以阿茲海默氏症小鼠初級細胞培養和動物模式評估具TrkB促效劑潛力化合物之治療效果

Abstract

阿茲海默氏症(Alzheimer' s disease, AD)是目前最常見的神經退化性疾病之一，至今尚無顯著有效的治療方法，該疾病會導致認知和行為問題，不論是對於個人或社會都造成極大的負擔。AD在病理學上主要有兩個病徵：類澱粉蛋白(β-amyloid peptides , Aβ)堆積成的斑塊(plaques)及Tau蛋白的過度磷酸化所導致的神經纖維糾結(neurofibrillary tingles, NFTs)。許多研究顯示AD病患腦中之腦源性神經滋養因子(Brain-derived neurotrophic factor, BDNF)濃度較正常人低，且此現象可讓更多的Aβ生成。BDNF結合其受體原肌球蛋白相關激酶B(tropomyosin-related kinase B, TrkB)可調節長期增益效應(long-term potentiation, LTP)、突觸可塑性和神經元分化等，因此增強BDNF/TrkB訊號傳遞成為AD治療之潛力策略。7,8-二羥基黄酮(7,8-dihydroxyflavon, 7,8-DHF)是目前已知的最佳TrkB促效劑，可與細胞膜上的TrkB受體結合並引發受體之同源二聚化而啟動下游之訊息傳導。本研究使用7種與7,8-DHF結構類似，以及8種與Coumarin結構相似之化合物，篩選出具潛力之TrkB促效劑。首先確定Aβ25-35對小鼠海馬迴初級細胞培養的有效傷害濃度為5 µM，以建立AD體外模式篩選平台；我們以此平台篩選出最具神經保護之化合物ZN014進入動物實驗。我們以透過立體定位注射Aβ25-35到小鼠海馬迴CA1區建立AD小鼠模型，再評估7,8-DHF與ZN014對AD動物模式之效用，用藥期間進行一系列行為實驗，結果顯示ZN014可有效改善Aβ25-35所導致的短期記憶受損。在病理與分子機制分析上，我們利用免疫組織化學染色和西方墨點法分析小鼠海馬迴組織，結果表示ZN014可改善Aβ25-35所導致的神經細胞缺失，也可以緩解小鼠海馬迴的神經發炎。ZN014可能是藉由降低神經發炎以保護神經，進而改善因Aβ25-35所導致的記憶受損。綜合上述結果ZN014也許可以開發成具有AD治療潛力的藥物。

Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases without significant therapeutics currently. The two main pathological features identified in AD are Aβ deposition and hyperphosphorylation of tau protein. Many studies have shown that abnormalities of neurotrophin, brain-derived neurotrophic factor (BDNF), occur in patients with AD which promote synthesis of Aβ peptides. BDNF in combination with its receptor tropomyosin-related kinase B (TrkB) can regulate long-term potentiation (LTP), synapse plasticity and neuronal differentiation. Currently 7,8-dihydroxyflavon (7,8-DHF) is known as the best TrkB agonist, which can bind to the TrkB receptor and trigger homodimerization of the receptor to initiate downstream signaling. This study evaluated 15 new compounds with similar structures to 7,8-DHF and coumarin using mouse hippocampal primary culture. First, we determined the effective concentration of Aβ25-35 in the mouse hippocampal primary culture to establish an in vitro model to identify the potential compounds for in vivo animal experiments. The AD mouse model was established using stereotactic injection of Aβ25-35 into the mouse hippocampus. We used a series of behavioral experiments to evaluate 7,8-DHF and ZN014, and the results showed that ZN014 can effectively ameliorate the short-term memory impairment caused by Aβ25-35. Immunohistochemical staining and western blot were conducted to analyze mouse hippocampus. The results showed that ZN014 can reduce the neuronal cell loss in the dentate gyrus caused by Aβ25-35, and it can also alleviate the neuroinflammation in the mouse hippocampus. ZN014 may reduce the level of neuroinflammation and further protect the neurons and improve memory under the damage of Aβ25-35. Combination of the above results, we suggest ZN014 might be potential in AD treatment.