Yuksel Melike, Biberoglu Kevser, Onder Seda, Akbulut K Gonca, Tacal Ozden
Department of Biochemistry, School of Pharmacy, Hacettepe University, 06100, Ankara, Turkey.
Department of Biochemistry, School of Pharmacy, Hacettepe University, 06100, Ankara, Turkey.
Biochimie. 2017 Jul;138:82-89. doi: 10.1016/j.biochi.2017.04.012. Epub 2017 Apr 27.
The excess accumulation of amyloid-β (Aβ) peptides derived from the sequential cleavage of amyloid precursor protein (APP) by secretases, is one of the toxic key events leading to neuronal loss in Alzheimer's disease (AD). Studies have shown that cholinergic activity may also be involved in the regulation of APP metabolism. In the current study, we have investigated the roles of toluidine blue O (TBO) and thionine (TH), newly recognized phenothiazine-derived cholinesterase inhibitors, on the metabolism of APP in Chinese hamster ovary cells stably expressing human APP751 and presenilin 1 (PS70 cells). We assessed the effects of both compounds on the levels of Aβ, soluble APP-α (sAPPα), intracellular APP and β-site APP-cleaving enzyme 1 (BACE1). After treatment of PS70 cells with TBO or TH without any side effect on cell viability, the levels of secreted Aβ40, Aβ42 and sAPPα were assayed by specific sandwich ELISAs while APP and BACE1 in cell lysates were analyzed using Western blot. The secreted Aβ40, Aβ42 and sAPPα in TBO- and TH-treated cells were found to be reduced in a dose-dependent manner compared to vehicle-treated cells. Results suggest that TH mitigated the Aβ pathology by lowering APP levels whereas reduced Aβ caused by TBO treatment seems to be the outcome of both less substrate availability and amyloidogenic APP processing. Taken together, our results represent the first report demonstrating that TBO and TH can affect amyloid metabolism in vitro.
淀粉样前体蛋白（APP）经分泌酶顺序切割产生的β淀粉样蛋白（Aβ）肽过量积累，是导致阿尔茨海默病（AD）神经元丢失的关键毒性事件之一。研究表明，胆碱能活性可能也参与APP代谢的调节。在本研究中，我们研究了新认识的吩噻嗪衍生的胆碱酯酶抑制剂甲苯胺蓝O（TBO）和硫堇（TH）对稳定表达人APP751和早老素1的中国仓鼠卵巢细胞（PS70细胞）中APP代谢的作用。我们评估了这两种化合物对Aβ、可溶性APP-α（sAPPα）、细胞内APP和β位点APP切割酶1（BACE1）水平的影响。在用TBO或TH处理PS70细胞且对细胞活力无任何副作用后，通过特异性夹心酶联免疫吸附测定法检测分泌的Aβ40、Aβ42和sAPPα的水平，同时使用蛋白质免疫印迹法分析细胞裂解物中的APP和BACE1。与载体处理的细胞相比，发现TBO和TH处理的细胞中分泌的Aβ40、Aβ42和sAPPα以剂量依赖性方式降低。结果表明，TH通过降低APP水平减轻了Aβ病理，而TBO处理导致的Aβ减少似乎是底物可用性降低和淀粉样生成性APP加工减少的共同结果。综上所述，我们的结果首次表明TBO和TH可在体外影响淀粉样蛋白代谢。
