死亡相关蛋白激酶1损伤海马前额叶皮层环路介导卒中后抑郁的机制研究

Post-stroke depression (PSD) is a common and complex post-stroke neuropsychiatric disorder, which not only delays functional recovery but also increases mortality, and currently lacks effective drug therapy. The pathogenesis of PSD is associated with impairment of the subcortical circuits of the prefrontal cortex, and alterations of synaptic plasticity, glutamate neurotransmitter and so on, but the exact mechanisms of PSD is still unknown. Our previous work indicates that the death associated protein kinase 1 (DAPK1) mediates neuronal death after stroke. Knockout DAPK1 gene or blocking of DAPK1 signal in PSD mouse model can not only alleviate cerebral ischemic injury, but also relieve post-stroke depression like behaviors. Based on the previous work, by using genetically modified mice to establish PSD model, combined with neural tracing, optogenetics, electric physiology and depression like behavior detection methods, the present study is proposed to investigate: (1)DAPK1 signal in the synapse of dCA1-mPFC neural circuits, (2) the structure and functions of dCA1-mPFC neural circuits after stroke, and (3) their relations with post-stroke depression like behaviors. Our study will further replenish the molecular and neural circuits mechanisms involved in post-stroke depression and provide new insights for the treatment of PSD.

卒中后抑郁(PSD)是一种常见的脑卒中后复杂神经精神疾病，严重影响预后，缺乏有效治疗。PSD发病机制与前额叶皮层下回路破坏，突触可塑性、谷氨酸递质改变等有关，但具体机制不明。我们前期发现：死亡相关蛋白激酶1（DAPK1）及下游信号介导卒中后神经元死亡，敲除DAPK1基因或阻断其信号，可减轻脑缺血损伤并缓解PSD。此外，PSD小鼠背侧海马（dCA1）到内侧前额叶皮层（mPFC）投射纤维丢失。本项目拟在此基础上，运用转基因小鼠建立PSD模型，结合病毒示踪、光遗传学、电生理以及抑郁样行为学检测方法，探究：（1）dCA1-mPFC神经突触DAPK1及其下游分子信号；（2）dCA1-mPFC突触结构与突触传递功能；（3）dCA1-mPFC神经环路与抑郁样行为的关系。本研究将揭示卒中后DAPK1信号通过损伤突触进而损伤dCA1-mPFC环路介导PSD的分子神经环路机制，为PSD的临床治疗提供新策略。

卒中后抑郁症（Post stroke depression, PSD）是严重影响患者卒中后生活质量和康复的常见并发症之一。目前PSD的治疗以对症支持疗法为主，尚无有效的治疗药物。PSD的发病机制尚不清楚。已知miRNA在大脑中广泛表达，在脑卒中和抑郁症等多种神经与精神疾病的发病机制中起关键作用。本研究项目通过建立PSD小鼠模型，从转录组测序结果出发，以miRNA为切入点，探究PSD的分子机制。我们首先建立小鼠脑卒中模型，卒中后通过旷场试验、高架十字迷宫试验、悬尾试验和强迫游泳试验，并结合无监督模糊聚类分析，筛选和验证具有PSD样表型的小鼠。通过正电子发射断层扫描确定PSD小鼠大脑继发病变部位。我们接着对继发病变脑组织进行RNA测序，结合qPCR验证确定差异表达miRNAs。进一步通过生信分析方法对目标miRNA的靶基因进行预测，结合基因功能富集分析、蛋白质免疫印迹、双荧光素酶试验和免疫荧光染色确定靶基因。通过向PSD小鼠海马区脑内定点注射腺相关病毒过表达靶基因以明确该靶基因对PSD小鼠抑郁样行为的调控作用。应用脑片膜片钳电生理技术进一步明确靶基因在PSD发病机制中的作用。我们发现：PSD小鼠海马组织RNA测序分析发现19个差异表达miRNAs，结合qPCR验证确定差异表达最显著miRNA为miR-34b-3p。通过生信分析方法对miR-34b-3p靶基因进行预测评分，结合基因功能富集分析、蛋白质免疫印迹实验、双荧光素酶试验和免疫荧光染色实验确定靶基因eIF4E。应用腺相关病毒AAV2/9-sheIF4E特异性抑制PSD小鼠海马神经元中eIF4E，PSD小鼠抑郁样行为改善。应用膜片钳电生理技术、免疫荧光实验和qPCR实验进一步明确eIF4E通过激活小胶质细胞使IL-1、IL-6和TNF-α分泌增多引起炎症反应从而导致PSD。综上结果，我们初步阐明缺血性卒中后海马中神经元miR-34b-3p的下调导致其靶基因eIF4E的表达增加，并通过激活小胶质细胞引起神经炎症和PSD。而下调海马神经元eIF4E的表达可抑制小胶质细胞的活化、减轻神经炎症并改善PSD样症状。