神經退化相關蛋白與轉譯後修飾在腦部及血清中之研究

Studies of Neurodegeneration-Related Proteins and Post Translational Modifications in Brain and Serum

蔡逸石(I-SHIH TSAI)

指導教授 ： 林詠&CEF

臺北醫學大學/醫學科技學院/醫學檢驗暨生物技術學系所/碩士(2016年)

摘要

高齡化時代來臨，伴隨而來的是神經退化性疾病的病患與日俱增，如阿茲海默症 (Alzheimer’s disease, AD)，進一步瞭解其病理機轉顯得迫切需要。已知AD患者有學習記憶能力喪失和認知功能障礙，並在早、中期時伴隨著憂鬱的症狀。疾病的病程與大腦中β類澱粉蛋白(Amyloid β, Aβ)斑塊沉積和神經纖維糾結有關，而類澱粉蛋白斑塊是源於類澱粉前驅蛋白(amyloid precursor protein, APP)的下游產物。亨丁頓關聯蛋白(Huntingtin-associated protein-1, HAP1) 是一個參與囊泡運輸的蛋白。目前已知HAP1會分別與APP及阿伯爾森幫手整合(Abelson helper integration site-1, AHI1) 形成穩定的複合體，進而一起調控微管依賴性囊泡之運輸。另一方面，也有研究指出AHI1及HAP1的缺失與憂鬱的症狀有著密切關聯。實驗初步結果利用共同免疫沉澱的技術證實了在新生仔鼠腦中APP及AHI1確實有交互作用；更進一步的，在Hap1基因剔除的新生仔鼠和異合子型(heterozygous or +/-)成年小鼠腦中，APP分解產物羧端片段(C-terminal fragments, APP-CTFs)相較於正常小鼠有上升的趨勢。核磁共振影像中發現Hap1+/-鼠大腦些微變小，雖然沒有顯著差異，但在行為學的研究中發現Hap1+/-鼠在記憶的表現上有變差的趨勢，憂鬱的症狀，和運動協調性不足。 這些結果顯示HAP1、AHI1和APP在AD的病程中有緊密的關係。進一步的血液分析，發現Hap1+/-鼠的血糖比野生型鼠高，在AD病人的血中也發現一致性的結果，其血糖較正常對照組高。當分析血清中的轉譯後修飾時，發現AD病人脂蛋白(Apo)-A1有較高的離胺酸羧乙基化(CEL)，這是一種糖毒素甲基乙二醛 (methylglyoxal, MGO)的修飾，病人血中也有較高的CEL自體抗體。總結而論，囊泡運輸蛋白、MGO修飾和自體免疫都可能參與了AD致病的過程，這些因子或可成為AD的診斷和治療的生物標誌。

關鍵字

阿茲海默症 ； 憂鬱症 ； 微管 ； 囊泡 ； 類澱粉前驅蛋白 ； 亨丁頓關聯蛋白 ； 阿伯爾森幫手整合位點蛋白 ； 甲基乙二醛 ； 自體抗體

並列摘要

The era of aged population with continuing pollution is accompanied by increasing patients with neurodegenerative disorders such as Alzheimer’s disease (AD). AD is characterized by progressive deterioration of memory and cognitive functions. Depression is also common among people with AD, especially during the early and middle stages. The disease process is associated with deposit of amyloid plaques and neurofibrillary tangles in the brain, which were mainly caused by amyloid beta (Aβ), a protein fragment originated from Amyloid precursor protein (APP) and microtubule-associated protein tau. Recent studies demonstrated that Huntingtin associated protein 1 (HAP1), a microtubule dependent vesicular trafficking protein, is abundantly expressed in neurons and associated with APP and Abelson helper integration site-1 (AHI1 or Jouberin), a protein related to depression. Getting the insights of APP processing, first, we discovered the interaction of APP and AHI1 in mouse brain using co-immunoprecipitation. Then, we observed that more APP fragments present in the Hap1-KO newborn and Hap1 heterozygous (+/-) adult mouse brains compared to wild-type (WT) brains. Further comparing the nuclear magnetic resonance images (MRIs), we found a slight reduction of Hap1+/- brain although it was not significant. When comparing the behaviors of the mice, we found a worse memory performance, depressive phenotype and deficient motor coordination of Hap1+/- mice. These results demonstrate the close relationship of HAP1, AHI1 and APP in AD pathogenesis. In further experiments using blood, we found higher glucose concentrations in Hap1+/- mice compared to WT mice. Consistently higher blood glucose concentrations were found in AD patients than in normal controls. When analyzing the post-translational modifications (PTMs) in the serum, we discovered that more N(ε)-(carboxyethyl)lysine (CEL), a product from methylglyoxal (MGO) modification, on apolipoprotein (Apo)-A1 in AD patients than in the controls. Additionally, more autoantibodies of CEL were found in the patients’ serum. All these results concluded that vesicle transport proteins, MGO modifications and autoimmunity might take roles in AD pathogenesis and could serve as biomarkers for AD diagnosis and treatment.

並列關鍵字

neurodegeneration ； depression ； Huntingtin-associated protein-1 (HAP1) ； amyloid precursor protein (APP) ； Abelson helper integration site-1 (AHI1) ； methylglyoxal (MGO) ； autoantibody

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