NO-mediated neuroprotection against alcohol: mechanism and potential therapy

描述(由申请人提供)：胎儿接触酒精会严重和永久性地损害发育中的大脑，并可能导致胎儿酒精谱系障碍(FASD)。尽管公众宣传活动和卫生局局长的警告，许多妇女继续在怀孕期间滥用酒精，FASD仍然是导致智力低下的最常见原因之一。仅在美国，FASD病例估计每年给经济造成54亿美元的损失。酒精扰乱大脑发育的最重要方式之一是通过杀死神经元。神经元丢失是FASD儿童小脑、行为问题和学习障碍的主要原因。利用药理学、分子和遗传学手段，我们发现由一氧化氮(NO)介导的一种特殊的信号通路可以保护发育中的神经元免受酒精毒性。利用体外系统，我们发现药物激活NO-cGMP-PKG通路可以防止酒精诱导的神经元死亡。相反，阻断这条通路会加重神经元的损失。编码神经元型一氧化氮合酶(NNOS)的基因过表达，在神经元内产生一氧化氮，保护神经元免受酒精诱导的死亡。这项建议的目的是将我们的发现推进到体外环境之外，以确定该途径是否可以类似地保护体内发育中的大脑免受酒精毒性的影响，并可用于治疗干预。第一个目的是研究过表达nNOS基因是否可以改善酒精诱导的神经元死亡和行为缺陷。这一目标的研究将检验在浦肯野细胞中异位表达nNOS是否可以保护这些细胞免受酒精诱导的细胞死亡和防止酒精诱导的小脑功能障碍。浦肯野细胞是酒精中毒最脆弱的神经元群体。第二个目的是研究NO-cGMP-PKG通路发出神经保护信号的机制。这方面的研究将确定NO-cGMP-PKG通路是否通过抑制氧化应激来保护发育中的神经元免受酒精诱导的死亡。第三个特定目的是检查磷酸二酯酶2抑制剂Bay60-7550是否可以通过激活NO-cGMP-PKG途径来保护发育中的大脑免受酒精中毒，从而抑制氧化应激。这项研究的完成可能有助于阐明酒精诱导的氧化应激在FASD中的作用，并可能确定NO-cGMP-PKG通路是预防FASD的有希望的靶点。

DESCRIPTION (provided by applicant): Fetal exposure to alcohol can severely and permanently damage the developing brain and can lead to fetal alcohol spectrum disorder (FASD). Despite public information campaigns and warnings from the Surgeon General, many women continue to abuse alcohol during pregnancy, and FASD remains one of the most common causes of mental retardation. In the US alone, FASD cases are estimated to cost the economy $5.4 billion annually. One of the most important ways in which alcohol disrupts brain development is by killing neurons. Loss of neurons contributes strongly to the microencephaly, behavior problems, and learning deficits in children with FASD. Utilizing pharmacological, molecular, and genetic approaches, we have discovered that a particular signaling pathway mediated by nitric oxide (NO) can protect developing neurons against alcohol toxicity. Using in vitro systems, we have found that pharmacological activation of the NO-cGMP-PKG pathway can prevent alcohol-induced neuronal death. Conversely, blockade of this pathway worsens neuronal losses. Over-expression of the gene encoding neuronal nitric oxide synthase (nNOS), which produces NO within neurons, protects neurons against alcohol-induced death. The objective of this proposal is to advance our findings beyond the in vitro setting, to determine whether the pathway can similarly protect the developing brain in vivo against alcohol toxicity and can be used as a therapeutic intervention. The first aim examines the possibility that overexpression of the nNOS gene can ameliorate alcohol- induced neuronal death and behavioral deficits. Studies in this aim will examine whether ectopically expressed nNOS in Purkinje cells, the most vulnerable neuronal population to alcohol toxicity, can protect those cells against alcohol-induced cell death and prevent alcohol-induced cerebellar dysfunction. The second aim examines the mechanism by which the NO-cGMP-PKG pathway signals its neuroprotective effects. Studies in this aim will determine whether the NO-cGMP-PKG pathway protects developing neurons against alcohol-induced death by inhibiting oxidative stress. The third specific aim examines whether the phosphodiesterase 2 inhibitor, Bay60-7550, can protect the developing brain against alcohol toxicity by activating the NO-cGMP-PKG pathway, thus inhibiting oxidative stress. Completion of this research may shed light on the role of alcohol-induced oxidative stress in FASD and may identify the NO-cGMP-PKG pathway as a promising target for preventive interventions against FASD.