α-突觸核蛋白聚集體在中樞神經系統和其他器官中的傳播

The propagation of α-synuclein aggregates in the central nervous system and other organs

張琳欣(Lam Yan Cheung)

指導教授 ： 王致恬

國立臺灣大學/生命科學院/分子與細胞生物學研究所/碩士(2020年)

https://doi.org/10.6342/NTU202002838

摘要

帕金森氏病（Parkinson’s disease）是一種影響中樞神經系統的慢性神經退化疾病，主要影響運動神經系統由中腦黑質（Substantia nigra pars compacta）的多巴胺神經元 ( Dopaminergic neurons)變性損失所致。在過去的十年的研究中，便秘被發現是帕金森氏病的常見症狀之一。 2003年，Heiko Braak的研究發現帕金森氏病患者的屍檢樣本中，路易體 (Lewy bodies)同時出現在大腦和胃腸道系統中，顯示帕金森氏病可能起源於腸道而非大腦。然而，目前帕金森氏病進展時程與α-突觸核蛋白(α-synuclein)的傳播之間的因果關係尚未明朗。因此，本論文探討人類致病性α-突觸核蛋白突變體是否可能在成年期帕金森氏病的大鼠模型中，依不同時程，從黑質傳播到其他神經組織或周圍器官。實驗方式為，將雄性Sprague-Dawley大鼠（〜250 g，2週齡）深度麻醉以進行立體定位手術，顯微注射2 μL含有三重突變的質體DNA（5 μg/μL）溶液到黑質中，以遞送人類致病性α-突觸核蛋白突變體的基因。使用自製鉑電極進行活體電穿孔，條件為五次電脈衝，每次持續時間為50毫秒，間隔為950毫秒，電場強度為133 V / cm。活體電穿孔後，通過免疫螢光染色和西方墨點法 (Western Blot) 分析大鼠中α-突觸核蛋白的聚集(aggregation)和多巴胺神經元的流失。結果顯示，轉染三重突變株後一至三個月，以免疫螢光染色標誌α-突觸核蛋白第129個胺基酸Serine的磷酸化 (pS129) (以pS129免疫反應性作為α-突觸核蛋白聚集體的標誌)，確認成年期帕金森氏病的大鼠模型中有α-突觸核蛋白聚集體；轉染三重突變株後三個月，黑質多巴胺神經元有明顯酵素流失並且α-突觸核蛋白聚集可出現在離黑質較遠的大腦區域，包括：紋狀體、嗅球、視丘、視神經、和視網膜，顯示腦中α-突觸核蛋白具有在神經系統內傳播的能力。最後，通過西方墨點法分析腸道和附睾脂肪的組織，發現轉染三重突變株後一至三個月之後，腸道也疑似呈現出pS129免疫反應性。這些結果表明，人類致病性α-突觸核蛋白突變體可以在成年期帕金森氏病的大鼠模型中，從黑質傳播到中樞神經系統及其他周圍器官。

關鍵字

三重突變α-突觸核蛋白 ； 帕金森氏症 ； 活體電穿孔 ； 成年期帕金森氏病的大鼠模型

並列摘要

Parkinson's disease (PD) is the second most common movement disorder, owing to degenerative loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc) of midbrain. Moreover, in the past decade, constipation has been found as one of the most common symptoms of PD. In 2003, Heiko Braak proposed that PD may originate in the gut rather than the brain, as evidenced by that Lewy bodies appeared in both brain and the gastrointestinal (GI) system in post-mortem samples of PD patients. However, it remains unclear what is the causal relationship between PD progression and the propagation of α-synuclein (α-Syn) aggregates (the main component of Lewy bodies) in the central and peripheral system. In our study, we examined whether the human pathogenic α-Syn mutant may propagate from the SNc to other neural tissues or peripheral organs in a rat model mimicking sporadic PD during adulthood. In this PD model, male Sprague-Dawley rats (~250 g, 2-week-old) were deeply anesthetized for stereotaxic surgery. The 2 μL-solution containing DNA plasmids (5 μg/μL) was microinjected into SNc to deliver genes expressing the human pathogenic α-Syn mutant. Five electric pulses were delivered by homemade platinum electrodes via ear bars, with duration of 50 msec, intervals of 950 msec, and electric field strength of 133 V/cm. After in vivo electroporation, the aggregation of α-Syn and DA neuronal dysfunction in the PD rats were examined by immunostaining and western analysis. First, the presence of α-Syn aggregates in the SNc were verified in the PD rats, by immunostaining the phosphorylated Ser129 (pS129) of α-Syn (the pS129 immunoreactivity as the hallmark of α-Syn aggregates). The results showed that DA neurons may become dysfunction after 3 months post transfection. Further, aggregation of α-Syn in can be found in remote brain regions such as striatum, olfactory bulbs, thalamus, optic nerves, and retinas, suggesting the ability of pathological aggregated α-Syn to propagate within the neural tissues. Finally, by using western analysis, we analyzed that the tissues from the gut and epididymis fat and found that the gut may likely display the pS129 immunoreactivity. These results suggest that the human pathogenic α-Syn mutant can be propagated from the rat midbrain to other neural tissues or to peripheral organs during PD progression.

並列關鍵字

The triple mutant of α-Synuclein ； Parkinson’s disease ； in vivo electroporation ； adult rat PD model

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