内侧前额叶磷酸化的突触体相关蛋白25在抑郁样行为中的作用及机制

Major depressive disorder (MDD) is a severe psychiatric illness characterized by high recurrence and suicide rate. Clinical and animal experiments have proved that depression can lead to elevated glutamate and abnormal expression of glutamate receptors in the medial prefrontal cortex (mPFC) . However, the molecular and synaptic plasticity mechanisms are still unclear. Studies have found that synaptosomal-associated protein of 25 kDa (SNAP25) can mediate presynaptic glutamate-containing vesicles release and regulate post-synaptic N-methyl-D-aspartate receptor (NMDAR) surface localization. Our previous studies have shown that there was consistent high expression of GluN2B-NMDAR with phosphorylated Ser187-SNAP25 protein in the mPFC of depressive mice. Based on it, this project plans to use chronic unpredictable mild stimuli to establish a mouse model of MDD by inhibiting the phosphorylated SNAP25 (pSNAP25) with interfering peptide to study the influence on glutamate levels, NMDAR membrane expression and structural plasticity of neurons. Immunofluorescence and immunogold electron microscopy will be used to confirm pSNAP25 distribution in the pre-synapse and post-synapse. Furthermore, whole-cell patch clamp will be adopted to study the functional regulation of pSNAP25 on presynaptic glutamate release and postsynaptic NMDAR membrane expression in the mPFC. This project is beneficial for clarifying the function of pSNAP25 in the regulation of glutamate system in depressive behavior and providing new molecular clues for intervention of MDD.

抑郁症是一种具有高复发率、自杀率特点的情感障碍性精神疾病。临床和动物实验都已证明抑郁症能导致内侧前额叶（mPFC）谷氨酸升高，谷氨酸受体表达异常，但其分子及突触可塑性机制尚不清楚。已知突触体相关蛋白25（SNAP25）不仅介导突触前谷氨酸的释放，而且调控突触后NMDAR的膜表达。我们前期研究发现，抑郁样小鼠mPFC内GluN2B蛋白水平与磷酸化的Ser187-SNAP25呈现一致性的高表达。据此，本课题计划采用慢性不可预期应激诱导小鼠抑郁样模型，在mPFC内，特异性干预SNAP25的磷酸化（pSNAP25），检测谷氨酸水平、NMDAR表达及突触结构可塑性的改变；利用免疫荧光和免疫电镜观察pSNAP25在突触前、后的表达分布；采用电生理学检测pSNAP25对谷氨酸突触（前后）可塑性的影响。以期阐明pSNAP25对谷氨酸系统功能的调控在抑郁样行为中的作用，为治疗抑郁症提供新的干预的分子线索。

谷氨酸系统的功能失衡参与了抑郁症的病理过程。研究发现抑郁症导致内侧前额叶皮层谷氨酸系统的异常，包括谷氨酸水平和谷氨酸受体表达异常。已知突触体相关蛋白25（SNAP25）作为囊泡运输家族的一员不仅能介导突触前谷氨酸的释放，并且能调控突触后谷氨酸受体的表达。目前仍不清楚SNAP25是否能通过调控谷氨酸系统异常参与抑郁行为。本项目采用慢性不可预期轻度应激建立小鼠抑郁模型，证明了腹内侧前额叶皮层（vmPFC）Ser187-SNAP25的磷酸化通过调控突触前谷氨酸的释放，参与介导抑郁行为。主要结果如下：1）抑郁小鼠vmPFC脑区被活化，且激活的神经元类型是谷氨酸能神经元。2）在单细胞水平，抑郁小鼠vmPFC兴奋性神经元数量增多，抑制性神经元数量减少。3）抑郁小鼠vmPFC脑区pS187-SNAP25表达增加，并且NMDA受体中的GluN2B亚基表达水平升高。氨基酸分析显示，vmPFC脑区兴奋性神经递质谷氨酸水平显著升高，抑制性神经递质γ-氨基丁酸（γ-ABA）水平下降。4）小鼠抑郁行为测试前，在其vmPFC脑区给予干扰肽特异性干预Ser187-SNAP25的磷酸化显著抑制了焦虑抑郁行为。5）抑郁小鼠vmPFC脑区特异性干预Ser187-SNAP25的磷酸化可显著逆转兴奋性神经递质谷氨酸水平的升高，GluN2B亚基表达水平也有下降的趋势。本研究首次证明小鼠vmPFC中pS187-SNAP25通过调控突触前谷氨酸的释放作用于抑郁行为的形成。该研究结果对揭示抑郁形成和干预的分子机制提供了新的依据。