Prevention of Alcohol Neurotoxicity by PDE4 Inhibitor

描述(申请人提供)：怀孕期间酗酒会严重影响胎儿，导致胎儿酒精综合症(FAS)。Fas是导致智力低下的主要原因，也是一个主要的公共卫生问题。在发育中的大脑中，神经元死亡是酒精最显著的病理效应之一。因此，确定能够预防酒精诱导的神经元死亡的药物是当务之急。我们已经证明，刺激一个特定的信号通路，cAMP-PKA-CREB通路，可以保护未成熟神经元免受酒精毒性。因为细胞内cAMP浓度的升高激活了这一途径，所以提高cAMP水平的药物可能会保护神经元免受酒精中毒。磷酸二酯酶通过降解cAMP来控制细胞内cAMP的浓度。在神经元内，cAMP被磷酸二酯酶4(PDE4)降解。抑制PDE4导致cAMP在神经元内积聚，并激活cAMP-PKA-CREB通路。因此，PDE4的抑制剂可以通过激活cAMP-PKA-CREB通路来保护神经元。罗利普兰是PDE4的特异性抑制剂。在啮齿动物神经元中，罗利普兰增加细胞内cAMP浓度并激活CREB。因此，我们假设罗利普兰可以通过增加细胞内cAMP水平来刺激cAMP-PKA-CREB途径来保护发育中的神经元免受酒精毒性。CAMP-PKA-CREB途径通过转录因子CREB发挥保护作用，通过改变基因表达促进细胞存活。神经元型一氧化氮合酶(NNOS)保护发育中的神经元免受酒精诱导的死亡，是cAMP-PKA-CREB通路的下游靶点。因此，我们假设罗利普兰和cAMP-PKA-CREB通路利用nNOS产生对酒精毒性的神经保护作用。在这项提案中，我们将确定罗利普兰是否在培养的神经元和活体中增加cAMP水平和激活CREB。我们将确定罗利普兰的预处理是否可以改善酒精诱导的培养神经元和发育中小鼠脑内神经元的死亡。我们将研究罗利普兰是否增加了小脑颗粒细胞和浦肯野细胞中nNOS的表达。我们将通过检测罗利普兰在野生型和nNOS-/-神经元中的促进存活作用，来确定nNOS在罗利普兰保护作用中的重要性。因此，这些研究将探讨罗利普兰作为神经保护剂对抗酒精神经致畸的有效性和机制。 公共卫生相关性：怀孕期间酗酒会对胎儿产生深远影响，并导致胎儿酒精综合症(Fas)，这是导致智力低下的主要原因，也是一个重大的公共卫生问题。我们发现，一种特殊的细胞内信号通路，称为cAMP通路，可以保护实验室培养的神经元免受酒精毒性。在这项研究中，我们建议用欧洲和日本使用的抗抑郁药物罗利普兰来治疗小鼠，以刺激cAMP途径，保护它们的脑细胞免受酒精中毒。

DESCRIPTION (provided by applicant): Alcohol abuse during pregnancy can profoundly affect the fetus, resulting in fetal alcohol syndrome (FAS). A leading cause of mental retardation, FAS is a major public health problem. In the developing brain, neuronal death is one of alcohol's most prominent pathologic effects. Thus, identification of agents that can prevent alcohol-induced neuronal death is a high priority. We have demonstrated that stimulation of a particular signaling pathway, the cAMP-PKA-CREB pathway, protects immature neurons against alcohol toxicity. Because elevated intracellular cAMP concentrations activate this pathway, agents that increase cAMP levels may protect neurons against alcohol toxicity. Phosphodiesterases control the intracellular concentration of cAMP by degrading it. Within neurons, cAMP is degraded by phosphodiesterase 4 (PDE4). Inhibition of PDE4 leads to the accumulation of cAMP and activation of the cAMP-PKA-CREB pathway in neurons. Thus, an inhibitor of PDE4 could protect neurons by activating the cAMP-PKA- CREB pathway. Rolipram is a specific inhibitor of PDE4. In rodent neurons, rolipram increases intracellular cAMP concentrations and activates CREB. Thus, we hypothesize that rolipram can protect developing neurons against alcohol toxicity by increasing intracellular cAMP levels to stimulate the cAMP-PKA-CREB pathway. The cAMP-PKA- CREB pathway exerts its protective actions via the transcription factor CREB, which alters gene expression to promote cellular survival. Neuronal nitric oxide synthase (nNOS) protects developing neurons against alcohol-induced death and is a downstream target of the cAMP-PKA-CREB pathway. Thus, we hypothesize that Rolipram and the cAMP-PKA-CREB pathway utilize nNOS to produce their neuroprotective effects against alcohol toxicity. In this proposal, we will determine whether rolipram increases cAMP levels and activates CREB, both in cultured neurons and in vivo. We will determine whether pretreatment with rolipram can ameliorate alcohol-induced death of cultured neurons and of neurons within the brains of developing mice. We will examine whether rolipram increases the expression of nNOS in cerebellar granule cells and Purkinje cells. We will determine the importance of nNOS for rolipram's protective effects, by examining rolipram's survival-promoting actions in wild type and nNOS-/- neurons. Thus, these studies will examine the efficacy and mechanism of rolipram as a neuroprotective agent against alcohol neuroteratogenicity. PUBLIC HEALTH RELEVANCE: Alcohol abuse during pregnancy can profoundly affect the fetus and cause fetal alcohol syndrome (FAS), which is a leading cause of mental retardation and a major public health problem. We have discovered that a particular intracellular signaling pathway, referred to as the cAMP pathway, can protect cultured neurons in the laboratory against alcohol toxicity. In this study we propose to treat mice with rolipram, an antidepressant drug used in Europe and Japan, to stimulate the cAMP pathway to protect their brain cells against alcohol toxicity.