尼古丁暴露和成瘾对海马神经干细胞发育的影响及Epac调控机制

Cigarette smoking exerts a tremendous negative impact on physical and mental health of smokers. Nicotine in cigarette or tobacco is the primary reinforcing components for tobacco addiction. Cumulating evidence suggests that nicotine exposure and addiction impair the proliferation, differentiation and migration of neural stem cells, but the underlying mechanism is not fully understood. Exchange protein directly activated by cAMP(Epac) is a multidomain cAMP mediator fine-tuning of diverse biological functions. The discovery of Epac has ignited a new surge of research of novel cAMP properties independent of protein kinase A and cyclic nucleiotide-gated channels. Previously, we found that chronic nicotine treatment increased Epac1/2 (2 isoforms of Epac) and its downstream target Rap1 expression in mouse hippocampus, meanwhile another in vitro study indicated that inhibition of Epac enhanced the redistribution of skeletal protein F-actin in embryoic neural stem cells of mice. Accordingly, we hypothesize that Epac signaling implements nicotine addiction-related neural stem cell aberrant development, behavior and cognition via increase in Epac/Rap1 expression and abnormality in cellular skeletal protein. To test our hypothesis, we will first conduct in vitro cell culture of embryonic hippocampal stem cells and explore the direct influence of nicotine on the proliferation, differentiation and migration of cultured stem cells, as well as the impact of pharmacological or genetic downregulation of Epac protein expression on neuronal function recovery. To further test our hypothesis, we will conduct in vivo animal study by developing antenatal/prenatal nicotine exposure mouse model and nicotine self-administration model. The Epac and Rap1 expression in the hippocampal dentate gyrus, neural stem cell development, the morphology of new-born neurons , body behavior and cognition in the offspring of maternal nicotine exposure mice and nicotine-addicted mice developed by nicotine self-administration paradigm will be examined. Once identify the alteration of Epac to nicotine exposure and addiction, we will downregulate Epac expression in the dentate gyrus with Epac blocker or viral vector-carried small hairpin RNA targeting Epac, and investigate the functional recovery. This study will elucidate the mechanism of Epac signaling cascade in the development of nicotine addiction and may open a new perspective of developing therapeutic approach against nicotine addiction.

吸烟或尼古丁成瘾抑制神经干细胞发育和机体行为认知的机制尚不清楚，cAMP下游效应元件Epac调控神经干细胞发育。我们发现尼古丁暴露小鼠海马Epac/Rap1上调；抑制Epac后小鼠胚胎神经干细胞actin重新分布。我们假说认为，Epac信号介导尼古丁暴露导致的成瘾等行为认知异常，其中的机制与Epac/Rap1异常增加而致细胞骨架蛋白变化和神经干细胞发育异常有关。据此，首先体外观察尼古丁对小鼠神经干细胞发育的直接影响及抑制或下调Epac后神经干细胞功能的恢复；进一步在体研究尼古丁暴露孕鼠的子代和尼古丁自我给药成年小鼠海马齿状回Epac/Rap1表达、神经干细胞发育、新生神经元结构及整体行为认知的变化，抑制或下调Epac后神经干细胞与个体功能的改善。本研究将首次阐明神经干细胞Epac信号在尼古丁暴露和成瘾中的调控机制，丰富尼古丁成瘾理论，为干涉治疗提供新的方向。

烟草和环境中的尼古丁、尼古丁替代疗法通过母亲影响发育早期子代海马的神经形成及长期的行为与认知。尼古丁成瘾影响海马神经形成和可塑性的机制尚不清楚。cAMP下游效应元件 Epac 调控神经干细胞发育。为检测Epac信号在尼古丁暴露的发育期及成年海马的改变，1）我们使用尼古丁母孕饮水暴露模型，检测新生子代0,5,10,20天海马中nAChR/Epac1/2/Rap1/CREB信号改变。发现尼古丁发子代出生个数减少；20天尼古丁海马Epac1/2/pCREB下调明显；使用基因芯片发现尼古丁暴露20天海马下调明显的信号通路有9条，与细胞因子及受体、趋化因子、T细胞分化及MAPK信号通路有关，上调明显的有5条。2）发现母亲来源（孕前、怀孕期间、哺乳期）的尼古丁青春期雌性子代有明显焦虑行为，且有性别差异；首次发现来源于母亲的尼古丁发育期暴露，在撤除长期慢性尼古丁作用后，尼古丁青春期子代海马神经形成受到明显影响，有瞬时的短期内明显增加且排列错乱、未成熟神经元DCX+可塑性改变；新生神经细胞长期存活未见差异，但存活有性别差异；首次发现尼古丁青春期子代海马组织内小胶质细胞数目增加且形态发生改变，CX3CL1/CX3CR1信号上调；尼古丁成年子代行为与记忆与对照组比较未能检测到明显差异。3）尼古丁处理原代小鼠小胶质细胞后，TNF-α分泌减少。4）首次发现急性尼古丁暴露导致成年小鼠位置偏爱行为的海马Epac信号变化，上调海马Epac2信号，雄性Rap1和CREB磷酸化上调，而雌性Rap1上调、CREB磷酸化下调。提示将来靶向Epac的药物，需要考虑性别不同可能带来不同的结果。5）利用体外小鼠海马神经干细胞，增殖培养条件下，尼古丁处理增加NPCs活力。随着尼古丁处理时间及浓度的增加，a7AChR表达上调，Epac1、Rap1表达下调，pCREB上调；高浓度尼古丁处理随着时间的增加，Epac2表达上调，pCREB上调，PKA与对照无明显差异；随着尼古丁处理时间及浓度的增加，p-p38 MAPK/p38 MAPK、pERK/ERK上调，pAKT/AKT下调。诱导分化条件下尼古丁处理3天，a7AChR、Epac1/2、Rap1、PKA未见明显变化；处理7天，Epac2表达下调，DCX+细胞数目增加，形态发生明显变化。本研究将首次阐明神经干细胞 Epac 信号在尼古丁暴露和成瘾中的调控机制。