Junctophilin 3和4在胚胎干细胞神经分化早期作用研究

发布时间：2018-05-14 00:02

本文选题：Junctophilins(JPs) + Megf10　；参考：《浙江大学》2016年博士论文

【摘要】：Junctophilin 3 (JP3) 和 Junctophilin 4 (JP4)在大脑皮层、海马及小脑高表达,是一类存在于可兴奋细胞中的膜结合蛋白。JP3,4能够连接细胞膜和内质网膜,形成膜连接复合物,对维持胞内钙稳态有着重要作用。研究发现,Jp3,4基因双敲除的小鼠出生后不但出现严重的发育迟缓及致死率高,而且呈现记忆认知功能损伤和运动协调功能失常,提示这两种蛋白在维持动物记忆和运动功能上起重要作用,并在胚胎神经分化发育过程中的不可或缺性。但其在胚胎发育过程的表达变化、功能特征以及功能差异尚未见报道。JP3,4能否在神经分化发育早期便参与神经系统的建立也亟待探索。果蝇表达的JP同源蛋白Undertaker (UTA),能通过维持吞噬细胞内Ca~(2+)平衡来参与凋亡细胞的清理。神经分化过程伴随大量细胞凋亡,未被及时清理的凋亡细胞则会发生二次坏死,导致细胞毒性并影响轴突的生长。凋亡的细胞会释放出"find-me"信号,快速吸引周围的细胞迁移,同时也会暴露出"eat-me"信号,保证吞噬细胞的识别和吞噬。然而,哺乳动物神经组织JPs是否也具有相同的功能迄今未见报道。除了需要严格和适宜的外部环境,神经元极化及轴突生长还受胞外信号的调控。凋亡细胞能够召唤周围细胞的增殖,但是否能对神经极化以及轴突生长产生影响尚未见报道。同时,JP3,4在连接凋亡、吞噬及神经极化之间扮演的角色也亟待阐明。在神经发育初期,未分化的突起通过竞争,最终只形成单一的轴突,而其他的突起将会停止生长或者变成树突,该过程使神经元在形态和功能上都形成了极化。神经元的极化及生长需要局部的Ca~(2+)信号和cAMP,形成局部极化。通过TRPC内流的Ca~(2+)能够启动轴突的特化,并激活下游CaMKI等级联信号,促使轴突形成。而在轴突切断的外周神经中,钙波会沿着轴突传向胞体,启动轴突的再生。此外,Ca~(2+)信号还可以和cAMP通路相互作用形成正反馈,放大cAMP信号。在诸多突起中,cAMP浓度最高的突起能够通过正反馈不断提升内部cAMP浓度,促使该突起长成轴突,而剩下突起则长成树突。然而,胞内cAMP信号如何被唤起,Ca~(2+)信号如何在局部区域被精准调控,基于JP3,4维持的胞内Ca~(2+)平衡是否与此相关联尚未完全明确。本论文第一部分将对以上问题做出探索。在轴突和树突的生长过程,会有过量的轴突分支、突触以及树突树形成,在形成神经环路后,多余的轴突分支将会被选择性修剪,以保证神经信号传递的专一性。吞噬受体Megf10能够参与中枢神经系统中突触的修剪与重塑,Megf10敲除小鼠则保留了过多数量的突触。果蝇Megf10的同源蛋白Draper已被发现能够参与果蝇胚胎发育过程的吞噬,而UTA则与Draper协同完成这一事件。因此,本论文第二部分(附)探索了JP3,4能否在胚胎干细胞分化神经过程中参与轴突的修剪及重塑。Junctophilin-3,-4连接吞噬参与神经极化作用研究本论文第一部分考察了JP3,4参与神经极化的分子机制,探索JP3,4正常表达与Ca~(2+)/cAMP形成局部极化的相关性。实验结果显示,JP3,4在ES细胞定向分化神经元早期有一过性高表达。在ES细胞沉默Jp3和Jp4,其神经分化早期表达显著降低,导致Ca~(2+)/cAMP极化正反馈通路受破坏,进而抑制神经极化。抑制JP3,4早期表达高峰使神经前体细胞胞内Ca~(2+)分布失去空间特异性,并导致Ca~(2+)瞬变能力显著减弱,同时cAMP含量明显下降,cAMP-LKB1-SAD/MAR K2极化通路激活受阻。提示JP3,4早期高表达对维持神经前体细胞胞内局部Ca~(2+)/cAMP极化正反馈通路有重要作用。与此同时,ES细胞定向分化神经早期JP3,4一过性高表达尚与吞噬作用密切相关。神经分化过程伴随大量凋亡,抑制JP3,4早期表达致使神经前体细胞清理凋亡细胞的效率显著下降。提示JP3,4在凋亡细胞清理过程需要被大量表达。进一步研究发现,JP3,4能够感应凋亡细胞的信号。凋亡细胞的刺激可快速提高JP4及吞噬受体Megf10的表达水平,并可促进JP3与RyR2、RyR3的结合,同时,JP4与Megf10的结合也显著上调。这些结果提示,除了维持内质网与细胞膜的空间距离,JP3与JP4在功能上各有分工,可分别通过JP3-RyR2,3相互作用及JP4-Megf10相互作用参与吞噬,保证神经极化适宜的微环境。最后,实验结果显示,凋亡细胞刺激可显著提升胞内cAMP的含量并激活cAMP-LKB1-SAD/MARK2极化通路,促进神经元极化。更为重要的是,凋亡细胞的刺激能够提升神经前体细胞胞浆Ca~(2+)瞬变能力,易化神经前体细胞极化。该结果提示神经分化发育过程,凋亡细胞可与神经前体细胞建立联系,不仅可诱导其吞噬,同时也能分泌信号促进神经极化及轴突生长,而JP3,4在其中起着桥梁作用。附：JP3,4参与神经分化过程轴突修剪研究本论文第二部分初步探索了JP3,4在神经分化过程参与轴突修剪。实验结果显示,通过慢病毒沉默吞噬受体Megf10可导致d 8+0 JP3的表达下降,而JP4的表达上调。而沉默JP3则导致Megf10的表达上调,沉默JP4导致Megf10的表达轻微下调,同时沉默JP3,4则会上调Megf10的表达。结合第一部分JP3,4与Megf10的免疫共沉淀结果,提示JP3,4与Megf10之间存在调控关系。在d 8+5用凋亡细胞刺激可提升JP4以及Megf10的表达。免疫荧光及电镜结果表明,在d 8+5,细胞的吞噬能力更强,能够同时吞噬2-3个凋亡细胞或者细胞碎片。此外,神经元之间被发现存在相互联系。进一步研究发现,在神经分化d 8+5,β-tubulinⅢ碎片被包裹于Megf10阳性的细胞中。同时,三维重构的结果也显示JP3,4阳性的细胞也能吞噬轴突碎片(β-tubulinⅢ日性)。此外,实验结果也表明,神经元阳性的细胞不会参与吞噬,而JP3,4及Megf10则大部分分布在神经元阳性细胞以及神经前体阳性细胞中,提示可能有表达JP3,4的胶质前体细胞参与轴突修剪。结论：1.ES细胞分化神经前体细胞过程,凋亡细胞能吸引吞噬细胞触发其吞噬作用,并促未成熟神经突极化。JP3和JP4在该过程维持胞内Ca~(2+)平衡并耦联这两个关键事件,确保神经突极化的完成。神经分化早期一过性高表达JP3和JP4,不仅为两种进化保守的膜连接蛋白属性,而且存在不同的分工及功能,这些功能在神经轴突形成极化中不可或缺。2.JP3和JP4在神经分化过程中后期参与轴突修剪,并分别与Megf10之间存在调控关系。Megf10及JP4能够参与轴突碎片的吞噬,从一定程度上揭示神经环路形成和完善过程的分子基础,为神经退行性疾病的修复认识提供有益的思路。
