新穎探針用於監測多巴胺動態

Novel Molecular Probes Monitor Dopamine Dynamics

洪至平(Chih-Ping Hung)

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(2014年)

摘要

多巴胺（DA）在大腦中作為重要的神經傳導物質，在動物的生理以及心理層面上扮演著舉足輕重的角色，而多巴胺系統的失衡現象也在多數神經疾病中被發現，包含了帕金森氏症（PD）。偵測腦中多巴胺神經細胞（Dopaminergic neuron）內的多巴胺濃度變化有助於釐清帕金森氏症的發病機制，因此有必要建立可在細胞規格下偵測多巴胺濃度變化的分子探針。多巴胺神經細胞中的單胺氧化酶（MAO）與黃素腺嘌呤二核苷酸（FAD）作輔因子代謝細胞中的多巴胺，藉以維持多巴胺濃度的恆定。其一的單胺氧化酶B（MAO-B）已被發現含有獨特的吸收光譜，我們利用此特性與綠色螢光蛋白結合，設計多巴胺探針。當單胺氧化酶B在氧化態時，吸收了400-500 nm波長的光波段，使綠色螢光蛋白無法被激發，我們稱為「屏蔽效應」。而當氧化多巴胺形成還原態時，屏蔽效應便會消失，使得綠色螢光蛋白得以被激發螢光。因此，綠色螢光蛋白訊號可以做為多巴胺濃度的讀值。我們的實驗結果顯示，增加神經細胞中的多巴胺濃度可確實激發多巴胺探針（MMG1）發出螢光，並且經過測試後，探針的酵素活性及基質結合能力不受設計過程而被破壞。未來將能應用此一多巴胺探針於患有帕金森氏症突變基因的實驗動物體中，確定多巴胺濃度變化是否參與帕金森氏症等多巴胺相關疾病的發病機制。

關鍵字

多巴胺 ； 分子探針 ； 單胺氧化酶B ； 遮蔽效應

並列摘要

Dopamine (DA) system impairment in the central nervous system links to both motor and mental dysfunction in human. However, it remains impossible to monitor the brain DA dynamic in cellular level. MAO-B (monoamine oxidase B) is an enzyme anchored on the mitochondrial outer membrane to catalyse amine oxidation, including DA; a process requires FAD as a cofactor. Because of MAO-B absorbance at 400-500 nm spectrums, MAO-B fused with a GFP chromophore cannot be elicited by 488 nm light if FAD remains in oxidized form, we called this phenomenon as “Shield effect”. Shield effect would be diminished upon the enzyme binds to the substrate, which would allow us to “visualize” the substrate binding of MAO-B in real time. Indeed, we have established such a molecular probe, MMG1, which could detect the intracellular fluctuation of DA concentration by monitoring GFP fluorescent readout. The L-DOPA (dopamine precursor)–induced fluorescence in a set of quantitative experiments using flow cytometry confirmed that MMG1 is sensitive to L-DOPA in a dose-dependent manner. To ultimately validate this DA probe, I constructed three MMG1 mutants, MMG1Y398S, MMG1Y435S, and MMG1C397A, which are defective in FAD or substrate binding. To further characterize this DA probe (MMG1), I characterized the enzyme kinetics of MMG1 and revealed the substrate binding affinity of MMG1 is comparable to native MAO-B. Together, this study provides the first in vivo tool that could monitor DA dynamics in cellular level.

並列關鍵字

Dopamine ； Molecular probe ； Monoamine oxidase B ； MAO-B ； Shield effect

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