内吞接头蛋白HIP1R在神经元树突发育和兴奋性突触形成中的作用

发布时间：2018-03-13 11:10

本文选题：HIP1R　切入点：树突发育　出处：《浙江大学》2016年博士论文　论文类型：学位论文

【摘要】：亨廷顿蛋白互作蛋白 1 相关蛋白(Huntingtin-interacting protein 1-related,HIP1R)、亨廷顿蛋白互作蛋白1(HIP1)以及在酵母中的同源蛋白Sla2p隶属同一个蛋白家族,都含有三个相似的结构域,即N末端的ENTH结构域、中间部位的coiled-coil结构域及C末端的talin-like结构域。这些结构域能分别与膜磷脂组分、网格蛋白(Clathrin)轻链以及F-肌动蛋白(F-actin)相互作用。近年来的研究表明,这些接头蛋白的功能可能与内吞体(Endosome)形成和转运有关,被归类为内吞接头蛋白(Endocytotic adaptor),但具体功能及生物学意义并不清楚。迄今为止仅有少数有关HIP1R的研究报告,提示其可能与某些肿瘤的发生有关,遗传学分析提示其可能是帕金森疾病的相关基因。我们也注意到有研究表明HIP1R在脑内各脑区均有较高水平的表达,其C末端还含有一个特有结构域负责与另一个内吞接头蛋白—皮层蛋白(Cortactin)相互作用。另一方面,神经元是形态上高度分化具有树突和轴突长突起的细胞,具有复杂和精致的信号处理机制,包括通过活化受体内吞形成信号内体并在细胞内转运介导信号转导的机制,因而进一步探讨内吞接头蛋白HIP1R在神经系统的作用并阐明其分子机制具有重要的意义。本博士学位论文采用免疫印迹、免疫组织和细胞化学、培养海马神经元的基因敲除、电生理等技术方法,研究发现:(1)HIP1R在大鼠的各个脑区广泛分布,不同发育阶段海马组织的出生后表达逐渐增加;海马神经元中有较高表达,呈颗粒状广泛分布,部分与PSD95共定位;(2)在shRNA敲减HIP1R表达的培养海马神经元中,发现其树突分支数目及总长度明显减少,分支复杂性明显降低,树突棘密度也明显降低;在HIP1R过表达的神经元结果与之相反,即树突分支、总长度及树突棘均增加;(3)采取遗传挽救(Rescue)和显性负向(Dominant negative)实验发现,含有与内吞接头蛋白皮层蛋白相互作用位点的HIP1R C末端是决定树突发育的关键功能区;(4)研究还发现,在HIP1R表达敲减的神经元中,AMPA受体和NMDA受体的表达明显降低,与之相一致,PSD95的密度也明显降低,微小兴奋性突触后电流(mEPSC)的电流峰值显著性降低;而GABA受体表达及mIPSC并未发生改变;提示HIP1R表达的敲减选择性损害了兴奋性突触的形成和功能。(5)在敲减HIP1R的培养的海马神经元中,表皮生长因子受体(EGFR)的早期内吞过程受损,EGFR-ERK通路信号传导受到破坏。综合以上研究结果表明,作为内吞接头蛋白HIP1R在海马神经元的树突生长发育以及兴奋性突触的形成过程中起着重要作用,其分子机制可能是HIP1R通过与皮层蛋白相互作用调节EGFR的内吞过程,影响EGFR-ERK通路的信号传导。
[Abstract]:Huntington protein interacting protein related protein 1 (Huntingtin-interacting protein, 1-related, HIP1R), Huntington protein interacting protein 1 (HIP1) and Sla2p homologous protein belong to the same family of proteins in yeast, there are three similar domains, namely ENTH domain N terminal, talin-like terminal coiled-coil domain domain and the middle part of the C. These domains respectively and membrane phospholipid components, clathrin light chain (Clathrin) and F- (F-actin) actin interaction. Recent studies show that these functions may be associated with endosomal adaptor proteins (Endosome) formation and transport, are classified as endocytosis joint protein (Endocytotic adaptor), but the specific function and biological significance is not clear. So far, only a few HIP1R related research reports, suggesting that it may occur with some tumor related genetic analysis. The may be associated with Parkinson disease. We also note that studies have shown that high level expression of HIP1R in different brain regions are in the brain, the C terminal also contains a unique domain responsible for another endocytic adaptor protein - protein interaction of cortex (Cortactin). On the other hand, neurons are morphological differentiation with dendrites and axons of cells with long processes, signal processing mechanism of the complex and sophisticated, including through activation of receptor endocytosis in the body and the formation mechanism of signal transport in the cells mediated signal transduction, thus further study has important significance and endocytic adaptor protein HIP1R to clarify the molecular mechanism in the nervous system the role of this doctoral dissertation. By Western blot, immunohistochemistry and immunocytochemistry in cultured hippocampal neurons, gene knockout, found electrophysiological study methods such as: (1) HIP1R in rats All regions are widely distributed in the hippocampus at different developmental stages after birth was increased; the higher the expression of hippocampal neurons in the granular, widely distributed, and the co localization of PSD95; (2) in shRNA on cultured hippocampal neurons reduced the expression of HIP1R, found that the dendritic branches and the total length was significantly reduced, branch of complexity is significantly reduced, the density of dendritic spines were significantly decreased; in the over expression of HIP1R neurons in the opposite, that is the total length of the dendritic branches, and dendritic spines were increased; (3) to save the genetic (Rescue) and dominant negative (Dominant negative) found that HIP1R and C terminal containing endocytic adaptor protein cortex protein interaction sites is a key function of determining region of dendritic development; (4) the study also found that the expression of HIP1R knockdown neurons, the expression of AMPA receptor and NMDA receptor significantly decreased, consistent with the PSD95. Is also significantly reduced, miniature excitatory postsynaptic currents (mEPSC) of the peak current decreased significantly; while the expression of GABA receptor and mIPSC did not change; prompt formation and function expression of HIP1R knockdown selective damage of excitatory synapses. (5) in HIP1R knockdown in cultured hippocampal neurons, epidermal growth factor receptor (EGFR) in the process of early swallowing impairment, EGFR-ERK signal pathway is damaged. The above research results show that plays an important role as an endocytic adaptor protein HIP1R on the growth of hippocampal neurons and dendritic development of excitatory synapses in the formation process of the molecular mechanisms of endocytosis may be adjusted by the EGFR and HIP1R cortex protein interaction, effects of signal transduction of EGFR-ERK pathway.

【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】：Q42

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