在細胞膜上偵測多巴胺釋放的分子探針

Molecular probe for detecting dopamine exocytosis

鐘婷琪(Jong, Ting Chi)

指導教授 ： 桑自剛

國立清華大學/生命科學院/生物科技研究所/碩士(2015年)

摘要

多巴胺為一種神經傳導物，在腦中由多巴胺神經製造。在哺乳動物及果蠅中，已經有研究指出多巴胺具有調控運動、睡眠、學習、與記憶等功能。如果多巴胺神經有缺失，便可能造成神經退化性疾病，例如：帕金森氏症。為了瞭解多巴胺造成的相關疾病及其對行為之調控，我們想要了解多巴胺的釋放與路徑。至今，多巴胺的釋放與路徑還未清楚了解，傳統偵測多巴胺的釋放的方法非常繁瑣，還未有適合的方式讓我們了解多巴胺的釋放路徑。因此，我們想發展出一種分子探針，可以在活體內觀察到多巴胺的釋放，並且解析度可以達到細胞等級。
在先前實驗室研究中，我們將人類酵素MAO B和GFP結合在一起，形成融合蛋白。由於MAO B的cofactor FAD會吸收波長488雷射的激發，因此融合蛋白中的GFP不會被488 雷射激發發出螢光，而我們將這個現象稱為「shield effect」。在本計畫中，我們發展出表現在細胞膜上的多巴胺分子探針。從目前免疫染色的結果中顯示，M498SG11探針是表現在細胞膜上的。未來，此探針仍須一些修改才能提高MAO B的活性，以達到偵測多巴胺的目的。在HEK 293細胞中成功表現了分子探針後，我們將做基因轉殖果蠅，希望可以觀察不同環境下果蠅腦中的多巴胺動態變化，了解大腦如何傳遞訊息。

關鍵字

多巴胺 ； monoamine oxidase

並列摘要

Dopamine circuit has been shown to associate with locomotion, sleep and arousal, decision making, and associative learning in mammals and fly. To understand the mechanism of dopaminergic-associated behaviors and disorders, we aim to visualize dopamine release in vivo. Currently, monitoring the dynamics and distribution of dopamine released in live is unattainable; the traditional ways to measure dopamine level in neurons is cumbersome and lacks of temporal and spatial resolutions. Our goal is to develop a probe that can switch fluorescence upon detecting dopamine release from dopaminergic neurons in vivo. By modifying our previously established cytosolic probe MMG1, in which we used intrinsic spectral properties of MAO B to switch GFP emission upon dopamine binding (we have coined this phenomena as “shield effect”) as an intracellular dopamine sensor, I have developed different membrane dopamine sensors that could potentially use for detecting dopamine release. Current results show that two full-length constructs, MSG11 (MAO B-ste2p-sfGFP11) and MSG (MAO B-ste2p-GFP), and two MAO B C-terminal truncated probes could be embedded on the cell membrane with expected topology. Additional tests are currently ongoing to validate the shield effect and enzyme activity of these sensors. My ultimate goal is to make the transgenic flies and test the probes in the brain. With this type of molecular probe on hand, we may have a better way to determine how brain process information in dopamine circuits under different environmental stimulations and behavioral responses.

並列關鍵字

dopamine ； monoamine oxidase ； fluorescence probe

參考文獻

Berke, J. D., and S. E. Hyman. 2000. “Addiction, Dopamine, and the Molecular Mechanisms of Memory.” Neuron 25 (3): 515–32.

Binda, Claudia, Paige Newton-Vinson, Frantisek Hubálek, Dale E. Edmondson, and Andrea Mattevi. 2002. “Structure of Human Monoamine Oxidase B, a Drug Target for the Treatment of Neurological Disorders.” Nature Structural Biology 9 (1): 22–26. doi:10.1038/nsb732.

Binda, Claudia, Jin Wang, Min Li, Frantisek Hubalek, Andrea Mattevi, and Dale E. Edmondson. 2008. “Structural and Mechanistic Studies of Arylalkylhydrazine Inhibition of Human Monoamine Oxidases A and B.” Biochemistry 47 (20): 5616–25. doi:10.1021/bi8002814.

Cabantous, Stéphanie, Thomas C. Terwilliger, and Geoffrey S. Waldo. 2005. “Protein Tagging and Detection with Engineered Self-Assembling Fragments of Green Fluorescent Protein.” Nature Biotechnology 23 (1): 102–7. doi:10.1038/nbt1044.

Chambers, Ian, Douglas Colby, Morag Robertson, Jennifer Nichols, Sonia Lee, Susan Tweedie, and Austin Smith. 2003. “Functional Expression Cloning of Nanog, a Pluripotency Sustaining Factor in Embryonic Stem Cells.” Cell 113 (5): 643–55.