褪黑素2受体、糖原合酶激酶-3、蛋白磷酸酯酶-2A在突触可塑性中的作用及机制研究

发布时间：2018-05-23 08:06

本文选题：MT2受体 + 糖原合酶激酶-3　；参考：《华中科技大学》2011年博士论文

【摘要】：[背景] 突触可塑性是学习记忆的生物学基础。轴突的正常发育和成熟所维系的轴-树突极化对功能性突触的形成和维持至关重要。褪黑素2(MT2)受体、糖原合酶激酶-3(Glycogen synthase kinase-3, GSK-3)和蛋白磷酸酯酶-2A(Protein phosphatase-2A, PP-2A)广泛分布于脑内,在神经系统发育和神经退行性病变中起重要作用。MT2受体属于G蛋白偶联受体(GPCR)超家族,负责将细胞外信号转至细胞内。MT2受体下调抑制成年海马神经干细胞的分化、长时程增强(Long term potentiation, LTP)和学习记忆能力；而激活MT2受体能够抑制海马区域γ-氨基丁酸能突触的传递。GSK-3和PP-2A属于下游信号转导枢纽,它们不但是脑内公认的调节tau蛋白磷酸化的主要蛋白激酶和蛋白磷酸酯酶,而且还参与神经元极性和突触可塑性的调节。据报道,GSK-3过度激活导致突触前神经递质释放减少进而引起LTP抑制和学习记忆障碍。本课题组前期研究结果显示,褪黑素(MT2受体的天然配体)可下调GSK-3和上调PP-2A的活性,从而改善AD样的tau蛋白过度磷酸化。然而,关于MT2受体与GSK-3和PP-2A的关系,以及三者对神经元轴突形成和突触可塑性的影响尚无报道。 [目的] 在离体和整体水平探讨MT2受体和GSK-3、PP-2A在神经元轴突形成及突触可塑性中的作用以及它们之间的联系;探讨GSK-3异常引起突触损伤的分子机制。 [方法] 在离体培养的原代海马神经元模型上,运用FM4-64释放实验研究突触前神经递质的释放及轴突功能；全细胞膜片钳检测钙内流及突触后微型兴奋性突触后电流(mEPSCs);全内反射荧光显微镜(TIRFM)用于观察囊泡的胞吐作用;免疫共沉淀和荧光共振能量转移(FRET)检测MT2与Akt及突触前囊泡释放相关蛋白之间的相互结合；免疫荧光检测轴突发育及相关蛋白在细胞内的分布情况；免疫印迹检测蛋白质的含量及磷酸化水平的改变。 [结果] (1)MT2受体对轴突形成及功能的影响及相关分子机制：激活MT2受体明显促进海马神经元功能性轴突的形成并增强突触传递,而抑制MT2受体则阻碍轴突分化,其机制如下：MT2受体通过Akt/GSK-3p/CRMP-2信号通路而不是aPKC信号通路发挥作用；MT2受体可与Akt直接结合从而抑制GSK-3活性：给予MT2受体C末端的多肽可以封闭MT2-Akt之间的结合,同时明显削弱了MT2受体激活所引起的GSK-3抑制、轴突形成和突触传递的增强效应；在整体水平上,MT2受体敲除通过下调PP2A活性引起tau蛋白过度磷酸化。 (2)GSK-3β对突触前神经递质释放的影响及相关分子机制：激活GSK-3β可抑制突触前囊泡的胞吐,其机制如下：激活GSK-3β通过磷酸化P/Q型Ca2+通道上含synprint位点的LⅡ-Ⅲ来抑制Ca2+内流；激活GSK-3β可抑制LⅡ-Ⅲ与Ca2+感受器(synaptotagmin)、突触小体相关蛋白25(SNAP25)和syntaxin的结合,突触囊泡膜相关蛋白(synaptobrevin)与SNAP25和syntaxin的结合以及synaptobrevin与synaptophysin I的解离从而抑制Ca2+依赖的SNARE复合物的形成来阻止突触前囊泡的胞吐。 (3)PP-2A对神经突起生长的影响：PP-2A是神经突起发育所必需的,上调PP-2A活性能够促进突起,尤其是轴突的生长。 [结论] MT2受体激活通过抑制GSK-3来促进神经元轴突生长和功能性突触的形成,而在整体水平上,MT2受体敲除则通过下调PP-2A活性引起AD样tau蛋白过度磷酸化；激活GSK-3通过磷酸化P/Q型钙离子通道而抑制突触前递质释放,而上调PP-2A活性则对神经突起尤其是轴突的生长有明显促进作用。
[Abstract]:[background]
Synaptic plasticity is the biological basis for learning and memory. The axis dendrite polarization maintained by the normal development and maturation of axons is crucial to the formation and maintenance of functional synapses. Melatonin 2 (MT2) receptors, glycogen synthase kinase -3 (Glycogen synthase kinase-3, GSK-3), and protein phosphatase -2A (Protein phosphatase-2A, PP-2A) are widely divided. In the brain, it plays an important role in the development of nervous system and neurodegenerative diseases. The.MT2 receptor belongs to the G protein coupled receptor (GPCR) superfamily. It is responsible for the transfer of extracellular signal to the down regulation of.MT2 receptor to inhibit the differentiation of adult hippocampal neural stem cells, the long-term potentiation (Long term potentiation, LTP) and the learning and memory ability. The active MT2 receptor inhibits the transmission of gamma aminobutyric acid synapses in the hippocampus,.GSK-3 and PP-2A belong to the downstream signal transduction hub. They are not only recognized as the major protein kinase and protein phosphatase that regulate the phosphorylation of tau proteins in the brain, but also participate in the regulation of neuronal polarity and synaptic plasticity. It is reported that GSK-3 is overactivated. The release of presynaptic neurotransmitters and the reduction of LTP inhibition and learning and memory disorders. The previous study in our group showed that melatonin (natural ligand of MT2 receptor) could down regulate the activity of GSK-3 and PP-2A, thus improving the overphosphorylation of AD like tau protein. However, the relationship between the MT2 receptor and GSK-3 and PP-2A, as well as the three The effects of neuronal axonal formation and synaptic plasticity have not been reported.
