漢丁頓關聯蛋白-1在神經細胞分化中調控斷裂點叢集區域蛋白的活性

Huntingtin associated protein 1 modulates Break point cluster region protein activity in neuronal differentiation

Shima Ahmad Salim(Shima Ahmad Salim)

指導教授 ： 林詠峯

臺北醫學大學/醫學科技學院/醫學檢驗暨生物技術學系/碩士(2013年)

https://doi.org/10.6831/TMU.2013.00029

摘要

突變的漢丁頓蛋白〈Huntintin;Htt〉會經由異常的蛋白相互作用影響神經細胞受體和囊泡的傳輸系統，而細胞內傳輸系統損壞可導致漢丁頓舞蹈症〈Huntinton’sDisease;HD〉。第一個被發現的Htt關聯蛋白〈HAP1〉，已知是參與細胞內傳輸的蛋白。最近研究表示，神經細胞失去HAP1會抑制微小管依賴性的運輸，導致小鼠下視丘神經纖維完整性和延展性的損壞，類似於HD的腦下視丘退化。從立體測量學的分析中, 我們注意到在HAP1缺失的小鼠腦中分化的阿立新〈orexin〉神經元有減少的現象。短暫轉染表達HAP1，再以流式細胞儀分析，則發現大量表達HAP1會減緩神經細胞的增生，這支持HAP1有促進神經元分化的角色。為了進一步了解HAP1如何參與微小管的運輸和神經元的分化，我們溶解HAP1缺失鼠和野生鼠的腦組織，並用紫杉醇〈taxol〉去沈澱其微小管複合體，再作蛋白質體分析，我們發現許多鳥嘌呤交換因子〈guanine exchange factors; GEFs〉，如斷裂點叢集區域蛋白〈Breakpoint Cluster Region Protein; BCR〉，是HAP1可能的夥伴蛋白。我們進一步用免疫染色觀察到BCR和HAP1在共轉染的Neuro2a細胞內有共同定位的分佈。最後，我們的西方墨點結果顯示 HAP1的缺失抑制了小鼠下視丘神經元內 BCR 以及下游效用蛋白的活化。因此，我們得出的結論是 HAP1 調控 BCR 功能，並促進小鼠下視丘的神經元分化和保護。

關鍵字

漢丁頓舞蹈症 ； 神經退化 ； 漢丁頓關聯蛋白-1 ； 斷裂點叢集區域蛋白 ； 下視丘

並列摘要

Mutant huntingtin (Htt) can affect vesicular and receptor trafficking via its abnormal protein interactions, suggesting that impairment of intracellular trafficking contributes to Huntington’s disease (HD). The first identified partner of Htt Associated Protein-1 (HAP1), is known to be involved in intracellular trafficking. Recent studies showed that Loss of HAP1 inhibits microtubule-dependent trafficking in neurons, and results in impairment of neurite integrity and extension in mouse hypothalamus, resembling hypothalamic degeneration in HD. In a stereological analysis, we noticed a reduction of differentiated orexin neurons in the HAP1-defficient mouse brain. Transient transfection followed by flow cytometric analysis demonstrated that HAP1 over-expression reduced neuronal cell proliferation, supporting a role for HAP1 in promoting neuronal differentiation. To further understand the mechanisms how HAP1 is involved in microtubule-dependent trafficking and neuronal differentiation, we performed a proteomic analysis using taxol-precipitated microtubules from HAP1-null and wild-type mouse brains. Guanine exchange factors (GEFs), such as Breakpoint Cluster Region Protein (BCR), were identified as potential HAP1-interacting partners. Then the co-localization of BCR and HAP1in Neuro2a co-transfected cells was observed by immunostaining in confocal microscopy. Finally our western blotting results showed that activation of BCR and its downstream effecters are inhibited in HAP1-defficient mouse hypothalamus. In conclusion, HAP1 modulates BCR function to promote neuronal differentiation and protection in mouse hypothalamus.

並列關鍵字

huntington ； desease ； HD ； neurodegeneration ； HAP1 ； BCR ； hypothalamus

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