Mechanism of Alcohol Induced Neurotoxicity

描述(申请人提供)：过量摄入酒精会增加血液中的乙醇浓度，并导致大脑发育障碍，表现为胎儿酒精综合征(Fas)。出生时患有Fas的儿童面部特征异常，伴有轻度至严重的智力低下。小脑是大脑中受酒精影响最敏感的区域之一。然而，乙醇对小脑的有害影响的潜在机制在很大程度上是未知的。我们的初步研究表明，在活体条件下，中剂量乙醇(血液乙醇浓度40 mM)抑制分化，高剂量乙醇(血液乙醇浓度80 mM)诱导小脑颗粒神经元(CGN)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，这表明其他因素(S)也可能参与其中。进一步观察到，在体外条件下，乙醇(40 mM和80 mM)与生理浓度的维甲酸(100 NM)存在类似于体内条件下观察到的有害作用。在这些研究的基础上，我们的工作假设是乙醇对CGN分化和存活的有害影响是通过维甲酸介导的。为了验证这一假说，我们将确定(1)体内乙醇暴露是否影响维甲酸受体的激活，以及(2)维甲酸受体拮抗剂的应用是否对乙醇对CGN分化和存活的有害影响具有保护作用。为了解决这个问题，被广泛用作胎儿酒精综合症啮齿动物模型的小鼠(出生后第7天)将暴露在乙醇中。小脑颗粒神经元将被分离并用于拟议的研究。维甲酸受体的转录活性将通过电迁移率改变分析来研究。如果乙醇干扰转录活动，这将表明维甲酸受体可能介导乙醇效应。我们的研究表明，Rho GTP酶在CGN的分化过程中起着至关重要的作用；体内暴露于乙醇会影响这些GTP酶的激活。受体激活受损可能会影响Rho GTP酶信号转导。为了测试这种可能性，将使用维甲酸受体拮抗剂，已知的是阻止激活。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，我们将采用体外细胞培养模型。在这个细胞培养模型中，CGN将暴露在乙醇(40 mM，80 mM)和维甲酸(100 NM)的存在下。Rho GTP酶的结构活性或显性阴性形式的表达是否能阻止乙醇对CGN的分化和存活的毒性效应，将被研究。

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.