Binge alcohol-induced neurodamage and omega-3 DHA Protection

描述(申请人提供)：慢性酒精(酒精)滥用，尤其是暴饮型酒精中毒，通过尚未解决的机制导致脑损伤。成年大鼠通过反复酗酒暴露在高血酒精水平下，会导致海马区(HC)和内嗅皮层(EC)神经变性，可以想象，这种变性是脑水肿、神经炎症和氧化应激(OXS)的下游。我们与NIAAA的Hee-yong Kim博士等人合作的研究利用了成年雄性大鼠模型和平行的体外模型(大鼠器官型HC-EC切片培养)，提供了证据表明，酗酒通过先前未知的神经炎症途径激活OXS和与水肿相关的磷脂连接的促氧化途径。可能在不同的细胞中被激活的特定蛋白包括水通道蛋白-4(AQP4)、聚(腺苷二磷酸-核糖)聚合酶-1(PARP-1)和某些磷脂酶A2(PLA2)亚型，这些亚型可以动员过量的ω6花生四烯酸(ARA)生成OXs。此外，ω-3-二十二碳六烯酸(DHA)可能通过几种机制在培养物中具有有效的抗炎和神经保护作用。我们将继续与Kim博士的实验室合作，通过扩展的神经炎症假说探索这些因素之间的信号转导：PARP-1和AQP4是关键的“第一反应”蛋白质，调控着不同的酗酒依赖神经炎症通路-PARP-1感知酒精产生的OXS最初的DNA氧化损伤/链断裂，变得高度升高，并触发PLA2衍生的过度ARA释放；而AQP4增加促进脑水肿、DHA丢失和Akt减少，从而引起更多的OXS，这两条途径都合并在神经损伤中；补充DHA然后通过其可能涉及其自由形式和脂结合形式的机制来促进这些途径的抗氧化抑制和神经保护。提出了三个目标：前两个目标使用酗酒处理的成人HC-EC切片培养(我们目前的研究中新开发的)，而目标3外推体外结果以符合 体内模型，成年雄性大鼠的重复暴饮式中毒(Majchrowicz模型)--这是一种神经退行性变的模型，首次在我们的实验室中出现，目前正被NIAAA的研究人员使用。目的1研究酒精如何调控PARP-1、AQP4和PLA2，并利用特定的抑制剂和RNA干扰技术研究PARP-1、AQP4和PLA2如何相互作用以及与神经退行性变有关；AIM 2探测补充DHA的机制，从而排除酒精依赖的神经炎性磷脂机制，同时实现神经保护，包括其膜磷脂掺入以及其代谢到保护性解析剂和神经保护素-1；以及AIM 3将目标1和2的结果扩展到大鼠酗酒模型，检验我们的酗酒/PARP-1“级联”假说和DHA在体内的神经保护机制。这一结果可能有助于深入了解新的神经炎性/神经毒性酒精机制，并阐明补充DHA作为酒精中毒治疗的辅助治疗在认知上可能是有益的。

DESCRIPTION (provided by applicant): Chronic alcohol (ethanol) abuse, especially binge-type alcoholism, causes brain damage by unresolved mechanisms. Adult rats exposed to high blood alcohol levels via repeated binges incur hippocampal (HC) and entorhinal cortical (EC) neurodegeneration that is conceivably downstream of brain edema, neuroinflammation and oxidative stress (OxS). Our collaborative studies with Dr. Hee-Yong Kim at NIAAA and others, which have utilized an adult male rat model and a parallel in vitro model (rat organotypic HC-EC slice cultures), provide evidence that binge alcohol activates OxS and edema-related, phospholipid-linked pro-oxidative pathways via previously unacknowledged neuroinflammatory routes. The specific proteins activated, quite likely in different cells, include aquaporin-4 (AQP4, poly (ADP-ribose) polymerase-1 (PARP-1), and certain phospholipase A2 (PLA2) isoforms that mobilize excessive ω6 arachidonic acid (ARA) to generate OxS. Furthermore, ω3 docosahexaenoic acid (DHA), possibly via several mechanisms, is potently anti-inflammatory and neuroprotective in the cultures. In continued collaboration with Dr. Kim's laboratory, we will explore the signaling between these factors with an expanded neuroinflammatory hypothesis: PARP-1 and AQP4 are the critical "1st response" proteins that govern distinct binge alcohol-dependent neuroinflammatory pathways-with PARP-1 sensing initial DNA oxidative damage/strand breaks from alcohol-generated OxS, becoming hyper-elevated, and triggering PLA2-derived excessive ARA release, while the increased AQP4 promotes brain edema, DHA loss and Akt reductions to elicit more OxS, with both paths coalescing in neurodamage; supplemented DHA then promotes antioxidative suppression of these pathways and neuroprotection via mechanisms that may involve both its free and lipid-bound forms. Three aims are proposed: the first two aims use binge alcohol-treated adult-age HC-EC slice cultures (newly developed in our current studies), while aim 3 extrapolates in vitro results to a fitting in vivo model, repetitive binge intoxication of adult male rats (Majchrowicz model)-a model in which neurodegeneration was first characterized in our laboratories and is being used by NIAAA investigators. AIM 1 examines how PARP-1, AQP4 and PLA2 are regulated by alcohol and relate to each other and to neurodegeneration, using specific inhibitors and RNA interference; AIM 2 probes supplemental DHA's mechanisms that preclude alcohol- dependent neuroinflammatory phospholipid mechanisms while achieving neuroprotection, including its membrane phospholipid incorporation as well as its metabolism to protective resolvins and neuroprotectin-1; and AIM 3 extends results from Aim 1 and 2 experiments to the rat binge model, examining our binge alcohol/PARP-1 "cascade" hypothesis and DHA's neuroprotective mechanism in vivo. The results could shed insight into novel neuroinflammatory/neurotoxic alcohol mechanisms, and clarify how DHA supplementation might be cognitively beneficial as adjunct therapy in alcoholism treatment.