Phosphorylation and the CNS Actions of Ethanol

描述(申请人提供)：我们研究的长期目标是验证这样一个假设，即翻译后修饰，如磷酸化，是酒精暴露下大脑区域特异性反应的主要贡献者。在第一轮资助中，我们发现N-甲基-D-天冬氨酸受体(NMDAR)对乙醇的敏感性是由非受体酪氨酸激酶Fyn(激活)和Src(抑制)的激活状态决定的，导致NMDAR的NR2B(Fyn)和NR2a(Src)亚单位的磷酸化状态发生变化。我们发现，这些信号变化导致NMDAR活性在切片制备和体内急性暴露于乙醇期间和之后发生深刻变化。最近，我们在背侧纹状体中发现了Fyn和NR2B-NMDAR在酒精暴露后长期促进含有NR2B的NMDAR介导的活动和酒精饮酒行为中的重要作用。NMDAR是乙醇在大脑中作用的重要中介，我们的结果表明，背侧纹状体Fyn激活状态的变化有助于突触可塑性的异常，这可能是行为表型发展的基础，如消费酒精的倾向。利用分子、生化、电生理和行为学等方法，我们试图阐明乙醇在大鼠背侧纹状体内激活Fyn的机制及其后果。由于FYN活性受磷酸化的正负调节，我们将确定酪氨酸磷酸酶PTPalpha和STEP在乙醇对体内FYN激活、NR2B磷酸化和长期促进NMDAR活性的作用中的作用。此外，我们计划研究Fyn背侧纹状体激活的可能的生理后果。最后，我们将确定Fyn、PTPalpha和STEP对大鼠可操纵性乙醇自我给药的贡献。我们的长期目标是了解与酒精成瘾相关的疾病状态发展的分子机制。确定新的细胞内靶标是酒精成瘾的媒介，这是开发酒精相关障碍的新干预措施的重要未来方向。 公共卫生相关性：酗酒是一种毁灭性的疾病，仅在美国每年就有大约1400万人受到影响。不幸的是，目前只有有限数量的药物被FDA批准用于治疗与疾病相关的不良表型。因此，迫切需要确定药物开发的新靶点。离子通道NMDAR作为治疗酒精中毒和其他神经系统疾病的潜在药物靶点引起了人们的极大兴趣。这些研究产生的结果可能使我们能够开发出调节大脑特定区域通道亚群活动的药物，从而可能导致开发新的、选择性的靶点来治疗酒精滥用。

DESCRIPTION (provided by applicant): The long-term goal of our research is to test the hypothesis that post-translational modifications such as phosphorylation are major contributors to brain region-specific responses to alcohol exposure. In the first round of funding we found that the sensitivity of the N-methyl-D-Aspartate receptor (NMDAR) to ethanol in specific brain regions is determined by the activation state of the non-receptor tyrosine kinases Fyn (activation) and Src (inhibition), leading to changes in the phosphorylation state of the NR2B (Fyn) and NR2A (Src) subunits of the NMDAR. We found that these signaling alterations lead to profound changes in the activity of the NMDAR during and after acute exposure to ethanol in slice preparations and in vivo. More recently, we identified an important role for Fyn and NR2B-NMDAR in the dorsal striatum in the long-lasting facilitation of NR2B-containing NMDAR-mediated activity after ethanol exposure and in ethanol drinking behavior. The NMDAR is an important mediator of ethanol's actions in the brain, and our results suggest that changes in the activation state of the Fyn in the dorsal striatum contribute to aberrant synaptic plasticity that may underlie the development of behavioral phenotypes such as the propensity to consume ethanol. Using molecular, biochemical, electrophysiological and behavioral approaches, we propose to elucidate the mechanism leading to, and are the consequences of, ethanol-mediated Fyn activation in the dorsal striatum of rats. As Fyn activity is both positively and negatively regulated by phosphorylation, we will determine the contribution of the tyrosine phosphatases, PTPalpha and STEP, to ethanol's actions on Fyn activation, NR2B phosphorylation and long-term facilitation of NMDAR activity in vivo. Additionally, we plan to investigate possible physiological consequences of dorsal striatal activation of Fyn. Finally, we will determine the contribution of Fyn, PTPalpha, and STEP to operant ethanol self-administration in rats. Our long-term goal is to understand the molecular mechanisms that contribute to the development of disease states associated with alcohol addiction. Identification of new intracellular targets that are mediators of alcohol addiction is an essential future direction for the development of novel interventions for alcohol related disorders.7 PUBLIC HEALTH RELEVANCE: Alcoholism is a devastating disease that affects approximately 14 million people per year in the USA alone. Unfortunately, only limited numbers of drugs are currently approved by the FDA to treat adverse phenotypes associated with the disease. Therefore, there is a great need to identify novel targets for medication development. The ion channel NMDAR has attracted great interest as a potential drug target to treat alcoholism and other neurological diseases. Results generated from these studies may enable us to develop agents that modulate the activity of a subset population of the channel in specific areas of the brain, and therefore may lead to the development of novel, selective targets to treat alcohol abuse.