脆性X智力低下蛋白调减circ_0000208的“miRNA海绵”作用引发PTSD恐惧记忆消退障碍的机制

One of the pathogenesis of posttraumatic stress disorder (PTSD) is fear memory extinction disorder, which is closely related to synaptic plasticity and synaptic protein expression. Our previous work found that the fragile X mental retardation protein (FMRP) and circ_0000208 were downregulated in the amygdala of PTSD rats. Upregulation of FMRP or circ_0000208 decreased postsynaptic density protein 95 (PSD95) expression in neurons stressed by lipopolysaccharide (LPS). Bioinformatics software predicted that there were FMRP and miR-153-3p binding sites in circ_0000208, and we briefly proved that circ_0000208 might be associated with miR-153-3p in the regulation of the expression of PSD95. We also found that miR-153-3p was upregulated in the amygdala of PTSD rats and downregulation of miR-153-3p could increase the expression of Sig-1R, which plays an important role in alleviating the anxiety-like behavior and cognitive impairments in a rat model of PTSD. Combined with our previous study, we speculate that the interaction between circ_0000208 and FMRP can enhance the regulation of miR-153-3p on target gene SIGMAR1, thus participating in the process of fear memory extinction in PTSD rats. This study first prepares a neuronal stress model of circ_0000208 gene silencing, and then detects the effect of circ_0000208 gene silencing on the promotion of long-term potentiation (LTP) and PSD95, synaptic GTPase-activating protein 1 (SynGAP 1), Synaptotagmin Ⅶ (syt7) and synapsin Ⅰ expression. Furthermore, we analyze the effects of circ_0000208 gene silencing on the expression of FMRP, miR-153-3p, Sig-1R and verifies target-binding effect and binding sites between FMRP and circ_0000208, circ_0000208 and miR-153-3p, miR-153-3p and SIGMAR1 to prove the regulatory mechanism of fear memory extinction disorder. Finally, we will verify the effect of application with the activator or inhibitor of Fmr1, circ_0000208 or miR-153-3p separately or in combination on synapse plasticity in amygdala and alleviating behavioral abnormality of PTSD rats. Then we will explore the regulatory mechanism on alleviating fear memory abnormality of PTSD rats by upregulation of circ_0000208. Thus, this study could not only elucidate the molecular mechanism by which FMRP regulates "miRNA Sponge" effect of circ_0000208 on synaptic plasticity impairment and synaptic protein expression to delay the fear-memory-extinction, but also provide theoretical basis for finding effective therapeutic targets of PTSD.

创伤后应激障碍(PTSD)的发病机制之一为恐惧记忆消退障碍，后者与突触可塑性损伤及突触蛋白表达密切相关。研究表明，脆性X智力低下蛋白(FMRP)是介导PTSD恐惧记忆的关键分子，但机制不清。我们前期研究发现PTSD模型大鼠杏仁体中FMRP和circ_0000208均显著降低，上调这两种分子均可降低PSD95的表达，且circ_0000208的作用可被miR-153-3p逆转。基于此，我们推测：FMRP调减circ_0000208水平，致使后者的“miRNA海绵”作用减弱，促进miR-153-3p对SIGMAR1的负性调控，从而促进突触蛋白表达以及突触可塑性损伤，引发PTSD恐惧记忆消退障碍。本项目拟应用circ_0000208基因沉默的神经元应激模型结合体内实验，旨在阐明FMRP通过调节circ_0000208的“miRNA海绵”作用从而延缓PTSD恐惧记忆消退的可靠机制。

创伤后应激障碍（PTSD）是由于经历对生命具有威胁的事件或严重创伤导致的一种与病理性恐惧和焦虑相关的精神疾病，其病理机制尚未完全研究清楚。课题组前期研究发现PTSD模型大鼠海马区脆性X智力低下（FMRP）水平下调，提示FMRP可能参与PTSD的病理生理过程，但具体机制需进一步研究。微小RNA（miRNA）通过调节靶基因或蛋白的表达，在多种生理和病理调控过程中发挥重要作用。通过生物学信息分析，我们预测到FMRP存在miR-142的结合位点，我们推测miR-142可能通过结合FMRP参与PTSD病理生理过程。本研究构建PTSD模型大鼠并通过侧脑室注射anti-miR-142，利用旷场试验、高架十字迷宫等实验检测了大鼠的行为学；通过免疫荧光染色、Western Blot或Elisa方法检测各组大鼠海马区PSD95、Synapsin I、Iba-1、TNF-α、IL-1β、IL-6、IL-10、Caspase-3、Bax、p-NF-κB p65的水平，并通过流式细胞术检测细胞凋亡水平，结合体外细胞实验，探讨抑制miR-142改善PTSD模型大鼠行为学异常的可能分子机制。结果发现anti-miR-142可以显著改善PTSD模型大鼠抑郁样行为、焦虑样行为及记忆异常，此作用可能通过上调海马区FMRP水平、降低促炎性细胞因子水平及细胞凋亡水平实现。本研究旨在为探索PTSD的防治新靶点提供理论和实验依据。