克林達黴素透過干擾細胞自噬作用誘發可溶性澱粉樣蛋白前驅蛋白-α表現

Clindamycin Upregulated Soluble Amyloid Precursor Protein α Through Autophagy Flux Impairment

高培鈞(Pei-Jun Kao)

指導教授 ： 康照洲

國立臺灣大學/醫學院/毒理學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.10361

摘要

克林達黴素為目前廣泛使用於感染症、瘧疾以及青春痘的抗生素，在實驗室前人的研究中發現，克林達黴素會造成細胞中ATP含量下降以及細線體的損傷；這兩個現象均為誘發細胞自噬作用的重要因子。細胞自噬作用為細胞藉由溶酶小體將細胞中非必要物質降解的機制。由於細胞自噬作用為細胞代謝物質的重要機轉，細胞自噬作用在生理上以及病理上扮演重要的角色。另一方面，對於細胞自噬作用的調控可能可以做為新的疾病治療標的或是藥物開發方向。近年的研究發現，阿茲罕默症的重要成因澱粉樣蛋白會經由富含澱粉樣蛋白前驅蛋白(amyloid precursor protein, APP)的器官中，不正常作用的自噬小體及胞吞小體產生。在本篇研究中，我們希望可以探討克林達黴素是否會影響神經膠細胞中的細胞自噬作用，並且進一步探討克林達黴素對於APP生合成或轉移修飾過程造成的影響。結果顯示，在神經膠細胞中投與克林達黴素會造成細胞自噬作用標的蛋白LC3-II/LC3-I的上升，以及造成自噬小體的堆積。比較克林達黴素與細胞自噬作用調控藥物的效果可以發現，克林達黴素會藉由抑制溶小體的活性造成細胞自噬作用的失調。進一步的研究發現，克林達黴素會造成APP朝向non-amyloidogenesis修飾，藉由活化裁切蛋白ADAM10，使得sAPPα上升。透過顯微鏡發現LC3與sAPPα位於相同的位置，且sAPPα將會被細胞自噬作用調控藥物所影響。綜合以上，我們發現在神經膠細胞中，克林達黴素的處理會藉由干擾細胞自噬作用導致sAPPα上升。

關鍵字

克林達黴素 ； 細胞自噬作用 ； 阿茲罕默症 ； 澱粉樣蛋白前驅蛋白 ； 可溶性澱粉樣蛋白前驅蛋白-alpha

並列摘要

Clindamycin is an antibiotic, which is widely used to treat infection, malaria, and acne. In our lab, pervious study has shown that clindamycin treatment would decrease intracellular ATP amount and cause mitochondrial damage, both are important factor in the induction of cell autophagy. Autophagy is the basic catabolic mechanism that involves cell degradation of unnecessary or dysfunctional cellular components through the actions of lysosomes. Due to the basic and important role of autophagy in protein and organelle degradation, autophagy connects with an astonishing number of human disease and physiology. On the other hand, regulation the autophagy flux in disease may be new treatment target or drug discovery. Recent evidence has shown that the amyloid beta peptide is generated from amyloid beta precursor protein (APP) during autophagy turnover of APP-rich organelles supplied by both autophagy and endocytosis. In this study, we are interested in whether clindamycin would affect glioma cell autophagy, and determent the drug effect on APP expression and trafficking. The result showed clindamycin treatment would induce LC3-II/LC3-I ratio and the autophagosome accumulation. Compare the effect of clindamycin and autophagy regulater show that clindamycin would lead the late stage of autophagy dysfunction through impair lysosome activity. In addition, we found clindamycin would lead Alzheimer’s disease maker protein amyloid precursor protein trafficking to non-amyloidogenesis pathway and up-regulation soluble amyloid precursor protein α(sAPPα) protein level by activated α-secretase, ADAM10 enzyme activity. Use microscopy, we determined LC3 and sAPPα are co-localized, and sAPPα level would regulated by autophagy regulater. Our data suggest that in glioma, clindamycin would up-regulate sAPPα level through autophagy flux impairment.

並列關鍵字

clindamycin ； autophagy ； alzheimer’s disease ； amyloid precursor protein ； sAPPα

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