[Objective]
To explore the role of MT2 receptor and GSK-3, PP-2A in the formation of axon and synaptic plasticity and the relationship between them, and to explore the molecular mechanism of synaptic damage caused by GSK-3 abnormalities in vitro and in whole level.
[method]
On the model of primary cultured hippocampal neurons in vitro, the release of presynaptic neurotransmitters and axon function were studied by FM4-64 release test. The whole cell patch clamp was used to detect the calcium influx and postsynaptic micro excitatory postsynaptic current (mEPSCs); the total internal reflection fluorescence microscopy (TIRFM) was used to observe the exocytosis of vesicles; immunoprecipitation and immunoprecipitation were used. Fluorescence resonance energy transfer (FRET) was used to detect the interaction between MT2 and Akt and the proteins associated with the release of presynaptic vesicles; immunofluorescence was used to detect the development of axon and the distribution of related proteins in the cells; the content of protein and the change of phosphorylation level were detected by immunoblotting.
[results]
(1) the effect of MT2 receptor on the formation and function of axon and its related molecular mechanism: activation of MT2 receptor obviously promotes the formation of functional axons of hippocampal neurons and enhances synaptic transmission, and the inhibition of MT2 receptors hinders axonal differentiation. The mechanism is as follows: MT2 receptor plays a role by Akt/GSK-3p/CRMP-2 signaling instead of aPKC signaling pathway. The MT2 receptor can bind directly with Akt to inhibit GSK-3 activity: the polypeptide given to the C terminal of the MT2 receptor can close the binding between MT2-Akt, and obviously weakens the GSK-3 inhibition caused by the activation of MT2 receptor, the formation of axon and the enhancement of synaptic transmission; at the overall level, MT2 receptor knockout by down regulation of PP2A activity causes tau protein. Excessive phosphorylation.
(2) the effect of GSK-3 beta on the release of presynaptic neurotransmitters and its molecular mechanism: activation of GSK-3 beta inhibits the exocytosis of presynaptic vesicles. The mechanism is as follows: activation of GSK-3 beta inhibits Ca2+ internal flow through the L II - III containing synprint loci on the phosphorylated P/Q Ca2+ channel, and activates GSK-3 beta to inhibit L II - III and Ca2+ receptor (synaptotagmin). The combination of synapse 25 (SNAP25) and syntaxin, the binding of synaptic vesicular membrane related protein (synaptobrevin) to SNAP25 and syntaxin, and the dissociation of synaptobrevin and synaptophysin I to inhibit the formation of Ca2+ dependent SNARE complexes to prevent the exocytosis of presynaptic vesicles.
(3) the effect of PP-2A on neurite outgrowth: PP-2A is necessary for neurite development. Up regulation of PP-2A activity can promote neurite outgrowth, especially axonal growth.
[Conclusion]
MT2 receptor activates the growth of neuronal axons and the formation of functional synapses by inhibiting GSK-3, while on the overall level, MT2 receptor knockout induces excessive phosphorylation of AD like tau protein by down regulation of PP-2A activity, and activates GSK-3 through the phosphorylated P/Q type calcium channel to inhibit the release of the pre inrush transmitter, while the up-regulation of PP-2A activity is to the deity. The growth of neurites is obviously promoted by neurites.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392.1

【相似文献】