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    題名: 大鼠紋狀體腦區 CK2/DARPP-32/GAD67 蛋白細胞訊息傳遞路徑對神經傳遞物質和運動行為影響之探討
    The influence of striatal CK2/DARPP-32/GAD67 signaling pathway on neurotransmitter and motor behavior of rats
    作者: 黃鉉豐
    Huang, Xuan Feng
    貢獻者: 趙知章
    Chao, Chi Chang
    黃鉉豐
    Huang, Xuan Feng
    關鍵詞: 紋狀體
    MSN 細胞
    蛋白激酶 CK2
    DARPP-32 蛋白
    麩胺酸脫羧酵素-67
    酪胺酸羥化酶
    多巴胺
    -丁氨基酪酸
    striatum
    medium spiny GABAergic neurons
    protein kinase CK2
    DARPP-32
    Glutamic acid decarboxylase 67
    -aminobutyric acid
    dopamine
    Tyrosine Hydroxylase
    日期: 2016
    上傳時間: 2016-03-01 10:41:29 (UTC+8)
    摘要: 蛋白激酶 CK2 是一種針對受質蛋白之絲胺酸/蘇胺酸進行磷酸化之多種功能的蛋白激酶，參與調節包括神經可塑性和神經保護等許多神經系統的功能，但是其分子層面的細胞機制目前尚未完全釐清。研究發現CK2在紋狀體腦區的表現量與活性皆高於其他腦區，而紋狀體之中型多刺狀GABA 神經元（medium spiny neuron, MSN）中DARPP-32 (Dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa) 蛋白的Ser102胺基酸被證實是 CK2 的磷酸化作用位置。許多研究文獻證實在多巴胺訊息傳遞路徑中， DARPP-32 蛋白之 Ser34/Thr75 的磷酸化現象參與藥物成癮相關生理行為的作用，然而 Ser102 的磷酸化作用與 MSN 細胞對運動行為調控的生理機制則仍待釐清。由於 MSN 細胞是藉由 -氨基丁酸 (GABA) 參與運動行為的控制，而負責GABA 生合成酵素之一的麩胺酸脫羧酵素-67 (GAD67) 的異常表現量被認為與巴金森氏症 (Parkinson’s disease) 引起之運動異常有相關性，但 GAD-67 的細胞調節機制仍待釐清，因此，論文研究的主軸是探討紋狀體 CK2 、 DARPP-32 和 GAD67 蛋白之間的訊息傳遞與神經傳遞物質和行為之間的關係。

    研究結果發現在大鼠紋狀體轉染野生型 CK2α DNA 質體會增加 DARPP-32 Ser102的磷酸化程度及 GAD67 mRNA 的表現量；而轉染 CK2 siRNA 則會降低 DARPP-32 蛋白含量和 Ser102 的磷酸化程度、 GAD67 蛋白含量和 mRNA 的表現量和紋狀體的 GABA 含量。轉染 DARPP-32 siRNA會降低 GAD67 蛋白含量及 mRNA 表現量和紋狀體的 GABA 含量；轉染突變型 DARPP-32 S102A DNA 質體（模擬胺基酸不能被磷酸化） 同樣會減少紋狀體的 GABA 含量。此外，共同轉染 CK2α DNA 和 DARPP-32 S102A DNA 質體會抑制單獨轉染 CK2α DNA 對提升紋狀體GABA和多巴胺含量增加的作用。在紋狀體給予 CK2 siRNA 觀察到黑質腦區 (substantia nigra) 酪胺酸羥化酶 (tyrosine hydroxylase, TH) 蛋白表現減少，給予 DARPP-32 siRNA 則觀察到黑質 TH Ser40 胺基酸磷酸化減少。在 rota-rod 運動行為測試中也可發現，轉染 CK2 siRNA 會抑制多巴胺受體致效劑 SKF38393 對促進運動能力的效果。綜合論文實驗的結果推測在紋狀體 MSN 細胞中，蛋白激酶 CK2 對 DARPP-32 蛋白 Ser102 磷酸化作用的細胞機制除了參與 GAD67 蛋白和神經傳遞物質 GABA 以及大鼠運動行為的生理調控外，亦可能回饋影響黑質腦區多巴胺神經細胞的 TH 蛋白含量和磷酸化程度。
    Protein kinase CK2 is a multifunctional serine/threonine protein kinase and involves in many neurophysiological functions including neuronal plasticity and neuroprotection, but its molecular mechanisms are not well investigated. Previous studies have shown that CK2 protein levels and activity are more elevated in the striatum than other brain areas. DARPP-32 (Dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa) is also highly enriched in striatal medium spiny GABAergic neurons and has been found the Ser102 residue is a phosphorylation site for CK2. Many studies have revealed that Ser34/Thr75 phosphorylation of DARPP-32 mediates dopamine signaling pathway which affects the physiological function and behavior in drug abuse. However, whether Ser102 phosphorylation by CK2 in the MSN controls motor behaviors is still unclear. Glutamic acid decarboxylase 67 (GAD67) which is one of the enzymes responsible for the synthesis of neurotransmitter GABA in the MSN and its dysfunction is presented relationship with Parkinson’s disease-induced behavior deficits. But the cellular regulatory mechanism of GAD67 is not fully studies. The aims of this proposal are to investigate the signaling relationship between CK2, DARPP-32 and GAD67 reflecting on neurotransmitter content in striatum and motor behavior in rats.
    The present results demonstrates that DARPP-32 Ser102 phosphorylation status and GAD67 mRNA levels are increased by wild-type CK2 plasmid DNA transfection. CK2 siRNA treatment also decreased DARPP-32 protein levels and Ser102 phosphorylation status, GAD67 protein and mRNA levels as well as GABA levels in the striatum. On the other hand, DARPP-32 siRNA transfection decreased GAD67 protein and mRNA levels, as also GABA levels in the striatum. TH Ser40 phosphorylation level in the substantia nigra. Striatal GABA levelds were decreased by transfection of mutant DARPP-32 S102A, which mimics the un-phosphorylated by CK2. Co-express CK2 and DARPP-32 S102A plasmid DNA reduced GABA level which was induced by CK2alone. The striatal CK2 or DARPP-32 siRNA transfection decreased Tyrosine Hydroxylase (TH) protein level or TH Ser40 phosphorylation level in the substantia nigra. Furthermore, DA agonist SKF38393 induced motor behavior promotion was inhibited by CK2 siRNA transfection. All the current results suggest that cellular signaling of DARPP-32 Ser102 phosphorylation by CK2 not only mediates GAD67 protein expression and biosynthesis of GABA neurotransmitter in striatum and motor behavior of rats, but also might affect TH protein level and phosphorylation status in the substantia nigra.
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    描述: 碩士
    國立政治大學
    神經科學研究所
    99754010
    資料來源: http://thesis.lib.nccu.edu.tw/record/#G0099754010
    資料類型: thesis
    顯示於類別:[神經科學研究所] 學位論文

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