以tau 聚集為目標的阿茲海默氏症治療策略

Abstract

阿茲海默氏症是一種漸近性的神經退化性疾病，主要病理特徵包 括細胞外間質的Aβ 胜肽堆積，及細胞內高度磷酸化tau 形成的神經 纖維糾結。由於tau 聚集與阿茲海默氏症進程具相關性，因此透過抑 制或消除tau 聚集或可保護受影響之神經細胞。本研究構築tau 蛋白 微管結合重複區域(tauRD)之野生型(K18)及第280 位置賴胺酸缺失的 促tau 聚集突變型(ΔK280)與DsRed 紅螢光蛋白融合之tauRD-DsRed基因，來建立誘導表現tauRD-DsRed 之Tet-On 293 與Tet-On SH-SY5Y 細胞，作為藥物篩檢平台，來篩檢可抑制tauRD 聚集的抑制物，並對 這些具聚集抑制性的化合物或中草藥、植物萃取物等，檢測其神經保 護機制。在Tet-On 293 細胞誘導tauRD-DsRed 蛋白表現後，ΔK280 突變型細胞螢光亮度較野生型細胞顯著減少。以剛果紅(正控制組)處 理細胞後，突變型細胞的紅螢光亮度較野生型者更能有效提升。對於 293 促tau 聚集突變型細胞，在細胞數無顯著影響下， 前處理 NC009-1、-2、-3、-6、-7 (吲哚基喹啉化合物)、NTNU-003、-008 (GSK-3β抑制劑類似物)、NH021、NTNU-043、-057、-059、-224、NTNU-309、 -313、-319、-331、-379 (中草藥或植物萃取液)等皆可顯著提升DsRed 紅螢光亮度。以維他命A 酸誘導ΔK280 促tau 聚集突變型SH-SY5Y 細胞神經分化後，螢光顯微影像分析顯示神經突觸生長性狀(包含總 生長及突起數、分支數)較野生型K18 細胞顯著下降。以NC009-1、 -7 及NTNU-008 前處理突變型SH-SY5Y 細胞後，可顯著提升神經突 觸總生長性狀，並可提升HSP27 蛋白(NC009-1、-7)或GRP 78、HSP27 蛋白(NTNU-008)的表現。Alzheimer's disease (AD) is a progressive neurodegenerative disorder associated with accumulation of extracellular amyloid-β and intracellular tau-associated neurofibrillary tangles (NFTs). The aggregation of tau in AD correlates with the clinical progression of the disease and inhibition or reversal of tau aggregation may protect the affected neurons. In this study wild type (K18) and pro-aggregation mutant (ΔK280) of repeat domain of tau (tauRD) was fused with DsRed (tauRD-DsRed fusion gene) and used to generate Tet-On 293 and SH-SY5Y cell clones as drug screening platforms. Inhibitors that retard or block ΔK280 tau aggregation can be distinguished by increasing fluorescence on Tet-On 293 cells. For those compounds/herbs with putative aggregate inhibitors, mechanisms of neuroprotection were determined. Upon induction with doxycycline, red fluorescence in ΔK280 tauRD-DsRed 293 cells was significantly reduced compared to that in wild type cells. As a positive control, congo red increased red fluorescence more effectively in ΔK280 tauRD-DsRed 293 cells than in wild type cells. Without significantly reducing cell numbers, pretreatment of NC009-1, -2, -3, -6, -7 (indolylquinoline compounds), NTNU-003, -008 (GSK-3 inhibitor-like compounds), NH021, NTNU-043, -057, -059, -224, -309, -313, -319, -331, -379 (herbal extracts) resulted in significant increased fluorescence on ΔK280 tauRD-DsRed cells. For SH-SY5Y cell clones, retinoic acid treatment generated cells resemblance to adult neurons. Fluorescent microscopy examination revealed that neurite outgrowth (including total outgrowth, process and branch) was significantly reduced in ΔK280 tauRD-DsRed cells compared to that in wild type cells. NC009-1, -7 and NTNU-008 significantly increased neurite total outgrowth in ΔK280 SH-SY5Y cells, accompanying with enhanced HSP27 (NC009-1 and -7) or GRP78 and HSP27 (NTNU-008) expression.