[Abstract]:Junctophilin 3 (JP3) and Junctophilin 4 (JP4) are highly expressed in the cerebral cortex, hippocampus and cerebellum. It is a kind of membrane binding protein.JP3,4 that exists in excitable cells to connect the cell membrane and the endoplasmic reticulum to form a membrane junction complex, which plays an important role in the maintenance of intracellular calcium homeostasis. The study found that the Jp3,4 gene knockout mice were born. It not only has serious developmental delay and high mortality rate, but also presents the impairment of memory cognitive function and movement coordination dysfunction, suggesting that these two proteins play an important role in maintaining animal memory and motor function, and are indispensable in the process of embryonic neural differentiation, but the expression changes and functions in the process of embryonic development. The characteristics and functional differences have not yet been reported that.JP3,4 can be involved in the establishment of the nervous system in the early stage of differentiation and development. The JP homologous protein Undertaker (UTA) expressed by the Drosophila can participate in the cleaning of apoptotic cells by maintaining the Ca~ (2+) balance within the phagocyte. The apoptotic cells cleaned two times, causing cytotoxicity and affecting the growth of the axons. The apoptotic cells release the "find-me" signal, rapidly attract the migration of the surrounding cells, and also expose the "eat-me" signal to ensure the recognition and phagocytosis of the phagocytes. However, the mammalian nerve tissue JPs is also the same. No reports have been reported so far. In addition to the strict and appropriate external environment, neuronal polarization and axon growth are regulated by extracellular signals. Apoptotic cells can summon the proliferation of peripheral cells, but the effect on nerve polarization and axonal growth has not yet been reported. At the same time, JP3,4 is connected to apoptosis, phagocytosis, and nerve polarization. In the early stages of neurodevelopment, undifferentiated protrusions, through competition, eventually form a single axon, and other protrusions will cease to grow or become dendrites. The process causes the formation and function of neurons to polarize. The polarization and growth of neurons require local Ca~ (2+) signals and cAMP, The local polarization is formed. The Ca~ (2+) of the TRPC internal flow can activate the specialization of the axon and activate the downstream CaMKI level combined signal to promote the formation of the axon. In the peripheral nerve cut off, the calcium wave will pass along the axon to the cell and initiate the regeneration of the axon. In addition, the Ca~ (2+) signal can also interact with the cAMP pathway to form positive feedback to magnify cA MP signals. In a number of protrusions, the highest concentration of cAMP can increase the internal cAMP concentration through positive feedback, prompting the protuberance to grow into an axon and the remaining protruding into a dendrite. However, how the intracellular cAMP signal is aroused, how the Ca~ (2+) signal is accurately regulated in the local region, and whether the intracellular Ca~ (2+) balance maintained by JP3,4 is based on the JP3,4 maintenance. The first part of this paper will explore the above problems. In the growth process of axon and dendrites, there will be an excess of axon branches, synapses, and dendritic trees. After the formation of the nerve