認知障礙疾病相關生物標記之病理與分子特性分析

Abstract

失智症為腦神經因老化或病變而退化造成認知功能的失調，而阿茲海默症與巴金森氏病為常見的神經退化性疾病。阿茲海默症是著名的認知退化疾病，而巴金森氏症雖主要造運動功能障礙，亦有相當比例之病患發展出非動作相關之認知障礙或失智症狀，使得用藥效果及病患本人與照護者生活品質的下降。目前仍然無法以藥物或神經再生等方式達到治癒神經退化性疾病的目標，早期診斷並給予相對應的藥物來延緩疾病的進程為現下主要的治療方針。但目前的認知障礙診斷耗費相當長的時間與醫療資源，因此本研究旨在透過阿茲海默症轉基因小鼠模式AppNL-G-F與巴金森氏症患者的血液檢體，找出於神經退化性疾病早期與巴金森氏症不同階段具鑑別度的生物標記，同時分析其分子的特徵與病理的關係。阿茲海默症主要病徵為認知功能障礙與β澱粉樣蛋白沉積，而近期的研究顯示於第22與23個胺基酸處形成二級結構的β澱粉樣蛋白具有高度沉積能力與神經毒性，為此我們以此毒性異構物的專一抗體24B3與48D4進行三明治法酵素免疫分析 (Sandwich ELISA) 於小鼠的腦與血液樣本中的含量，來鑑別其作為生物標記的可能性。另外，本實驗室也在此AppNL-G-F小鼠不會有tau蛋白病變的特性下，觀察到小腦亦會有跳躍子表現量上升現象，其符合老化與神經退化性疾病具有基因體不穩定的關聯性。故我們也利用此小鼠探討跳躍子、β澱粉樣蛋白沉積與小鼠行為三項指標間的關聯性。結果顯示此小鼠在六個月大時出現社交記憶的障礙、跳躍子在海馬迴與小腦後葉的失調及β澱粉樣蛋白的沉積，但跳躍子失調與β澱粉樣蛋白沉積的關係並不如跳躍子與tau蛋白病變之間有顯著關聯。而巴金森氏症於併發認知障礙後將影響用藥成效並增加死亡率，但病情往往在完善的醫療診斷結束前發展至下一階段，使臨床上的精準投藥執行不易，為了快速區分巴金森氏症患者認知功能狀況，能夠跨越血腦障壁的胞外囊泡成為找尋生物標記的主要來源。本團隊透過比較健康人、巴金森氏症患者、巴金森氏症輕度認知功能障礙患者與巴金森氏失智症患者的血漿胞外囊泡中的蛋白質體分析，發現Thousand And One Amino Acid Protein Kinase 1 (TAOK1)在巴金森氏症併發認知功能障礙患者的樣本中顯著上升，因此本研究以TAOK1與胞外囊泡專一性抗體CD9為目標發展診斷巴金森氏失智症患者的三明治法酵素免疫分析套組。我們利用細胞培養的人類神經母細胞瘤細胞系SH-SY5Y分泌的胞外囊泡進行套組建立，並發現胞外囊泡內TAOK1可能有轉錄後修飾的現象，我們也利用病患樣本確認其實際應用的可能性。總結兩項神經退化性疾病的研究，以來自血液甚至於胞外囊泡的蛋白質作為神經退化性疾病的生物標記搭配三明治法酵素免疫分析有其可行性，但疾病可能造成蛋白質的構型改變與特異的轉錄後修飾，如何在檢驗過程中防止生物標記流失並準確標定還需要進一步的探索。

Dementia is a syndrome involving cognitive impairment such as memory loss, which mainly affects elderly people, and the number of patients is rapidly increasing all over the world. Since the regenerative capacity of neuron is limited, early diagnosis is important for treatment of dementia. Here, two different types of dementia were investigated. Alzheimer’s disease (AD) is the most prevalent dementia, whose pathological hallmarks are the accumulation of amyloid beta (Aβ) protein and intracellular hyperphosphorylated tau protein. Recent study identified toxic conformer of Aβ protein with a turn at positions 22 and 23, which showed especially high aggregative ability and neurotoxicity. Since it might have pathological role as well as serving as a potential biomarker, two different sandwich ELISA were developed using 24B3 and 48D4 antibodies targeting the oligomer of this conformer. On the other hand, genomic instability has been associated with aging and neurodegenerative diseases. Our previous study implied dysregulation of transposable element (TE) in cerebellum of AppNL-G-F mouse, which is an AD model mouse harboring three AD familial mutation on amyloid precursor protein (APP). In this study, I aimed to characterize Aβ related TE dysregulation and to evaluate novel antibodies as potential early diagnostic agents in early stage of AD using AppNL-G-F mice which have aberrant Aβ but not Tau accumulation. In addition, the correlations of the TE expression level and toxic Aβ quantity with cognitive and behavioral alterations were investigated. As a result, TE dysregulation was observed in 6-month-old AD mice hippocampus and posterior part of cerebellum, suggesting epigenetic alteration in these regions. However, the correlation between TE dysregulation and Aβ accumulation was not as significant. In addition, social memory impairment in early stage of AD without objective memory impairment was also indicated in 6-month-old AD mice. Parkinson disease (PD) is a typical motor neuron disorder. However, significant portion of PD patients also develop dementia, causing serious problems such as reduced quality of life and increased mortality for patients. Characteristic pathology of PD is intraneuronal formation of Lewy body composed of α-synuclein. Since currently available diagnostic methods for Parkinson disease dementia (PDD) are labor-intensive, establishing biomarker for PDD is required. Our previous proteomic profiling from patient plasma extracellular vehicles (EVs) indicated dysregulation of “thousand-and-one amino acid kinase 1 (TAOK1) protein” in PDD patient compared to PD patients with normal cognition ability and healthy controls. Since it might serve as biomarker for PDD, we aimed to develop a sandwich ELISA for quantifying TAOK1 in plasma EVs. Here, exosome enriched EV fraction was successfully collected and applied for ELISA establishment. In addition, modification of TAOK1 in EV was implicated.