Ca2+信号通道在间歇低氧诱导认知功能障碍中的机制以及钙通道阻滞剂干预作用的探讨

Cognitive impairment is frequently associated with the patients of obstructive sleep apnea syndrome (OSAS), leading to a significant impact on the quality of life. The underlying mechanisms of OSAS-related cognitive deficits are not well understood, and chronic intermittent hypoxia (CIH) has been considered as one of the most important contributing factors for this syndrome both clinically as well as mechanistically. Changes in the synaptic plasticity, including long-term potentiation (LTP) as one of the major cellular mechanisms that underlie learning and memory, and the integrated Ca2+ signaling involved in hippocampal synaptic plasticity may play important role in the CIH-induced phenotype of cognitive impairment observed in OSAS..In our most recent research project supported by the small sponsors from National Natural Science Foundation of China（NO.81170070, 14,0000 RMB）, we have successfully established a CIH mouse model demonstrating significant impairment of spatial learning and memory in these animals that is characteristic of OSAS-related cognitive deficits. These phenotypic changes were associated with neuronal autophagy and apoptosis most prominent in the hippocampal CA1 region demonstrated by electron microscopic examination, and the hippocampal expression of NR1/CaMKII proteins also decreased significantly in CIH-treated animals compared to the controls. Such behavioral, histological and biochemical findings support our hypothesis that CIH-induced cognitive dysfunction involves signal transduction pathways in the hippocampal region leading to both neurodegeneration and impaired synaptic plasticity..The present project proposal therefore aims to further investigate the role of Ca2+ signaling in mediating neurocognitive impairment in male ICR mouse exposed to CIH. Using whole-cell patch-clamp recordings and Ca2+ fluorescence imaging, we will measure whole cell currents activated through voltage-dependented Ca2+ channels (VDCC) vs. ligand-gated Ca2+ channels (LGCC) in acute hippocampal brain slices, and the intracellular Ca2+ dynamics of acutely isolated CA1 pyramidal neurons from these animals. The roles of VDCC and LGCC in CIH-mediated changes in signaling transduction & neurodegeneration will be investigated with respective antagonists by delineating their effects on 1) activity and focal expression of Ca2+-dependent PKC, ERK and CREB mediating learning and memory;2) intracellular calcium concentration；and 3) the temporal changes in spatial learning-memory ability. Finally, the relative contribution of NMDAR and L-type Ca2+ channels in mediating Ca2+ influx in targeted hippocampal neurons will also be evaluated..Through these series of planned experimental investigations, we aim to evaluate the involvement of Ca2+ signaling and downstream pathways in CIH-induced cognitive impairment.This study will provide novel experimental insights for better understanding and management of neurocognitive dysfunction in OSAS .

阻塞性睡眠呼吸暂停综合征（OSAS）造成认知功能障碍的机制尚不明确，其核心损伤是慢性间歇低氧（CIH），学习记忆的重要机制是海马长时程增强(LTP)，Ca2+信号通道在其中起重要作用。本课题获得2011年NSFC面上项目小额资助（81170070），结果发现，在CIH作用下，海马神经元Ca2+通道藕联的NMDAR1及CaMKⅡ的表达发生改变，CA1区出现凋亡和自噬损伤，小鼠空间学习记忆能力障碍。本研究拟进一步采用全细胞膜片钳技术，观察CIH下海马脑片两种不同机制钙通道（电压依赖性及配体门控性钙通道）特性的改变，以及细胞内钙超载现象；同时，应用两种钙通道的阻滞剂干预后，观察细胞内[Ca2+]浓度的变化，分析下游学习记忆标志性蛋白Erk1/2、CREB的表达和PKC磷酸化水平的改变以及对小鼠学习记忆能力的影响，为探讨钙通道在CIH诱导OSAS认知障碍中的机制，并寻找可能的治疗靶点提供实验依据。

阻塞性睡眠呼吸暂停综合征（OSAS）重要的病理生理损伤机制是夜间间歇低氧，其导致的神经认知功能损害严重地影响了患者的生活质量，导致记忆力衰退、反应迟钝，甚至带来生产事故和交通意外。建立慢性间歇低氧（CIH）的动物模型，探讨OSAS认知障碍的机制及可能的治疗靶点，有着重要的科学意义。 .本课题在2011年NSFC面上项目一年期小额资助（81170070）所获结果的基础上，进一步采用全细胞膜片钳技术，研究CIH作用下，海马CA1区脑片NMDAR藕联的钙通道及电压依赖性钙通道的电生理特征改变，以及细胞内钙超载现象；同时，应用两种钙通道的阻滞剂（NMDA受体拮抗剂盐酸美金刚、电压钙通道拮抗剂尼莫地平）干预后，观察通道特性的改变，分析钙受体通道蛋白、下游学习记忆标志性蛋白及凋亡蛋白的表达改变，以及对小鼠空间学习记忆能力等行为学的影响。. 2011年课题成熟建立并验证了CIH小鼠模型，本课题多次重复进行了2周及4周的造模。行为学研究发现，旷场实验中CIH组小鼠自发活动度增高，水迷宫逃避潜伏期延长，目的象限时间百分率及穿越平台次数均少于对照组，学习记忆能力明显受损。CIH组细胞内[Ca2+]i浓度升高，凋亡蛋白cleaved caspase-9、caspase-3和cleaved PARP高表达，抑制了下游信号通路ERK1/2、CREB蛋白活性。美金刚干预组，抑制了细胞内钙超载和凋亡蛋白的表达，小鼠行为学有逆转表现。膜片钳电生理研究发现，CIH组CA1区神经元突触前膜谷氨酸释放增多，谷氨酸受体(NR2B)表达增高，产生兴奋性毒性作用，神经元动作电位阈值显著升高、自发兴奋性突触后电流（sEPSC）频率及振幅均显著升高，神经元电生理功能受损，美金刚能阻断NMDA受体过度激活，降低兴奋性毒性。CIH组神经元高电压依赖性Ca2+通道电流密度显著升高，半数最大激活电位降低、最大失活电位升高，使Ica（HVA）的稳态激活曲线及失活曲线均发生明显移动，尼莫地平干预组呈扭转现象。研究结果提示，Ca2+介导的神经元兴奋性毒性及细胞内钙超载，诱导神经元凋亡，抑制下游信号通路记忆蛋白的活性，参与了CIH导致的认知功能损伤。

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