本文選題：Junctophilins(JPs) + Megf10　； 參考：《浙江大學(xué)》2016年博士論文

【摘要】：Junctophilin 3 (JP3) 和 Junctophilin 4 (JP4)在大腦皮層、海馬及小腦高表達(dá),是一類存在于可興奮細(xì)胞中的膜結(jié)合蛋白。JP3,4能夠連接細(xì)胞膜和內(nèi)質(zhì)網(wǎng)膜,形成膜連接復(fù)合物,對維持胞內(nèi)鈣穩(wěn)態(tài)有著重要作用。研究發(fā)現(xiàn),Jp3,4基因雙敲除的小鼠出生后不但出現(xiàn)嚴(yán)重的發(fā)育遲緩及致死率高,而且呈現(xiàn)記憶認(rèn)知功能損傷和運(yùn)動協(xié)調(diào)功能失常,提示這兩種蛋白在維持動物記憶和運(yùn)動功能上起重要作用,并在胚胎神經(jīng)分化發(fā)育過程中的不可或缺性。但其在胚胎發(fā)育過程的表達(dá)變化、功能特征以及功能差異尚未見報道。JP3,4能否在神經(jīng)分化發(fā)育早期便參與神經(jīng)系統(tǒng)的建立也亟待探索。果蠅表達(dá)的JP同源蛋白Undertaker (UTA),能通過維持吞噬細(xì)胞內(nèi)Ca~(2+)平衡來參與凋亡細(xì)胞的清理。神經(jīng)分化過程伴隨大量細(xì)胞凋亡,未被及時清理的凋亡細(xì)胞則會發(fā)生二次壞死,導(dǎo)致細(xì)胞毒性并影響軸突的生長。凋亡的細(xì)胞會釋放出"find-me"信號,快速吸引周圍的細(xì)胞遷移,同時也會暴露出"eat-me"信號,保證吞噬細(xì)胞的識別和吞噬。然而,哺乳動物神經(jīng)組織JPs是否也具有相同的功能迄今未見報道。除了需要嚴(yán)格和適宜的外部環(huán)境,神經(jīng)元極化及軸突生長還受胞外信號的調(diào)控。凋亡細(xì)胞能夠召喚周圍細(xì)胞的增殖,但是否能對神經(jīng)極化以及軸突生長產(chǎn)生影響尚未見報道。同時,JP3,4在連接凋亡、吞噬及神經(jīng)極化之間扮演的角色也亟待闡明。在神經(jīng)發(fā)育初期,未分化的突起通過競爭,最終只形成單一的軸突,而其他的突起將會停止生長或者變成樹突,該過程使神經(jīng)元在形態(tài)和功能上都形成了極化。神經(jīng)元的極化及生長需要局部的Ca~(2+)信號和cAMP,形成局部極化。通過TRPC內(nèi)流的Ca~(2+)能夠啟動軸突的特化,并激活下游CaMKI等級聯(lián)信號,促使軸突形成。而在軸突切斷的外周神經(jīng)中,鈣波會沿著軸突傳向胞體,啟動軸突的再生。此外,Ca~(2+)信號還可以和cAMP通路相互作用形成正反饋,放大cAMP信號。在諸多突起中,cAMP濃度最高的突起能夠通過正反饋不斷提升內(nèi)部cAMP濃度,促使該突起長成軸突,而剩下突起則長成樹突。然而,胞內(nèi)cAMP信號如何被喚起,Ca~(2+)信號如何在局部區(qū)域被精準(zhǔn)調(diào)控,基于JP3,4維持的胞內(nèi)Ca~(2+)平衡是否與此相關(guān)聯(lián)尚未完全明確。本論文第一部分將對以上問題做出探索。在軸突和樹突的生長過程,會有過量的軸突分支、突觸以及樹突樹形成,在形成神經(jīng)環(huán)路后,多余的軸突分支將會被選擇性修剪,以保證神經(jīng)信號傳遞的專一性。吞噬受體Megf10能夠參與中樞神經(jīng)系統(tǒng)中突觸的修剪與重塑,Megf10敲除小鼠則保留了過多數(shù)量的突觸。果蠅Megf10的同源蛋白Draper已被發(fā)現(xiàn)能夠參與果蠅胚胎發(fā)育過程的吞噬,而UTA則與Draper協(xié)同完成這一事件。因此,本論文第二部分(附)探索了JP3,4能否在胚胎干細(xì)胞分化神經(jīng)過程中參與軸突的修剪及重塑。Junctophilin-3,-4連接吞噬參與神經(jīng)極化作用研究本論文第一部分考察了JP3,4參與神經(jīng)極化的分子機(jī)制,探索JP3,4正常表達(dá)與Ca~(2+)/cAMP形成局部極化的相關(guān)性。實驗結(jié)果顯示,JP3,4在ES細(xì)胞定向分化神經(jīng)元早期有一過性高表達(dá)。在ES細(xì)胞沉默Jp3和Jp4,其神經(jīng)分化早期表達(dá)顯著降低,導(dǎo)致Ca~(2+)/cAMP極化正反饋通路受破壞,進(jìn)而抑制神經(jīng)極化。抑制JP3,4早期表達(dá)高峰使神經(jīng)前體細(xì)胞胞內(nèi)Ca~(2+)分布失去空間特異性,并導(dǎo)致Ca~(2+)瞬變能力顯著減弱,同時cAMP含量明顯下降,cAMP-LKB1-SAD/MAR K2極化通路激活受阻。提示JP3,4早期高表達(dá)對維持神經(jīng)前體細(xì)胞胞內(nèi)局部Ca~(2+)/cAMP極化正反饋通路有重要作用。