本文選題：MT2受體 + 糖原合酶激酶-3��； 參考：《華中科技大學》2011年博士論文

【摘要】：[背景] 突觸可塑性是學習記憶的生物學基礎。軸突的正常發(fā)育和成熟所維系的軸-樹突極化對功能性突觸的形成和維持至關重要。褪黑素2(MT2)受體、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)廣泛分布于腦內(nèi),在神經(jīng)系統(tǒng)發(fā)育和神經(jīng)退行性病變中起重要作用。MT2受體屬于G蛋白偶聯(lián)受體(GPCR)超家族,負責將細胞外信號轉(zhuǎn)至細胞內(nèi)。MT2受體下調(diào)抑制成年海馬神經(jīng)干細胞的分化、長時程增強(Long term potentiation, LTP)和學習記憶能力；而激活MT2受體能夠抑制海馬區(qū)域γ-氨基丁酸能突觸的傳遞。GSK-3和PP-2A屬于下游信號轉(zhuǎn)導樞紐,它們不但是腦內(nèi)公認的調(diào)節(jié)tau蛋白磷酸化的主要蛋白激酶和蛋白磷酸酯酶,而且還參與神經(jīng)元極性和突觸可塑性的調(diào)節(jié)。據(jù)報道,GSK-3過度激活導致突觸前神經(jīng)遞質(zhì)釋放減少進而引起LTP抑制和學習記憶障礙。本課題組前期研究結(jié)果顯示,褪黑素(MT2受體的天然配體)可下調(diào)GSK-3和上調(diào)PP-2A的活性,從而改善AD樣的tau蛋白過度磷酸化。然而,關于MT2受體與GSK-3和PP-2A的關系,以及三者對神經(jīng)元軸突形成和突觸可塑性的影響尚無報道。 [目的] 在離體和整體水平探討MT2受體和GSK-3、PP-2A在神經(jīng)元軸突形成及突觸可塑性中的作用以及它們之間的聯(lián)系;探討GSK-3異常引起突觸損傷的分子機制。 [方法] 在離體培養(yǎng)的原代海馬神經(jīng)元模型上,運用FM4-64釋放實驗研究突觸前神經(jīng)遞質(zhì)的釋放及軸突功能；全細胞膜片鉗檢測鈣內(nèi)流及突觸后微型興奮性突觸后電流(mEPSCs);全內(nèi)反射熒光顯微鏡(TIRFM)用于觀察囊泡的胞吐作用;免疫共沉淀和熒光共振能量轉(zhuǎn)移(FRET)檢測MT2與Akt及突觸前囊泡釋放相關蛋白之間的相互結(jié)合；免疫熒光檢測軸突發(fā)育及相關蛋白在細胞內(nèi)的分布情況；免疫印跡檢測蛋白質(zhì)的含量及磷酸化水平的改變。 [結(jié)果] (1)MT2受體對軸突形成及功能的影響及相關分子機制：激活MT2受體明顯促進海馬神經(jīng)元功能性軸突的形成并增強突觸傳遞,而抑制MT2受體則阻礙軸突分化,其機制如下：MT2受體通過Akt/GSK-3p/CRMP-2信號通路而不是aPKC信號通路發(fā)揮作用；MT2受體可與Akt直接結(jié)合從而抑制GSK-3活性：給予MT2受體C末端的多肽可以封閉MT2-Akt之間的結(jié)合,同時明顯削弱了MT2受體激活所引起的GSK-3抑制、軸突形成和突觸傳遞的增強效應；在整體水平上,MT2受體敲除通過下調(diào)PP2A活性引起tau蛋白過度磷酸化。 (2)GSK-3β對突觸前神經(jīng)遞質(zhì)釋放的影響及相關分子機制：激活GSK-3β可抑制突觸前囊泡的胞吐,其機制如下：激活GSK-3β通過磷酸化P/Q型Ca2+通道上含synprint位點的LⅡ-Ⅲ來抑制Ca2+內(nèi)流；激活GSK-3β可抑制LⅡ-Ⅲ與Ca2+感受器(synaptotagmin)、突觸小體相關蛋白25(SNAP25)和syntaxin的結(jié)合,突觸囊泡膜相關蛋白(synaptobrevin)與SNAP25和syntaxin的結(jié)合以及synaptobrevin與synaptophysin I的解離從而抑制Ca2+依賴的SNARE復合物的形成來阻止突觸前囊泡的胞吐。 (3)PP-2A對神經(jīng)突起生長的影響：PP-2A是神經(jīng)突起發(fā)育所必需的,上調(diào)PP-2A活性能夠促進突起,尤其是軸突的生長。 [結(jié)論] MT2受體激活通過抑制GSK-3來促進神經(jīng)元軸突生長和功能性突觸的形成,而在整體水平上,MT2受體敲除則通過下調(diào)PP-2A活性引起AD樣tau蛋白過度磷酸化；激活GSK-3通過磷酸化P/Q型鈣離子通道而抑制突觸前遞質(zhì)釋放,而上調(diào)PP-2A活性則對神經(jīng)突起尤其是軸突的生長有明顯促進作用。
[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.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R392.1

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