Ethanol, Insulin/IGF Signaling and Neuronal Migration

在前一次赠款期间进行的研究表明，慢性妊娠 暴露在乙醇中会损害发育中的大脑中的神经元迁移，而这种影响 乙醇与关键靶基因天冬氨酸的表达和功能降低有关。 肝素羟基酶(AAH)，它在细胞运动中具有被证明的作用。我们确认了3个 乙醇损伤AAH表达和功能的机制：1)酒精暴露引起 胰岛素和胰岛素样生长因子(IGF)抵抗，通过抑制下游信号传导 调节AAH mRNA的PI3K-Akt、Erk MAPK和CDK-5通路；2)乙醇增加 GSK-3的活性和高水平的GSK-3可能导致AAH磷酸化增加 使AAH更容易被Caspase降解；3)乙醇抑制AAH AAH促进细胞运动所需的催化活性。我们假设AAH 通过羟化Notch促进神经元的运动，Notch然后经历切割和 转位到细胞核，在那里它调节基因的表达。我们的首要目标是 说明酒精损害神经元迁移的机制，重点是GSK-2的作用 3？介导的磷酸化和伴随的AAH蛋白表达的抑制，催化 活跃性和能动性。具体目标1是表征GSK-3介导的效应 AAH的磷酸化对AAH蛋白表达、合成、降解和催化的影响 活动。具体目标2将研究GSK-3活性增加和磷酸化的作用 AAH作为乙醇损伤的AAH蛋白表达、AAH羟基酶活性、 以及神经元的运动性。具体目的3是评估GSK-3的磷酸化作用。 Aah对Notch信号的影响，并将这些影响与下游基因的损伤联系起来 在FASD中的表达和CNS神经元迁移。此外，由于初步 研究表明，AAH可以与Notch进行身体上的互动(这可能对 羟化)，我们将检测GSK-3对AAH磷酸化的影响。 AAH与Notch的相互作用、Notch裂解、Notch易位到细胞核，以及 下游刺激Notch调控的靶基因，例如Hes-1，p21/Waf-1，或 早老素-1。我们计划利用体内和体外分级酒精暴露模型来模拟 真实的生活条件。我们预计这些调查将产生关于 乙醇抑制AAH表达和功能的机制 FASD患者中枢神经系统神经元迁移受损的后果。

Studies performed during the previous grant period established that chronic gestational exposure to ethanol impairs neuronal migration in the developing brain, and that this effect of ethanol is associated with reduced expression and function of a critical target gene, aspartyl- aparaginyl-¿-hydroxylase (AAH), which has a demonstrated role in cell motility. We identified 3 mechanisms of ethanol-impaired AAH expression and function: 1) ethanol exposure causes insulin and insulin-like growth factor (IGF) resistance, inhibiting downstream signaling through PI3K-Akt, Erk MAPK, and Cdk-5 pathways that regulate AAH mRNA; 2) ethanol increases GSK-3¿ activity, and high levels of GSK-3¿ cause increased AAH phosphorylation, possibly rendering AAH more susceptible to degradation by Caspases; and 3) ethanol inhibits AAH's catalytic activity which is required for AAH to promote cell motility. We hypothesize that AAH promotes neuronal motility by hydroxylating Notch, which then undergoes cleavage and translocation to the nucleus where it regulates gene expression. Our overarching goal is to demonstrate mechanisms of ethanol-impaired neuronal migration, focusing on the role of GSK- 3¿-mediated phosphorylation and attendant inhibition of AAH protein expression, catalytic activity, and motility. Specific Aim 1 is to characterize the effects of GSK-3¿-mediated phosphorylation of AAH on AAH protein expression, synthesis, degradation, and catalytic activity. Specific Aim 2 will examine the role of increased GSK-3¿ activity and phosphorylation of AAH as a mediator of ethanol-impaired AAH protein expression, AAH hydroxylase activity, and neuronal motility. Specific Aim 3 is to evaluate the effects of GSK-3¿ phosphorylation of AAH on Notch signaling, and link those effects to the impairments in downstream gene expression and CNS neuronal migration that occur in FASD. Moreover, since preliminary studies showed that AAH can physically interact with Notch (which may be important for hydroxylation), we will examine the effects of GSK-3¿-phosphorylation of AAH on the physical interactions between AAH and Notch, Notch cleavage, Notch translocation to the nucleus, and downstream stimulation of the Notch-regulated target genes, e.g. Hes-1, p21/Waf-1, or presenilin-1. We plan to utilize graded in vivo and in vitro ethanol exposure models to mimic real life conditions. We expect these investigations to generate new information about the mechanisms by which ethanol inhibits AAH expression and function, and reveal the consequences with respect to the impairments in CNS neuronal migration that occur in FASD.