觉醒肽orexin兴奋内嗅皮层神经元升高海马Aβ水平的效应及机制研究

The accumulation of Aβ in the brain initiates the Alzheimer's disease (AD). Recently, several reports suggest that orexin, a hypothalamic awakening peptide, increases the levels of Aβ in the hippocampus. However, the underlying mechanisms are largely unknown. Based on the preliminary result of this project, we have found that orexin excites entorhinal cortex (EC) neurons and facilitates its Aβ generation. The generated Aβ is then secreted into hippocampus via axons of the perforant pathway, and thus involved in the increase of hippocampal Aβ levels. Therefore, in the present study, we first plan to further determine whether the orexin-induced Aβ generation in EC neurons is the primary source of Aβ accumulation in hippocampus. Next, whether the calcium signal-elicited Arc expression pathway is involved in the generation of Aβ induced by the excitation effects of orexin on EC neurons will be determined. Third, based on the acquired knowledge of the involved molecular pathway, we plan to interefer with them to investigate the changes of functional activities of hippocampal neurons and dendritic spines changes with two photon microscope techniques. Combining these results with the related senile plaques deposition analysis and behavioral test results, the possibly specific targets for sleep deprivation-induced AD will be further investigated. This project will provide new insights for the improvement of abnormal sleep/wake-induced cognitive deficits and the targeted prevention and treatment of AD.

Aβ在脑内的集聚是阿尔茨海默氏病（Alzheimer's Disease, AD）发病的始动环节。新近文献报道，觉醒肽orexin兴奋大脑可致海马Aβ水平升高，但具体机制尚不清楚。基于我们前期研究发现orexin兴奋内嗅皮层神经元产生Aβ，并经穿通纤维通路突触前末梢在海马分泌，升高海马Aβ水平，本课题拟首先明确orexin促进内嗅皮层神经元生成Aβ是海马Aβ水平升高的主要来源；其次，探索orexin对内嗅皮层神经元的兴奋效应是否经Ca2+信号上调Arc分子的表达以促进Aβ生成；在阐明orexin兴奋内嗅皮层升高海马区Aβ水平的完整分子信号途径基础上，利用AD小鼠进行实验干预，通过双光子显微镜观察海马神经元功能活动及树突棘变化，结合老年斑沉积和行为学的检测结果探索出睡眠剥夺引发AD关键分子靶点，为改善睡眠-觉醒异常造成的认知功能障碍以及针对性防治AD提供新线索。

神经活动与AD的发生发展备受关注，关于睡眠觉醒周期变化可致海马Aβ水平出现波动。基于内嗅皮层和海马环路参与学习记忆功能，并在AD大脑中出现显著的病理性改变，因此探索觉醒肽orexin对内嗅皮层的兴奋效应和机制对于深入了解海马Aβ水平改变导致AD病人认知功能进行性下降具有重要意义。. 本项目发表综述阐述Aβ通过相应受体在脑内发挥相应毒性作用，了解了突触活动调节脑内Aβ水平具有关键性作用。具体研究orexin-A对内嗅皮层浅层主要投射神经元膜电位、膜电流的作用及其机制；orexin-A对内嗅皮层浅层三种中间神经元活动的影响；OX1R,GABA能神经元、谷氨酸能神经末梢在内嗅皮层浅层的分布及其相互关系；以癫痫模型研究异常神经兴奋与AD发展的潜在机制；研究腹膜透析对AD小鼠血液中Aβ水平的影响。. 本项目采用电生理技术、免疫荧光、免疫电镜、Western blot、微透析等多种技术手段，研究了内嗅皮层浅层中有呈散在分布的orexin-1受体表达，且中间神经元表达较多；vglut1的表达多于vglut2；免疫电镜实验显示中间神经元的突触前兴奋性突触末梢内有orexin-1 受体的表达。电生理实验证实orexin-A对内嗅皮层浅层主要投射神经元的膜电位的超极化效应具有浓度依赖性；OX1R可能参与orexin-A在内嗅皮层浅层主要投射神经元引起的膜电位超极化效应；orexin-A对内嗅皮层浅层主要投射神经元的兴奋性产生抑制效应，其机制依赖于抑制性的突触传递； orexin-A增加PV、5-HT受体和SST等标记的三种中间神经元放电活动，促进GABA神经递质的释放，进而对兴奋性神经元产生间接的抑制作用；orexin-A增加兴奋性突触末梢向中间神经元释放的谷氨酸，进而提高中间神经元的兴奋性，增强其放电。. 综上，本项目系统研究orexin兴奋内嗅皮层的效应机制，为了解orexin通过内嗅皮层-海马环路调控海马Aβ水平提供实验依据，有可能通过睡眠节律的角度理解AD发生发展并为其防治提供新的思路和策略，从而具有广阔的应用前景。