Alcohol Antagonists

描述（由申请人提供）： 酒精使用障碍（AUD）在退伍军人中常见，并产生严重的发病率和死亡率。酒精会在整个生命周期中损害神经系统，导致成年人严重的神经系统疾病和妊娠期接触酒精的儿童的胎儿酒精谱系障碍。这两个生命阶段都与退伍军人有关，因为退伍军人在育龄妇女中所占比例越来越大。该提案扩展了正在进行的工作，旨在确定药物，以防止或逆转酒精对生命各个阶段神经系统的毒性作用。酒精在大脑中引起发育毒性，部分是通过阻断L1神经细胞粘附分子介导的细胞粘附。酒精阻断L1粘附后L1的多种作用，包括L1介导的轴突生长（L1 MAO）和L1激活Src家族激酶（SFK）信号传导。酒精还通过SFK阻断另外两个重要的生长因子：netrin-1和GDNF的信号传导。酒精与L1胞外结构域（L1-ECD）上的结合口袋相互作用，L1-ECD是从细胞膜突出并与其他细胞上的L1分子结合的分子部分。L1胞质结构域（L1-CD）中特定残基的磷酸化不会阻断L1粘附，但可能通过改变L1-ECD中的醇结合口袋的构象而消除了乙醇对L1粘附的抑制。重要的是，某些小分子和肽也可以阻断酒精对L1粘附的抑制，这些酒精拮抗剂可以防止酒精对发育中大脑的损伤。小肽NAPVSIPQ（NAP）保护神经系统免受各种各样的伤害，包括产前酒精暴露。NAP在飞摩尔浓度（低至每四倍体一份）下阻断酒精对L1粘附、L1 MAO和L1信号传导的影响。NAP如此强大的作用机制尚不清楚。在这里，我们将研究NAP如何阻断酒精在小脑颗粒神经元（CGN）和小脑皮质切片培养物中的作用，这些细胞来自出生后，青少年和成年大鼠[以及出生后早期酒精暴露的体内模型]。有三个目标：1。以确定NAP是否通过调节胞质结构域的磷酸化来阻断乙醇对L1粘附的抑制; 2.了解NAP是否阻断L1、netrin-1和GDNF对SFK激活的乙醇抑制; 3.以确定NAP是否在整个生命周期内预防或逆转酒精小脑神经毒性。在不存在和存在酒精、L1、netrin-1和GDNF的情况下，将培养物暴露于酒精不同的时间段。我们将系统地突变L1-CD中的磷酸化位点，以模拟这些位点的永久磷酸化或去磷酸化。然后将这些构建体用酒精和NAP处理，以鉴定使NAP不能阻断酒精作用的特定突变。然后，我们将测试NAP是否调节激酶或磷酸酶的活性，这些激酶或磷酸酶作用于所确定的位点。酒精通过阻断多种生长因子对SFK的激活来抑制CGN中轴突的生长。我们将确定NAP是否阻断酒精对L1，netrin-1和GDNF在出生后第7天（P7）大鼠的成熟CGN和小脑切片中激活SFK的影响。然后，我们将确定NAP是否拮抗酒精对出生后早期（P7），青少年（P40）和成人（P80）小脑切片中神经元存活，神经元网络和生长因子信号传导的影响[出生后早期暴露于乙醇的大鼠和小鼠小脑]。这些研究将有助于描述NAP（一种有效的酒精拮抗剂）的作用机制，并为开发预防和逆转大脑中酒精神经毒性的治疗方法奠定基础。

DESCRIPTION (provided by applicant): Alcohol use disorders (AUD) occur commonly in Veterans and produce serious morbidity and mortality. Alcohol damages the nervous system across the lifespan, leading to crippling neurological disorders in adults and fetal alcohol spectrum disorders in children with gestational exposure to alcohol. Both stages of life are relevant to Veterans, since returning Veterans comprise a growing proportion of women of childbearing age with AUDs. This proposal extends ongoing work directed at identifying medications that will prevent or reverse the toxic effects of alcohol on the nervous system at all stages of life. Alcohol causes developmental toxicity in the brain partly by blocking cell adhesion mediated by the L1 neural cell adhesion molecule. Alcohol blocks diverse actions of L1 that follow L1 adhesion, including L1-mediated axon outgrowth (L1MAO) and L1 activation of Src Family Kinase (SFK) signaling. Alcohol also blocks signaling through SFK for two other important growth factors: netrin-1 and GDNF. Alcohol interacts with a binding pocket on the extracellular domain of L1 (L1-ECD) - the portion of the molecule that protrudes from the cell membrane and binds to L1 molecules on other cells. Phosphorylation of specific residues in the L1 cytoplasmic domain (L1-CD) does not block L1 adhesion, but abolishes ethanol inhibition of L1 adhesion, presumably by altering the conformation of the alcohol binding pocket in the L1-ECD. Importantly, certain small molecules and peptides also block alcohol inhibition of L1 adhesion, and these alcohol antagonists prevent alcohol-induced damage to the developing brain. The small peptide NAPVSIPQ (NAP) protects the nervous system against a wide array of insults, including prenatal alcohol exposure. NAP blocks the effects of alcohol on L1 adhesion, L1MAO, and L1 signaling at femtomolar concentrations (as low as one part per quadrillion). The mechanism by which NAP acts so potently is unknown. Here we will investigate how NAP blocks the effects of alcohol in cultures of cerebellar granule neurons (CGN) and slices of cerebellar cortex from postnatal, adolescent, and adult rats [and in an in vivo model of early postnatal alcohol exposure.] There are three objectives: 1. to determine whether NAP blocks ethanol inhibition of L1 adhesion by modulating phosphorylation of the cytoplasmic domain; 2. to learn whether NAP blocks ethanol inhibition of SFK activation by L1, netrin-1, and GDNF; 3. to determine whether NAP prevents or reverses alcohol cerebellar neurotoxicity across the lifespan. Cultures will be exposed to alcohol for varying periods of time in the absence and presence of alcohol, L1, netrin-1, and GDNF. We will systematically mutate phosphorylation sites in the L1-CD to simulate permanent phosphorylation or dephosphorylation of these sites. These constructs will then be treated with alcohol and NAP to identify specific mutations that render NAP incapable of blocking the effects of alcohol. We will then test whether NAP modulates the activity of kinases or phosphatases that act at the identified sites. Alcohol inhibits axon outgrowth in CGNs by blocking the activatio of SFK by diverse growth factors. We will determine whether NAP blocks the effects of alcohol on the activation of SFK by L1, netrin-1, and GDNF in mature CGNs and cerebellar slices from postnatal day 7 (P7) rats. We will then determine whether NAP antagonizes the effects of alcohol on neuronal survival, neuronal networks, and growth factor signaling in slices from early postnatal (P7), adolescent (P40), and adult (P80) cerebellum and [in cerebellum from rats and mice exposed to ethanol during the early postnatal period.] These studies will help characterize the mechanism of action of NAP, a potent alcohol antagonist, and will lay the groundwork for the development of treatments to prevent and reverse alcohol neurotoxicity in the brain.