Taipei Medical University Institutional Repository:Item 987654321/6786
English  |  正體中文  |  简体中文  |  全文笔数/总笔数 : 45069/58245 (77%)
造访人次 : 2368149      在线人数 : 170
RC Version 7.0 © Powered By DSPACE, MIT. Enhanced by NTU Library IR team.
搜寻范围 查询小技巧：
  • 您可在西文检索词汇前后加上"双引号"，以获取较精准的检索结果
  • 若欲以作者姓名搜寻，建议至进阶搜寻限定作者字段，可获得较完整数据
    •  进阶搜寻


    jsp.display-item.identifier=請使用永久網址來引用或連結此文件: http://libir.tmu.edu.tw/handle/987654321/6786


    题名: 生長相關蛋白GAP-43 Ser41突變株影響發育期大腦皮質神經元中Gephyrin及GABAA受器交互作用之探討
    The Effect of GAP-43 Ser41 Mutants on the Gephyrin- GABAA Receptor Interaction in Developing Cortical Neurons
    作者: 宋伊平
    Yi-Ping Song
    Contributors: 醫學科學研究所
    Keywords: 抑制性神經傳導物
    生長相關蛋白
    抑制性神經傳導物受器聚集蛋白
    GABAA receptor
    GAP-43
    gephyrin
    cotrical neuron
    Date: 2009
    Issue Date: 2009-09-14 15:17:45 (UTC+8)
    Abstract: 神經生長相關蛋白(Growth-associated protein-43; GAP-43)是一種高度表現在發育時期神經細胞軸突生長錐的膜蛋白，GAP-43負責神經發育及修復時突觸的生長、分支，改變前突觸膜(presynaptic membrane)構造以利釋放神經傳導物。本實驗室先前在蛋白質體研究中利用MALDI-TOF發現GAP-43會與一受器聚集蛋白(receptor clustering protein) - gephyrin結合。Gephyrin的功能是聚集突觸後膜上的氯離子通道神經傳導物受器，包括GABAA receptor與glycine receptor，用以增加抑制型突觸傳遞效能。GABAA receptor為大腦中最主要的抑制神經傳導物受器，是由五個次單元組成的氯離子通道，已知gephyrin會經由與GABAA receptor的 γ2 subunit(GABAARγ2)結合來調控受器的聚集，先前研究發現，投予蛋白質激酶C (protein kinase C) 抑制劑抑制GAP-43活性後， GAP-43與gephyrin的結合有明顯地增加，故推測GAP-43的磷酸化可能會改變gephyrin與GABAARγ2的結合，進而影響GABAA receptor在後突觸膜上的表現。
    在本研究中，我使用初代培養的胚胎期大腦皮質神經細胞(Cortical neurons)為實驗系統，在發育時期的cortical neurons中投予100 nM PKC inhibitor( Ro318220)處理，發現會減少GABAARγ2和gephyrin的結合，且在細胞膜上的GABAARγ2表達量減少。為了模擬不同活性的GAP-43，我們建立了兩個GAP-43 S41位置之突變株(mutant)：將GAP-43 S41置換為Aspartic acid (S41D)的擬磷酸化mutant，及GAP-43 S41置換為Alanine(S41A)的擬去磷酸化mutant。過度表達GAP-43S41D可增加GABAAR與gephyrin的結合能力，且促進GABAARγ2在細胞膜上的表現；而過度表現GAP-43S41A會減少GABAAR與gephyrin的結合能力及細胞膜上GABAARγ2的量。最後，我們發現以離子通道阻斷劑阻斷神經電活性，也會增加gephyrin與GABAARγ2的結合。 因此，本研究證實了神經發育及再生相關生長蛋白GAP-43的活性會影響GABAA receptor在抑制型突觸形成過程中的表現與分佈，此作用可能是透過GAP-43的不同活化態會影響gephyrin與GABAARγ2的結合程度所致。GAP-43會藉由其PKC磷酸化態與gephyrin的結合與否，調控gephyrin聚合GABAA receptor的程度，及GABAA receptor在細胞表面的分布，此作用可能進而影響GABA突觸的發育與成熟後的傳導效能。

