NMDA受体2B亚基酪氨酸残基的磷酸化动态调节介导快速抗抑郁作用及其机制研究

The major depressive disorder is a common, recurrent and disabling mental illness. The existing antidepressants have the uncertainty of initial therapeutic effect, delayed-onset time of effects and obvious side effects. The further deep research is needed to find out the core of its pathogenesis. Our previous study has shown that chronic unpredictable mild stress (CUS) leads to a significant increase in levels of extracellular glutamate, which spill over to the extrasynapses, and results in an increase of the expression of the GluN2B-containing NMDA receptors (NR2B), in the medial prefrontal cortex of rats. We used ifenprodil, a selective NR2B antagonist, TC-DAPK6, an inhibitor of DAPK1 which phosphorylated NR2B and an interference peptide that blocked NR2B interaction with DAPK1. All of them had a rapid antidepressant effect. Our previous results proved that the elevated expression of NR2B in the extrasynapses in the mPFC plays a key role in the pathogenesis of depression. The functional NMDA receptor activity is dynamic regulated through protein phosphorylation to achieve synaptic stability under physiological condition. How does CUS affect NMDA receptor dynamic regulation, and whether the NMDA receptor dynamic regulation mediates rapid antidepressant action? There has been no reported. This project will use the chronic unpredictable stress model of depression; gene intervention techniques were used to overexpress Src-family tyrosine kinase Fyn and conditional knockdown phosphatase STEP61 in the mPFC; A modified classical subcellular fractionation approach was utilized in order to separate plasma membrane fractions into synaptic and extrasynaptic membranes of the mPFC of rats; The expression changes of subunits of NMDA receptor, the Src-family tyrosine kinase which phosphorylate subunits of NMDA receptor and phosphatase to dephosphorylate in these subcellular components will be detected by using western blotting; pharmacological technology, sleep deprivation technology and the behavioral testing of animal will be used to verify dynamic regulation of NMDA receptor is involved in the pathophysiological mechanism of depression and mediates the rapid antidepressant effects. The expected results will initially put forward the theory of dynamic regulation of NMDA receptor mediates the rapid antidepressant effects.

抑郁症是一种常见且易复发和致残性的精神疾病，现有抗抑郁药存在初次治疗疗效不确定，且起效慢并有明显副反应的困境，急需深入研究其发病机制。我们的前期研究慢性应激(CUS)导致大鼠前额叶皮层(mPFC)细胞外谷氨酸溢出至突触外，含2B亚基的NMDA受体(NR2B)在突触外表达升高；采用NR2B拮抗剂、磷酸化NR2B的DAPK1抑制剂和阻断NR2B与DAPK1相互作用的干扰肽，均起到快速抗抑郁效果，证明mPFC突触外NR2B表达升高是导致抑郁症的关键环节。生理条件下NMDA受体通过蛋白磷酸化动态调节实现突触稳定，这种动态调节是否受CUS影响，是否介导快速抗抑郁作用，尚无报道。本项目采用CUS抑郁模型，基因干预技术过表达激酶Fyn和条件性敲低磷酸酶STEP61，睡眠剥夺、药理学干预技术等，检测NMDA受体亚基、激酶及磷酸酶表达及模型动物抑郁样行为变化，验证NMDA受体磷酸化动态调节介导快速抗抑郁作用

抑郁症已被WHO列为全球造成致残和非致命性健康损失最大的疾病。然而，其发病机制尚不清楚，导致其诊疗现状面临艰巨的挑战。氯胺酮抗抑郁作用在动物30分钟起效，在人3-4小时起效，起效时间如此快速，应是脑内细胞中已有的某物质，经过药物刺激使其释放或转移，而非通过新合成蛋白质实现。NMDA 受体2B亚基磷酸化动态调节使其具有在突触膜和突触外膜之间迁移的特性，为快速抗抑郁作用提供了物质基础。采用SD大鼠，建立CUS抑郁模型，以mPFC为目标脑区，发现突触外膜上STEP61蛋白表达较对照组明显下降，NR2B表达较对照组明显升高，其他分子FYN、NR2A、p - NR2B（1336）和p - NR2B(1472)，CUS组和CON组无显著差异。说明CUS导致了突触外NR2B的聚集。突触内，CUS组STEP61和p - NR2B（1336）显著升高，说明STEP61和p - NR2B（1336）在突触内外的表达紊乱，同时CUS导致大鼠抑郁样行为。氯胺酮不仅逆转了CUS导致的大鼠抑郁样行为，还恢复了突触外膜STEP61的表达，降低了突触膜STEP61表达（WB和免疫共定位）。同时，氯胺酮降低了突触膜上p - NR2B（1336）表达，升高p - NR2B（1472）表达，使其恢复平衡。采用AAV-shPtpn5敲降大鼠mPFC的STEP61蛋白，与AAV-Scramble组比较，AAV-shPtpn5组表现出显著的抑郁样和焦虑样行为。同时，突触外膜NR2B水平显著增加，突触内膜上则没有变化。NR2B受体的1472位点磷酸化在突触内外膜上均升高。氯胺酮可以逆转AAV-shPtpn5组的抑郁样行为，降低突触外膜、升高突触内膜1472位点NR2B的磷酸化。说明mPFC的STEP61蛋白敲降，导致突触外膜NR2B表达增加，1472位点NR2B磷酸化激活增加，神经细胞毒性损伤，导致AAV-shPtpn5组大鼠出现抑郁样行为。氯胺酮降低了突触外膜上NR2B受体1472位点的磷酸化，抑制了突触外NR2B的激活，发挥快速抗抑郁样作用。综上，我们的结果证明了突触内外NR2B的磷酸化动态平衡调节参与了大鼠抑郁样行为发生的新机制，并证明了STEP61对NR2B磷酸化的调节在抑郁发生中的作用，为将来基于此开发新型抗抑郁剂提供了科学基础。此外，我们还进行抑郁症发病机制的其它研究内容，详见报告正文。