Ethanol, Insulin/IGF Signaling and Neuronal Migration

描述（由申请人提供）：在之前的资助期间进行的研究表明，妊娠期长期暴露于乙醇会损害发育中的大脑中的神经元迁移，并且乙醇的这种影响与关键靶基因天冬氨酸-天冬氨酸-2-羟化酶（AAH）的表达和功能降低有关，该基因在细胞运动中具有已证实的作用。我们确定了乙醇损伤AAH表达和功能的3种机制：1)乙醇暴露导致胰岛素和胰岛素样生长因子（IGF）抵抗，抑制通过PI3K-Akt、Erk MAPK和Cdk-5途径调节AAH mRNA的下游信号传导；2)乙醇增加GSK-32活性，高水平的GSK-32导致AAH磷酸化增加，可能使AAH更容易被Caspases降解；3)乙醇抑制AAH促进细胞运动所必需的催化活性。我们假设AAH通过羟基化Notch来促进神经元运动，Notch随后被切割并易位到细胞核，在那里它调节基因表达。我们的总体目标是证明乙醇损伤神经元迁移的机制，重点关注GSK- 32介导的磷酸化和随之而来的AAH蛋白表达、催化活性和运动性抑制的作用。具体目的1是表征gsk -32介导的AAH磷酸化对AAH蛋白表达、合成、降解和催化活性的影响。特异性目的2将检验GSK-32活性的增加和AAH磷酸化作为乙醇损伤的AAH蛋白表达、AAH羟化酶活性和神经元运动的中介的作用。具体目的3是评估GSK-32磷酸化AAH对Notch信号的影响，并将这些影响与FASD中下游基因表达受损和中枢神经系统神经元迁移联系起来。此外，由于初步研究表明AAH可以与Notch相互作用（这可能对羟基化很重要），我们将研究AAH的gsk -32磷酸化对AAH与Notch之间的物理相互作用、Notch切割、Notch易位到细胞核以及Notch调控靶基因（如Hes-1、p21/Waf-1或早老素-1）的下游刺激的影响。我们计划利用分级体内和体外乙醇暴露模型来模拟现实生活条件。我们希望这些研究能够产生关于乙醇抑制AAH表达和功能的机制的新信息，并揭示在FASD中发生的中枢神经系统神经元迁移损伤的后果。公共卫生相关性：在美国，怀孕期间酗酒是最常见的可预防的先天性认知运动缺陷的原因，从智力迟钝到注意缺陷多动障碍。酒精诱导的认知运动障碍与中枢神经系统（CNS）神经元存活、生长、运动和可塑性的主要障碍有关。我们的研究重点是乙醇如何抑制天冬氨酸-天冬氨酸-2-羟化酶（AAH）的表达和功能，这是一种在发育过程中调节神经元迁移的重要分子/酶。我们的研究是新颖的，可能为早期发现和治疗人类子宫内乙醇暴露引起的先天性中枢神经系统异常提供新的策略。

DESCRIPTION (provided by applicant): 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-2-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-32 activity, and high levels of GSK-32 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- 32-mediated phosphorylation and attendant inhibition of AAH protein expression, catalytic activity, and motility. Specific Aim 1 is to characterize the effects of GSK-32-mediated phosphorylation of AAH on AAH protein expression, synthesis, degradation, and catalytic activity. Specific Aim 2 will examine the role of increased GSK-32 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-32 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-32-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. PUBLIC HEALTH RELEVANCE: In the USA, alcohol abuse during pregnancy is the most common preventable cause of congenital cognitive-motor deficits that range from mental retardation to attention deficit hyperactivity disorders. Alcohol-induced cognitive-motor impairments are associated with major disturbances in neuronal survival, growth, motility, and plasticity in the central nervous system (CNS). Our research focuses on how ethanol inhibits expression and function of aspartyl-aparaginyl-2-hydroxylase (AAH), an important molecule/enzyme that regulates neuronal migration during development. Our research is novel and could lead to new strategies for early detection and treatment of congenital CNS abnormalities caused by in utero ethanol exposure in humans.