與此同時,ES細(xì)胞定向分化神經(jīng)早期JP3,4一過性高表達(dá)尚與吞噬作用密切相關(guān)。神經(jīng)分化過程伴隨大量凋亡,抑制JP3,4早期表達(dá)致使神經(jīng)前體細(xì)胞清理凋亡細(xì)胞的效率顯著下降。提示JP3,4在凋亡細(xì)胞清理過程需要被大量表達(dá)。進(jìn)一步研究發(fā)現(xiàn),JP3,4能夠感應(yīng)凋亡細(xì)胞的信號。凋亡細(xì)胞的刺激可快速提高JP4及吞噬受體Megf10的表達(dá)水平,并可促進(jìn)JP3與RyR2、RyR3的結(jié)合,同時,JP4與Megf10的結(jié)合也顯著上調(diào)。這些結(jié)果提示,除了維持內(nèi)質(zhì)網(wǎng)與細(xì)胞膜的空間距離,JP3與JP4在功能上各有分工,可分別通過JP3-RyR2,3相互作用及JP4-Megf10相互作用參與吞噬,保證神經(jīng)極化適宜的微環(huán)境。最后,實驗結(jié)果顯示,凋亡細(xì)胞刺激可顯著提升胞內(nèi)cAMP的含量并激活cAMP-LKB1-SAD/MARK2極化通路,促進(jìn)神經(jīng)元極化。更為重要的是,凋亡細(xì)胞的刺激能夠提升神經(jīng)前體細(xì)胞胞漿Ca~(2+)瞬變能力,易化神經(jīng)前體細(xì)胞極化。該結(jié)果提示神經(jīng)分化發(fā)育過程,凋亡細(xì)胞可與神經(jīng)前體細(xì)胞建立聯(lián)系,不僅可誘導(dǎo)其吞噬,同時也能分泌信號促進(jìn)神經(jīng)極化及軸突生長,而JP3,4在其中起著橋梁作用。附：JP3,4參與神經(jīng)分化過程軸突修剪研究本論文第二部分初步探索了JP3,4在神經(jīng)分化過程參與軸突修剪。實驗結(jié)果顯示,通過慢病毒沉默吞噬受體Megf10可導(dǎo)致d 8+0 JP3的表達(dá)下降,而JP4的表達(dá)上調(diào)。而沉默JP3則導(dǎo)致Megf10的表達(dá)上調(diào),沉默JP4導(dǎo)致Megf10的表達(dá)輕微下調(diào),同時沉默JP3,4則會上調(diào)Megf10的表達(dá)。結(jié)合第一部分JP3,4與Megf10的免疫共沉淀結(jié)果,提示JP3,4與Megf10之間存在調(diào)控關(guān)系。在d 8+5用凋亡細(xì)胞刺激可提升JP4以及Megf10的表達(dá)。免疫熒光及電鏡結(jié)果表明,在d 8+5,細(xì)胞的吞噬能力更強(qiáng),能夠同時吞噬2-3個凋亡細(xì)胞或者細(xì)胞碎片。此外,神經(jīng)元之間被發(fā)現(xiàn)存在相互聯(lián)系。進(jìn)一步研究發(fā)現(xiàn),在神經(jīng)分化d 8+5,β-tubulinⅢ碎片被包裹于Megf10陽性的細(xì)胞中。同時,三維重構(gòu)的結(jié)果也顯示JP3,4陽性的細(xì)胞也能吞噬軸突碎片(β-tubulinⅢ日性)。此外,實驗結(jié)果也表明,神經(jīng)元陽性的細(xì)胞不會參與吞噬,而JP3,4及Megf10則大部分分布在神經(jīng)元陽性細(xì)胞以及神經(jīng)前體陽性細(xì)胞中,提示可能有表達(dá)JP3,4的膠質(zhì)前體細(xì)胞參與軸突修剪。結(jié)論：1.ES細(xì)胞分化神經(jīng)前體細(xì)胞過程,凋亡細(xì)胞能吸引吞噬細(xì)胞觸發(fā)其吞噬作用,并促未成熟神經(jīng)突極化。JP3和JP4在該過程維持胞內(nèi)Ca~(2+)平衡并耦聯(lián)這兩個關(guān)鍵事件,確保神經(jīng)突極化的完成。神經(jīng)分化早期一過性高表達(dá)JP3和JP4,不僅為兩種進(jìn)化保守的膜連接蛋白屬性,而且存在不同的分工及功能,這些功能在神經(jīng)軸突形成極化中不可或缺。2.JP3和JP4在神經(jīng)分化過程中后期參與軸突修剪,并分別與Megf10之間存在調(diào)控關(guān)系。Megf10及JP4能夠參與軸突碎片的吞噬,從一定程度上揭示神經(jīng)環(huán)路形成和完善過程的分子基礎(chǔ),為神經(jīng)退行性疾病的修復(fù)認(rèn)識提供有益的思路。
[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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R741

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 劉海英,張慶俊,趙宗茂;人臍血干細(xì)胞的特性及其神經(jīng)分化的研究進(jìn)展[J];河北醫(yī)藥;2004年02期

2 王翠琴;張廣宇;彭越;彭濤;賈延R，

本文編號：1885402