Ethanol, Insulin/IGF Signaling and Neuronal Migration

在上一个资助期进行的研究表明， 暴露于乙醇会损害发育中大脑的神经元迁移， 乙醇与一个关键靶基因的表达和功能降低有关， 肝素-β-羟化酶（AAH），其在细胞运动中具有已证实的作用。我们发现3 乙醇损伤AAH表达和功能的机制：1）乙醇暴露导致 胰岛素和胰岛素样生长因子（IGF）抵抗，抑制下游信号传导， 调节AAH mRNA的PI 3 K-Akt、Erk MAPK和Cdk-5通路; 2）乙醇增加 GSK-3 <$活性和高水平的GSK-3 <$导致AAH磷酸化增加，可能是 使AAH更容易被半胱天冬酶降解;和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 通过羟基化Notch促进神经元运动，然后Notch进行切割， 转运到细胞核，在那里它调节基因表达。我们的首要目标是 证明乙醇损伤神经元迁移的机制，重点是GSK的作用， 3-介导的磷酸化和伴随的AAH蛋白表达抑制，催化 活性和运动性。具体目的1是表征GSK-3介导的 AAH磷酸化对AAH蛋白表达、合成、降解和催化 活动具体目标2将检查GSK-3活性和磷酸化增加的作用 AAH作为乙醇损伤的AAH蛋白表达，AAH羟化酶活性， 和神经元运动性。具体目标3是评估GSK-3？磷酸化对 AAH对Notch信号传导的影响，并将这些影响与下游基因的损伤联系起来 表达和CNS神经元迁移。此外，由于初步 研究表明，AAH可以与Notch发生物理相互作用（这可能对 羟基化），我们将研究GSK-3 <$-磷酸化AAH对身体的影响。 AAH和Notch之间的相互作用，Notch切割，Notch易位到细胞核，和 下游刺激Notch调节的靶基因，例如Hes-1、p21/Waf-1或 早老素-1。我们计划利用分级的体内和体外乙醇暴露模型来模拟 真实的生活条件。我们希望这些调查能够产生关于 乙醇抑制AAH表达和功能的机制，并揭示了 FASD中发生的CNS神经元迁移损伤的后果。

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.