MicroRNA-195/microRNA-153协同调节长期脑低灌注大鼠乙酰胆碱递质释放的分子机制

Impairment of cholinergic function is a common clinical feature of senile dementia at the early stage, while the molecular mechanism is unknown. Our previous studies have been demonstrated that rats suffering from chronic brain hypoperfusion (CBH) induced by bilateral common carotid artery occlusion (2VO) could promote the initiation of Aβ and Tau hyperphosphorylation through inhibiting the expression of miR-195. As a preclinical stage, whether CBH could result the impairment of cholinergic function has not been reported. Research contents: 1. To observe the distribution of synaptic vesicle and the level of miR-195, miR-153, ACh synthesis related proteins and synaptic fusion related proteins in hypocampi and cortices of 2VO rats and VaD patients; 2. To investigate the regulation of miR-195 and miR-153 on target genes; 3. To evaluate the distribution of synaptic vesicle and the level of miR-195, miR-153, ACh synthesis related proteins and synaptic fusion related proteins as well as the changes of fEPSP and EPSCs in hypocampi and cortices of gene editing mice with knockdown of miR-195 or overexpression of miR-153, and 2VO rats with stereotaxic injection of the lentiviral vectors containing pre-miR-195 or pre-AMO-miR-153. This study is the further study based on our previous published results. Our aim is to disclose the microRNAs mechanism involvement of dementia induced by CBH and provide the research evidence for future clinical drug discovery.

胆碱功能下降是老年痴呆患者早期重要的病理生理特征，但分子机制不清。我们前期研究证明大鼠双侧颈总动脉结扎（2VO）引起长期脑低灌注（CBH）可通过抑制miR-195表达促进Aβ生成和Tau蛋白过度磷酸化。CBH是否可引起胆碱功能下降未见报道。研究内容：1. 评价2VO大鼠及VaD患者海马和皮层胆碱能神经元突触小泡分布、miR-195、miR-153、ACh合成和突触融合相关蛋白表达；2. 阐明miR-195和miR-153对目的蛋白靶向调控作用；3. 检测敲减miR-195和过表达miR-153基因编辑小鼠和2VO大鼠局部海马注射含miR-195和AMO-miR-153慢病毒载体对ACh合成和突触融合相关蛋白及CA1区fEPSP和EPSCs的影响。本研究是课题组前期原创性研究的系列研究，旨在系统阐明miRNAs在CBH诱导痴呆发生中的分子机制，为临床药物研发提供实验依据。

胆碱功能下降是老年痴呆患者早期重要的病理生理特征，但分子机制不清。在课体组前期研究基础上，本研究联合使用网络数据库、慢病毒转染、miR-masking、电生理及相关分子生物学技术探讨在实验性CBH大鼠痴呆模型中miR-195联合miR-153在CBH引起的胆碱能神经递质释放中的作用及其机制。研究发现：（1）大鼠双侧颈总动脉结扎（2VO）引起miR-153表达升高，通过抑制SNAP-25、VAMP-2、Syntaxin-1A、Syt-1表达抑制突触囊泡的释放过程，从而损伤大鼠突触前可塑性；（2）CBH大鼠基底前脑MS/VDB-海马背侧CA1神经环路突触传递功能受损，突触前神经递质释放障碍；（3）CBH大鼠基底前脑MS/VDB-海马背侧CA1神经环路GABA能神经元功能受损， mAChR与nAChR功能发生障碍；（3）CBH能够诱发大鼠海马小胶质细胞活化和极化。其机制为CBH大鼠海马区下调的miR-195通过激活神经元与小胶质细胞之间CX3CL1-CX3CR1信号通路，促进小胶质细胞向M1表型极化的过程中。外源性给予miR-195能够改善CBH诱导的小胶质细胞向M1表型极化。本研究为CBH诱发痴呆的治疗提供了一个有价值的治疗靶点。