Mechanism of Alcohol Induced Neurotoxicity

描述（由申请方提供）：过量摄入酒精会增加血液乙醇浓度，并诱导表现为胎儿酒精综合征（FAS）的脑发育障碍。FAS患儿面部特征异常，伴有轻度至重度精神发育迟滞。小脑是大脑中受乙醇影响最敏感的区域之一。然而，乙醇对小脑有害作用的机制在很大程度上是未知的。我们的初步研究表明，在体内条件下，中等剂量的乙醇（血液乙醇浓度为40 mM）抑制分化，高剂量的乙醇（血液乙醇浓度为80 mM）诱导小脑颗粒神经元（CGNs）的凋亡。但在体外条件下，需要更高浓度的乙醇才能产生类似的结果，这表明可能还涉及其他一些因素。进一步观察到，在体外条件下，乙醇（40 mM和80 mM）在生理浓度的视黄酸（100 nM）存在下具有与在体内条件下观察到的类似的有害作用。基于这些研究，我们的工作假设是，乙醇对CGN的分化和存活的有害影响是由视黄酸介导的。为了检验这一假设，我们将确定（1）乙醇暴露在体内是否影响视黄酸受体的激活和（2）是否管理视黄酸受体拮抗剂保护免受乙醇对CGN的分化和存活的有害影响。为了解决这个问题，将广泛用作胎儿酒精综合征啮齿动物模型的大鼠幼仔（出生后第7天）暴露于乙醇。分离小脑颗粒神经元并用于拟定研究。视黄酸受体的转录活性将通过电迁移率变动分析来研究。如果乙醇干扰转录活性，这将表明视黄酸受体可能介导乙醇的影响。我们的研究表明，Rho GTP酶在CGN的分化中起着至关重要的作用;并且，体内乙醇暴露影响这些GTP酶的活化。受体活化受损影响Rho GT3信号传导似乎是合理的。为了测试这种可能性，将使用已知可防止激活的视黄酸受体拮抗剂。研究将确定拮抗剂是否阻止乙醇对Rho GTP酶激活的影响，并保护CGN免受乙醇的有害影响。为了研究Rho GTP酶的作用，我们将使用体外细胞培养模型。在该细胞培养模型中，CGN将在视黄酸（100 nM）存在下暴露于乙醇（40 mM，80 mM）。将研究Rho GTP酶的组成型活性或显性阴性形式的表达是否防止乙醇对CGN的分化和存活的毒性作用。

DESCRIPTION (provided by applicant): Excessive intake of alcohol increases blood ethanol concentration and induces brain developmental disorders manifested as fetal alcohol syndrome (FAS). Children born with FAS have abnormal facial features with mild to sever mental retardation. Cerebellum is one of the most sensitive areas in the brain that is affected by ethanol. However, mechanisms underlying the deleterious effects of ethanol in cerebellum are largely unknown. Our preliminary studies demonstrated that moderate dose of ethanol (blood ethanol concentration of 40 mM) inhibits differentiation, and high dose of ethanol (blood ethanol concentration 80 mM) induces apoptosis in cerebellar granular neurons (CGNs) under in vivo conditions. But under in vitro conditions, higher concentration of ethanol is required for producing similar results, suggesting that some other factor(s) might also be involved. It was further observed that under in vitro conditions ethanol (40 mM and 80 mM) in the presence of physiological concentration of retinoic acid (100 nM) had similar deleterious effects as observed under in vivo conditions. Based on these studies, it is our working hypothesis that harmful effects of ethanol on differentiation and survival of CGNs are mediated by retinoic acid. To test this hypothesis we will determine whether (1) ethanol exposure in vivo affects activation of retinoic acid receptors and (2) whether administration of retinoic acid receptor antagonists protect against harmful effects of ethanol on differentiation and survival of CGNs. To address the issue, rat pups (postnatal day 7) which are widely used as a rodent model of fetal alcohol syndrome, will be exposed to ethanol. Cerebellar granular neurons will be isolated and used for the proposed studies. The transcriptional activities of retinoic acid receptors will be studied by electromobility shift assay. If ethanol interferes in transcriptional activities, it will suggest that retinoic acid receptors might be mediating ethanol effects. Our studies demonstrated that Rho GTPases play a crucial role in differentiation of CGNs; and, exposure of ethanol in vivo affected the activation of these GTPases. It is plausible that impaired activation of receptors affects Rho GTPase signaling. To test this possibility retinoic acid receptor antagonists, known to prevent the activation, will be used. Studies will determine whether antagonists prevent the effects of ethanol on activation of Rho GTPases and protect CGNs against harmful effects of ethanol. To study the role of Rho GTPases we will use in vitro cell culture model. In this cell culture model CGNs will be exposed to ethanol (40 mM, 80 mM) in the presence of retinoic acid (100 nM). Whether, expression of constitutively active or dominant negative forms of Rho GTPases prevent toxic effects of ethanol on differentiation and survival of CGNs will be investigated.