域在出生后第4-5天（P4-5）对乙醇最敏感，但在P7-9对这些影响具有抵抗力。对于这些研究，我们将使用双管齐下的体内< >体外方法。P4和P7新生大鼠将通过蒸汽吸入暴露于乙醇，培养的小脑颗粒细胞将用于平行操作分析。待使用的技术包括用于表征Bax活化和亚细胞定位的蛋白质印迹分析，以及用于评估蛋白质-蛋白质相互作用的ELISA程序。将通过MTT试验在培养的细胞中进行细胞存活试验。此外，蛋白质纯化和圆二色性（CD）方法将用于检查蛋白质结构。具体的实验将定义（1）乙醇对JNK激酶活化的影响; 14-3- 3Bax锚定蛋白的JNK磷酸化;随后的Bax活化和插入线粒体膜;（2）乙醇对Bid蛋白切割和随后的tBid：Bax二聚化的影响;和（3）线粒体膜的Bax破坏途径，即，通过线粒体通透性转换孔或通过Bax形成膜通道。在这些系列研究中，颗粒细胞模型系统将使我们能够进行操作评估，并测量细胞死亡，以确认感兴趣的事件的重要性。此外，纯化的P4和P7 Bax将在两个年龄进行蛋白质构象的CD分析，以及乙醇对这种构象的影响。

DESCRIPTION (provided by applicant): The objectives of the proposed research will be to define cellular mechanisms critical to the devastating consequences produced by exposure to ethanol during the development of the nervous system. Such exposure can lead to the fetal alcohol syndrome (FAS) or alcohol-related birth defects (ARBD). Improved understanding of these mechanisms will make it possible to eventually devise therapeutic strategies for preventing or mitigating ethanol neurotoxicity. Of particular interest in these studies is the effect of ethanol on proteins of the Bcl-2 survival-regulatory gene family. Members of this family can inhibit apoptosis (e.g., Bcl-2, Bcl-xl) or promote it (e.g., Bax, Bad, Bid). Proposed experiments will focus on the Bax protein, an apoptosis agonist strongly linked to ethanol-induced cell death. These relationships are being explored in developing cerebellum, which is highly susceptible to ethanol during the early postnatal period. This region is maximally vulnerable to ethanol on postnatal days 4-5 (P4-5), but is resistant to these effects by P7-9. For these studies, we will use a two-pronged in vivo < > in vitro approach. P4 and P7 neonatal rats will be exposed to ethanol via vapor inhalation, and cultured cerebellar granular cells will be used for parallel manipulative analyses. Techniques to be used include Western blot protein analyses for characterizing Bax activation and subcellular localization, and the ELISA procedure to assess protein-protein interactions. Cell survival assays will be made in the cultured cells, via the MTT assay. In addition, protein purification and circular dichroism (CD) methodologies will be used to examine protein structure. Specific experiments will define (1) ethanol influences on activation of the JNK kinase; JNK phosphorylation of the 14-3-3 Bax anchoring protein; subsequent Bax activation and insertion into the mitochondrial membrane; (2) ethanol effects on cleavage of the Bid protein, and subsequent tBid:Bax dimerization; and (3) the pathway of Bax disruption of the mitochondrial membrane, i.e., via the mitochondrial permeability transition pore or by Bax formation of membrane channels. In each of these series of studies, the granule cell model system will enable us to perform manipulative assessments, and to measure cell death in order to confirm the importance of the events of interest. In addition, purified P4 and P7 Bax will be subjected to CD analyses of protein conformation at the two ages, and ethanol effects on this conformation.