林炎壽Yenshou Lin李智翰Chih-Han Lee2019-09-052019-7-162019-09-052014http://etds.lib.ntnu.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=id=%22GN060043058S%22.&%22.id.&http://rportal.lib.ntnu.edu.tw:80/handle/20.500.12235/104062阿茲海默症是世界上最普及的一種失智症，其中有兩個主要病徵與該疾病漸進的神經退化失調有關：細胞外由類澱粉蛋白貝塔(Aβ)組成的斑塊，以及細胞內由過度磷酸化的tau所導致的神經原纖維纏結。近期研究指出Aβ蛋白會減低第一型與第二型囊泡麩氨酸轉運體(vGLUT1/2)在前突觸麩氨酸性區的表現，以及抑制麩氨酸被星狀細胞回收清除。這使得過多的麩氨酸累積在突觸間隙而後引發神經興奮毒性。然而Aβ蛋白過度活化N-甲基-D-天門冬胺酸受體(NMDAR)的現象可被人工合成的NMDAR拮抗藥物美金剛(memantine)所改善。因此找出麩氨酸受體的拮抗劑被視為Aβ蛋白所引發之異常現象的治療策略。於本研究中，我們試圖以小鼠初級大腦皮質神經細胞篩選中草藥以找出能改善Aβ蛋白導致的過量麩氨酸神經傳遞的非人工合成之天然成分。由免疫細胞化學法得知，vGLUT1/2染色呈現幾乎一致的麩氨酸性神經細胞群，突觸結合蛋白(synaptotagmin)染色亦在這些細胞之間呈現神經網絡的形式。而該初級神經細胞的特徵也透過西方墨點法中的抗體訊號如第三型貝塔微管蛋白、PSD-95蛋白以及α-氨基烴甲基嘿挫丙酸型受體(AMPAR)得以確認。為了在篩選中草藥的過程中能偵測並量化受Aβ蛋白刺激所造成的膜電位變化，DiBAC4(3)，一個慢反應電位敏感染劑，被用來做為藥物篩選的工具。DiBAC4(3)可進入去極化細胞並與胞內或膜蛋白結合隨後引發增加的紅螢光強度。去極化程度越高紅螢光越強，反之過極化則導致紅螢光減弱。為了建立DiBAC4(3)的中草藥篩選平台，我們對初級神經細胞刺激氯化鉀、麩氨酸以及Aβ蛋白後發現DiBAC4(3)螢光強度如預期般地增強。此外我們藉由DiBAC4(3)方法發現於Aβ蛋白刺激前加入memantine可減弱Aβ蛋白所導致的去極化現象。由上述結果可知DiBAC4(3)的中草藥篩選平台已被成功建立。在篩藥之前，我們亦先量測了處理不同濃度的中草藥對細胞存活的半抑制濃度(IC50)以便日後篩藥。在我們近期研究中，我們找到一些能抑制Aβ蛋白所引發的過度去極化現象的中草藥，也希望之後能找出該中草藥之有效成分如何參與阿茲海默病理現象中的麩氨酸受體訊息傳遞機轉。Alzheimer’s disease (AD), the most prevalent form of dementia worldwide, is a progressive neurodegenerative disorder characterized by the presence of two hallmark lesions: extracellular amyloid β (Aβ)-containing neuritic plaques and intracellular phospho-tau-positive neurofibrillary tangle. Recent studies have revealed that Aβ reduces levels of vGLUT1/2 in presynaptic glutamatergic terminals and inhibits clearance of glutamate by astrocytes. This leads to increased concentration of glutamate at the synaptic cleft and subsequently trigger excitotoxicity. However, the Aβ-induced activation of NMDA receptor can be ameliorated by the synthetic NMDA antagonist, memantine. Hence, glutamate receptor antagonists have been regarded as a therapeutic strategy in Aβ-induced abnormality. In this study, we intend to find natural ingredients instead of synthetic drugs by screening Chinese herbal medicines (CHMs) for attenuating Aβ-induced abnormal neurotransmission in mouse primary cortical neurons. Judging from vGLUT1/2 staining in immunocytochemistry, we obtained an almost uniform population of glutamatergic neurons. Synaptotagmin staining showed the formation of neuronal network within these neurons. The property of these primary cortical neurons was characterized by Western blot analysis by using antibodies such as type III β-tubulin, PSD95, and AMPAR. In order to observe the changes of membrane potential after Aβ stimulation, bis-(1,3-dibutylbarbituric acid)-trimethineoxonol (DiBAC4(3)), a slow-response voltage-sensitive fluorescent dye, was employed to perform the drugs screening. DiBAC4(3) can enter depolarized cells where it binds to intracellular proteins or membrane proteins as to exhibit enhanced fluorescence and a red spectral shift. Increased depolarization results in additional influx of DiBAC4(3) anionic dye and an increase in fluorescence. Conversely, hyperpolarization is indicated by a decrease in fluorescence. After treating mouse primary cortical neurons with KCl, glutamate, or Aβ, the fluorescence of DiBAC4(3) was increased as expected. Moreover, we found that pretreatment of memantine can ameliorate the Aβ-induced depolarization by assessing changes of DiBAC4(3) fluorescence intensity. These results indicated that the platform of drug screening has been established. Meanwhile, we measured the cell viabilities of primary neurons treated with CHMs to assess their IC50. We found some effective CHMs against the Aβ-induced excessive depolarization in neurons. The molecular mechanisms of effective compounds from CHMs involved in signaling in AD pathology remain to be further investigated.阿茲海默症類澱粉蛋白貝塔麩氨酸受體美金剛DiBAC4(3)中草藥Alzheimer's diseaseamyloid βglutamate receptorsmemantineDiBAC4(3)Chinese Herbal Medicines