返回
类型 基础研究 预答辩日期 2017-11-30
开始(开题)日期 2015-09-15 论文结束日期 2017-07-18
地点 李文正楼北225 论文选题来源 国家自然科学基金项目     论文字数 5.6 (万字)
题目 果蝇Neurexin调控突触囊泡募集和释放的机制研究
主题词 果蝇,神经肌肉接头,微丝蛋白,突触囊泡
摘要 突触的形成,发育,成熟是一个高度复杂和动态的过程,突触囊泡作为神经递质的运载体在神经元之间的信号传递中扮演者十分重要的作用。干扰突触囊泡的转运和神经递质的释放将导致大脑功能的异常,引发一系列的神经疾病。之前关于突触囊泡与突触前膜接近,锚定,融合以及释放等过程已经研究的很多。研究发现许多突触相关分子参与这一过程的调控,例如Synaptotagmins, Synapsins, Synaptobrevins和Munc18等。大量的研究同样显示,突触囊泡运载着神经递质沿着骨架分子在轴突中转运,到达突触末端形成不同的囊泡池,靠近突触前膜准备释放的叫做准备释放池(RRP),远离突触前膜供应释放的叫做储存池(RP)。这些特异的结构对于正常的神经递质释放是必须的。然而,突触囊泡是如何募集在突触的特定区域,维持正常的神经信号传递的目前还不清楚。 研究表明突触粘附分子Neurexin(NRX)和Neuroligin(NLG)在突触前后膜上相互作用形成异源的二聚体,它们在突触的形成、发育、成熟、可塑性等过程中扮演者着十分重要的作用。医学研究发现nrx和nlg基因的突变与自闭症等精神疾病存在相互关系。之前的研究阐述了NRX参与调控突触的结构和功能,但是其中涉及到的详细的分子机制并不清楚。我们利用果蝇的神经肌肉接头(NMJ)为模型,研究果蝇NRX (DNRX) 参与调控囊泡的轴突转运,突触末端分布以及释放等过程的分子机制,进一步揭示NRX在突触中的重要功能。 NRX 是一类一次跨膜的神经细胞粘附分子,它广泛表达在中枢以及外周神经系统。我们的研究发现在体缺失DNRX导致突触末端囊泡弥散分布以及自发状态下囊泡释放频率增高。为了研究DNRX调控突触囊泡分布以及释放的分子机制,我们在dnrx突变体背景下,用actin组装相关分子挽救囊泡缺陷的表型,结果显示DNRX通过F-actin调控突触囊泡的募集和释放。利用生物化学的方法,我们发现了一种抑癌基因编码的蛋白Scribble能够通过它的四个PDZ结构域和DNRX的PDZ结合位点直接的相互作用。另外,结果显示Scribble可以作为一座桥梁介导DNRX和Dpix之间的连接。 Dpix是一个Rac/Cdc42特异的鸟苷酸交换因子,它可以通过激活Rac1促进actin的聚集。为了进一步验证DNRX是通过PDZ结合位点调控突触囊泡的募集和释放,接下来我们构建了缺失PDZ结合位点(缺失c末端7个氨基酸)的DNRX的转基因果蝇(DNRXΔPDZ)。我们发现在dnrx突变体背景下,突触前过表达DNRXΔPDZ却不能挽救dnrx突变体中F-actin和突触囊泡缺陷的表型。最后,我们检测了野生型,dnrx突变体以及DNRX和Scribble下调后活性形式Rac1(Rac1-GTP)的水平,结果显示缺失或下调DNRX和Scribble都将导致Rac1-GTP水平降低。为了进一步验证Rac1参与DNRX对突触前F-actin和突触囊泡的调控,我们将功能异常的Rac1(dominant nagetive Rac1,Rac1DN)整合到dnrx突变体背景下,通过突触前过表达观察能否挽救dnrx突变体中观察到的表型缺陷,免疫染色和电生理结果显示Rac1DN同样无法挽救dnrx突变体中F-actin和突触囊泡缺陷的表型。 总结,我们发现了DNRX和Scribble/Dpix相互作用形成一个复合体通过激活Rac1从而调控突触前actin的聚集,维持突触囊泡在突触末端的正常分布以及释放。另外,这一发现将加深我们对神经疾病,例如自闭症的病理机制的理解。
英文题目 Neurexin Directly Interacts with the Scribble-Pix Complex to Stimulate F-actin Assembly for Synaptic Vesicle Clustering
英文主题词 Drosophila, Neuromuscular junction, Actin,Synaptic vesicle
英文摘要 The synapse is a highly specialized and dynamic structure, which is involved in regulating neurotransmission. As the vehicles of neurotransmitters, synaptic vesicles (SVs) are essential for passing of neural signals from one neuron to another. Disruptions in SV trafficking and release will lead to various forms of neurological disorders. The process of synaptic vesicles priming, docking and fusion with presynaptic membrane has been described thoroughly, and a lot of molecules involved in this process have been identified, such as Synaptotagmins, Synapsins, Synaptobrevins and Munc18. It was also reported that SVs can cluster in specific synaptic compartment to form distinct pools and this well-regulated form is significant for normal neurotransmitter release. However, how SV cluster to particular region and maintain normal neurotransmitter release is not very clear yet. Previous studies have showed that Neurexin (NRX) and Neuroligins (NLGs) can form heterodimer at synaptic membrane and it is essential for the formation, development, maturity and plasticity of synapse. Medical research has also found that the mutation of nrx and nlg are associated with mental disorders, such as autism. Previous studies have showed that NRX is involved in the regulation of the structure and function of synapses, however, the detailed molecular mechanism is not very clear. We used Drosophila NMJ as a model system and revealed the significant role of Drosophila NRX (DNRX) in modulating SV clustering and release at synaptic terminals. NRX is a kind of synaptic adhesion