loop, the redundant axon branches will be selectively pruned to ensure the specificity of neural signal transmission. Phagocytic receptor Meg. F10 can participate in the pruning and remodeling of synapses in the central nervous system, and Megf10 knockout mice retain an excessive number of synapses. The Megf10 homologous protein Draper of the Drosophila melanogaster has been found to be able to participate in the phagocytosis of the Drosophila embryo development process, and UTA completes this event with Draper. In this paper, the second part of this paper explores JP3,4 energy. The pruning and remodeling of axon in the process of embryonic stem cell differentiation and remodeling.Junctophilin-3, -4 connection phagocytosis participates in the nerve polarization. The first part of this thesis investigates the molecular mechanism of JP3,4 involved in the nerve polarization, and explores the correlation between the normal expression of JP3,4 and the local polarization of Ca~ (2+) /cAMP. The experimental results show that JP3,4 is in ES The expression of Jp3 and Jp4 in ES cells decreased significantly in the early stage of neural differentiation, which resulted in the destruction of the Ca~ (2+) /cAMP polarization positive feedback pathway and the inhibition of the nerve polarization. The inhibition of the peak of the early expression of JP3,4 caused the loss of spatial specificity of the Ca~ (2+) distribution in the cell cell of the nerve precursor and led to Ca~. (2+) transient capacity decreased significantly, while the content of cAMP decreased significantly and the cAMP-LKB1-SAD/MAR K2 polarization pathway was hindered. It was suggested that early JP3,4 expression had an important role in maintaining the local Ca~ (2+) /cAMP polarization positive feedback pathway in the cell of neural precursor cells. At the same time, the high expression of JP3,4 1 in the early stage of the differentiation of ES cells to the differentiated nerve was still associated with phagocytosis. It is closely related. The process of neural differentiation is accompanied by a large number of apoptosis, and the inhibition of early expression of JP3,4 leads to a significant decrease in the efficiency of neural precursor cells cleaning apoptotic cells. It suggests that JP3,4 needs to be expressed in a large number of apoptotic cells. Further studies have found that JP3,4 can induce apoptotic cells signal. The stimulation of apoptotic cells can be quickly extracted. The expression level of high JP4 and phagocytic receptor Megf10 can promote the binding of JP3 to RyR2, RyR3, and the combination of JP4 and Megf10 significantly up-regulated. These results suggest that, in addition to maintaining the spatial distance between the endoplasmic reticulum and the cell membrane, JP3 and JP4 are functionally divided in function, and can be involved in swallowing by JP3-RyR2,3 interaction and JP4-Megf10 interaction, respectively. At last, the experimental results show that apoptotic cells stimulate the content of intracellular cAMP and activate the cAMP-LKB1-SAD/MARK2 polarization pathway and promote the polarization of neurons. More importantly, the stimulation of apoptotic cells can enhance the transient