Ethanol-induced disruption of kinase signaling pathways in brain development

摘要 胎儿酒精综合症(FAS)是美国智力残疾的主要原因之一。这个 美国疾病控制与预防中心估计，每1000名活产儿中有0.2-1.5名患有FASD，这是一种以紊乱为特征的综合征 胎脑发育与出生后智力残疾(ID)连接中断，包括更改 树突状结构、轴突寻径和白质束是Fas常见的表现，被认为是 是ID的主要贡献者。然而，酒精的细胞和生物靶点是多样的，目前还不清楚 是否有共同的潜在分子机制产生这些干扰。身份识别 共同的分子机制(S)将使我们能够更深入地了解这种疾病，为研究提供参考 遗传易感性，并为神经保护策略提供分子靶点。这项建议符合我们的要求 研究发现，急性乙醇(Etoh)暴露破坏了胚胎皮质神经元中的Src激酶活性。SRC是一种 位于多个信号通路中心位置的关键的非受体酪氨酸激酶，包括 控制脑层形成和树突发生的Reelin-Dab1信号通路。我们发现急性 乙醇暴露可激活Src并诱导许多蛋白质的磷酸化，包括必不可少的Dab1 Reelin信号通路中的接头蛋白。值得注意的是，这种磷酸化的戏剧性增加 随后是持续的去磷酸化反应，其中Reelin效应器的磷酸化包括 Dab1、Src本身和肌动蛋白切断蛋白n-cofilin恢复到基线水平或更低。在扩展期间 去磷酸化阶段，Reelin信号通路不再能被体外应用ITS激活 里兰，这是雷林。在本提案的目标1中，我们将确定Reelin-Dab1沉默在体内是否发生在 孕妇服用乙醇的剂量。然后我们将确定Src的遗传缺陷是否会阻止 磷酸化和去磷酸化反应。从基因上建立关键的激酶，启动 体内的乙醇反应将对未来的神经保护努力至关重要。我们和其他人已经证明了 Reelin-Dab1信号控制高尔基体在树突形成中的部署。在AIM 2中，我们将检查源 激活和失活会扰乱高尔基体的位置和功能。被破坏的高尔基人功能预计会 影响膜添加、糖基化、分泌和适当表达的许多蛋白质， 对神经发生和神经功能的潜在长期负面影响。在AIM 3中，我们将 确定EphA3信号通路是否同样受到Src失调的干扰。EphA3是一种 受体酪氨酸激酶是轴突和白质束发育所必需的。我们确定了 EphA3的活化位点作为乙醇诱导的Src失调的靶点增加了EphA3 激活然后沉默可能导致FASD相关的白质干扰。总而言之，这些 研究将确定乙醇依赖的Src失调在改变发育信号中的作用 在对大脑发育至关重要的途径上。

ABSTRACT Fetal Alcohol Syndrome (FAS) is one of the leading causes of intellectual disability in the United States. The CDC estimates that 0.2-1.5 per 1000 live births are children with FASD, a syndrome characterized by disrupted fetal brain development and postnatal intellectual disability (ID). Disrupted connectivity including altered dendritic structure, axonal pathfinding and white matter tracts are common findings in FAS and are thought to be major contributors to ID. However, the cellular and biological targets of alcohol are diverse and it is not clear whether there are common underlying molecular mechanisms producing these disruptions. Identification of common molecular mechanism(s) would enable a deeper understanding of this disorder, inform studies of genetic susceptibilities and provide molecular targets for neuroprotective strategies. This proposal pursues our finding that acute ethanol (EtOH) exposure disrupts Src kinase activity in embryonic cortical neurons. Src is a critical non-receptor tyrosine kinase that sits at central positions in multiple signaling pathways including the Reelin-Dab1 signaling pathway which controls brain layer formation and dendritogenesis. We found that acute EtOH exposure activates Src and induces phosphorylation of many proteins including Dab1, an essential adaptor protein in the Reelin-signaling pathway. Remarkably, this dramatic increase in phosphorylation is followed by a sustained dephosphorylation response in which the phosphorylation of Reelin effectors including Dab1, Src itself and the actin severing protein n-cofilin return to baseline levels, or below. During the extended dephosphorylation phase, the Reelin-signaling pathway can no longer be activated by in vitro application of its ligand, Reelin. In AIM 1 of this proposal, we will determine whether Reelin-Dab1 silencing occurs in vivo after maternal dosing with EtOH. We will then determine whether genetic deficiency in Src prevents the phosphorylation and dephosphorylation responses. Genetically establishing the critical kinase that initiates the EtOH response in vivo will be essential for future neuroprotective efforts. We and others have shown that Reelin-Dab1 signaling controls Golgi-deployment in the forming dendrite. In AIM 2 we will examine whether Src activation and inactivation disrupts Golgi location and function. Disrupted Golgi function would be expected to impact membrane addition, glycosylation, secretion and appropriate expression of many proteins, with potential long term negative consequences on neuritogenesis and neuronal function. In AIM 3 we will determine whether the EphA3 signaling pathway is similarly disrupted by Src dysregulation. EphA3 is a receptor tyrosine kinase that is required for axonal and white matter tract development. We identified the activation site of EphA3 as a target of EtOH-induced Src dysregulation raising the possibility that EphA3 activation and then silencing may contribute to FASD-related white matter disruptions. Collectively, these studies will determine the contribution of EtOH-dependent Src dysregulation to altered developmental signaling in pathways critical for brain development.