molecule, it is widely expressed in the central and peripheral nervous system. Our results, for the first time, demonstrated that DNRX regulates the clustering and spontaneous release frequency of SVs through presynaptic F-actin. Mechanistically, we employed integrative approaches and demonstrated that DNRX can directly bind to Scribble, a tumor suppressor protein, via its c terminal PDZ-binding motif. Following, we have also revealed that Scribble bridges DNRX to Dpix, a Rac/Cdc42-specific guanine nucleotide exchange factor (GEF). Dpix can activate Rac1, which is involved in pathways that promote actin polymerization and a well-regulated function of the brain. To determine the specific motif of DNRX that participate in the regulation of F-actin and SV, according to the previous result we constructed the transgenic fly with the deletion of PDZ-binding motif (DNRXΔPDZ), after driving DNRXΔPDZ at presynapse in dnrx mutant we have found that DNRXΔPDZ could not rescue the defeat of F-actin assembly and SV release observed in dnrx mutant. Finally, we have found that the level of Rac1-GTP is decreased after reducing the expression of DNRX or Scribble, it indicates DNRX regulates Rac1-GTP level via Scribble/Dpix complex physiologically. To further confirm the function of Rac1 in this process, we got the donimat nagetive Rac1 (Rac1DN) and intergrated it into dnrx mutant background, after inducing Rac1DN at presynapse in dnrx mutant we still could not find any rescue effect in F-actin assembly and SV release. In summary, we have provided important new insights into the function of DNRX in modulating pre-synaptic F-actin through the Scribble/Dpix complex, and how it regulates presynaptic SV clustering and release via activating Rac1. On the other hand, the findings will deepen the understanding for the pathological mechanism of neurological disorders such as autism.
学术讨论
主办单位时间地点报告人报告主题
东南大学 2016-09-02 李文正楼北225 钱进军 Functional studies on one intronic miR-932 hosted in Drosophila Neuroligin2
东南大学 2016-12-12 李文正楼北225 屠仁军 Mechanism and function of Drosophila Neuroligin 2 proteolytic cleavage
东南大学 2017-01-16 李文正楼北225 芮梦龙 Neurexin and Muscular Dystrophy
东南大学 2017-03-29 李文正楼北225 吴俊 Proteolytic cleavage of Drosophila Neuroligin3 is essential for maintaining locomotor activity
东南大学 2017-04-17 李文正楼南213 Wang hongyu New animal models for studying regulation of lipid metabolism
东南大学 2017-05-31 李文正楼北225 芮梦龙 Neurexin Interacts with Scribble-Pix Complex to Regulate Synaptic Vesicle Clustering via F-actin
东南大学 2017-06-12 李文正楼北225 芮梦龙 Neurexin Interacts with Dystroglycan and Laminin C to Sustain Normal Muscle Architecture and Function in Drosophila
东南大学 2017-06-26 李文正楼北225 芮梦龙 Neurexin Directly Interacts with the Scribble-Pix Complex to Stimulate F-actin Assembly for Synaptic Vesicle Clustering
     
学术会议
会议名称时间地点本人报告本人报告题目
第四届亚太果蝇大会 2017/05/08-11 日本.大阪 The Neuronal Protein Neurexin Directly Interacts with the Scribble-Pix Complex to Stimulate F-actin Assembly for Synaptic Vesicle Clustering
2013江苏省遗传学会年会 2013/11/23-24 中国.徐州 Neurexin Interacts with the Scribble-Pix Complex to Regulate SV Clustering via F-actin
     
代表作
论文名称
The Neuronal Protein Neurexin Directly Interacts with the Scribble-Pix Complex to Stimulate F-actin
 
答辩委员会组成信息
姓名职称导师类别工作单位是否主席备注
陈大华 正高 教授 博导 中国科学院动物研究所
赵允 正高 教授 博导 中国科学院生物化学与细胞生物学研究所
韩俊海 正高 教授 博导 东南大学
方明 正高 教授 博导 东南大学
周子凯 副高 副教授 博导 东南大学
      
答辩秘书信息
姓名职称工作单位备注
刘力娟 其他 讲师 东南大学