capacity of the cytoplasm Ca~ (2+) of the neural precursor cells and facilitate the refinement of the nerve precursor. The results suggest that the neuronal differentiation and development process, the apoptotic cells can establish a connection with the neural precursor cells, not only can induce its phagocytosis, but also secrete signals to promote the nerve polarization and axon growth, and JP3,4 plays a bridge role. Appendix: JP3,4 participates in the study of axonal pruning in the process of nerve differentiation in the second part of this paper JP3,4 was involved in axon pruning in the process of neural differentiation. The experimental results showed that the expression of D 8+0 JP3 decreased and the expression of JP4 was up regulated by lentivirus silencing, while silent JP3 led to the up regulation of Megf10 expression, and the silence JP4 led to a slight downregulation of Megf10 expression, while silence JP3,4 would increase the expression of Megf10. Combined with the immunoprecipitation results of JP3,4 and Megf10 in the first part, it is suggested that there is a regulatory relationship between JP3,4 and Megf10. The stimulation of D 8+5 with apoptotic cells can enhance the expression of JP4 and Megf10. The results of immunofluorescence and electron microscopy show that the phagocytosis of the cells is stronger in D 8+5, and 2-3 apoptotic cells or cell fragments can be phagocytic at the same time. Interneurons were found to be interconnected. Further studies found that the fragments of D 8+5, beta -tubulin III were wrapped in Megf10 positive cells, and the results of three-dimensional reconstruction also showed that JP3,4 positive cells could also phagocytic axon fragments (beta -tubulin III diurnal). Furthermore, the results of the experiment also showed that the neurons positive were cells. Not involved in phagocytosis, while JP3,4 and Megf10 are mostly distributed in the positive neurons and positive neurons of the nerve precursor, suggesting that the glial precursor cells expressing JP3,4 may participate in axon pruning. Conclusion: the 1.ES cells differentiate into the neural precursor cells, and the apoptotic cells can attract phagocytes to trigger their phagocytosis and promote the immature. Neuroprotrusion.JP3 and JP4 maintain the balance and coupling of the intracellular Ca~ (2+) in this process to ensure the completion of the polarization of the nerve process. The early and overexpression of JP3 and JP4 in the early differentiation of nerve differentiation is not only the two evolutionary conserved membrane connexin properties, but also the different division of labor and function. These functions are polarized in the axon. .2.JP3 and JP4 are indispensable for axon pruning in the middle and late stages of neural differentiation, and the regulation relationship between Megf10 and.Megf10 can participate in the phagocytosis of axon fragments. To a certain extent, it reveals the molecular basis of the formation of the nerve loop and the improvement of the process, and provides a useful idea for the restoration of neurodegenerative diseases.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R741

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