    Growth-associated protein 43 (GAP-43) is a membrane protein highly expressed in the growth cone of developing and regenerating neurons. GAP-43 can be activated by protein kinase C (PKC) upon phosphorylation at serine 41 (S41) to promote neurite outgrowth. During neuronal development, GABAA receptors are clustered by gephyrin at γ2 subunit (GABAARγ2) to enhance their synaptic transmission efficacy. In our previous study, we found that blockade of PKC activity by PKC inhibitor RO318220 induce the association between GAP-43 and gephyrin. In the present study, we further found that PKC inhibitor decreased GABAA receptors on cell surface presentation. This evidence suggest that PKC phosphorylation of GAP-43 may regulate the gephyrin-mediated GABAA receptor clustering. To test this hypothesis, we established a wild type GAP-43 expression construct and two GAP-43 mutants by replacing the GAP-43 Ser41, the PKC phosphorylation site with aspartic acid (S41D) or alanine (S41A) to mimic its phosphorylated or dephosphorylated forms, respectively, and transfected these mutants into developing cortical neurons at 4 days in vitro for their overexpression. The results show that the GAP-43S41D-transfected neurons exhibited long and more arborizing neuritis, whereas GAP-43S41A-transfected ones show retracted neurites. In addition, GAP-43S41D overexpression was accompanied by increase in the interaction of GABAARγ2 and gephyrin, GABAARγ2 aggregation in the cell periphery, and the surface expression of GABAAR as revealed by biotinylation assay, whereas GAP-43S41A transfection gave rise to opposite results. Finally, we found that blocked of neuronal activity by voltage-gated sodium channel blocker tetrodotoxin and calcium channel blocker nifedipine also increase the association between gephyrin and GABAARγ2. Together, the results suggest that PKC-mediate GAP-43 phosphorylation promotes the gephyrin-GABAARγ2 interaction and enhances the surface expression of GABAAR. This event depends on neuronal activity, further suggest that the activity-dependent neuroplasticity plays a pivotal role in the establishment of GABAergic synapse and inhibitory neurotransmission.
    Data Type: thesis
    Appears in Collections:[醫學科學研究所] 博碩士論文

    Files in This Item:

    File Description SizeFormat
    摘要.doc32KbMicrosoft Word100View/Open
    摘要.pdf147KbAdobe PDF184View/Open
    摘要.ppt115KbMicrosoft Powerpoint145View/Open
    摘要.ps599KbPostscript59View/Open


    All items in TMUIR are protected by copyright, with all rights reserved.

    著作權聲明 Copyright Notice

    • 本平台之數位內容為臺北醫學大學所收錄之機構典藏，包含體系內各式學術著作及學術產出。秉持開放取用的精神，提供使用者進行資料檢索、下載與取用，惟仍請適度、合理地於合法範圍內使用本平台之內容，以尊重著作權人之權益。商業上之利用，請先取得著作權人之授權。

      The digital content on this platform is part of the Taipei Medical University Institutional Repository, featuring various academic works and outputs from the institution. It offers free access to academic research and public education for non-commercial use. Please use the content appropriately and within legal boundaries to respect copyright owners' rights. For commercial use, please obtain prior authorization from the copyright owner.

    • 瀏覽或使用本平台，視同使用者已完全接受並瞭解聲明中所有規範、中華民國相關法規、一切國際網路規定及使用慣例，並不得為任何不法目的使用TMUIR。

      By utilising the platform, users are deemed to have fully accepted and understood all the regulations set out in the statement, relevant laws of the Republic of China, all international internet regulations, and usage conventions. Furthermore, users must not use TMUIR for any illegal purposes.

    • 本平台盡力防止侵害著作權人之權益。若發現本平台之數位內容有侵害著作權人權益情事者，煩請權利人通知本平台維護人員([email protected])，將立即採取移除該數位著作等補救措施。

      TMUIR is made to protect the interests of copyright owners. If you believe that any material on the website infringes copyright, please contact our staff([email protected]). We will remove the work from the repository.

    Back to Top
    DSpace Software Copyright © 2002-2004  MIT &  Hewlett-Packard  /   Enhanced by   NTU Library IR team Copyright ©   - Feedback