Ethanol and Bcl-2 Gene Interactions in the Developing CNS

描述（由申请人提供）：拟议研究的目标将是定义神经系统发育过程中暴露于乙醇所产生的破坏性后果的关键细胞机制。这种暴露可导致胎儿酒精综合征（FAS）或酒精相关出生缺陷（ARBD）。对这些机制的进一步了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。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的影响。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、Bid）。拟议的实验将集中在Bax蛋白上，这是一种与乙醇诱导的细胞死亡密切相关的凋亡激动剂。这些关系正在探索发育中的小脑，这是高度敏感的乙醇在出生后的早期阶段。该区域在出生后4-5天（P4-5）最容易受到乙醇的影响，但P7-9对这些影响有抵抗力。对于这些研究，我们将采用体内< >的双管齐下的体外方法。将P4和P7新生大鼠通过蒸汽吸入暴露于乙醇中，培养的小脑颗粒细胞将用于平行操作分析。使用的技术包括用于表征Bax活化和亚细胞定位的Western blot蛋白分析，以及用于评估蛋白相互作用的ELISA程序。细胞存活试验将在培养细胞中进行，通过MTT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激酶激活的影响；14-3-3 Bax锚定蛋白的JNK磷酸化研究随后Bax激活并插入线粒体膜；(2)乙醇对Bid蛋白裂解及随后的tBid:Bax二聚化的影响；(3) Bax破坏线粒体膜的途径，即通过线粒体通透性过渡孔或通过Bax形成膜通道。在这一系列的研究中，颗粒细胞模型系统将使我们能够进行操纵性评估，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.