Department of Chemistry, Chung Yuan Christian University, Chung Li, Taiwan.
Taiwan J Obstet Gynecol. 2012 Dec;51(4):554-64. doi: 10.1016/j.tjog.2012.09.010.
This study aims to determine the effects of curcumin and demethoxycurcumin on the internal ribosome entry site of the amyloid-β precursor protein (APP) and tau protein through a bi-cistronic reporter assay for screening of anti-Alzheimer's disease agents.
A bi-cistronic assay was performed wherein the expression of the first cistron, a β-galactosidase gene under the control of a cytomegalovirus promoter, represents the canonical cap-dependent mechanism of translation initiation; while the second cistron involves the utilization of the APP or the tau IRES elements to drive the expression of secreted alkaline phosphatase (SEAP) under a cap-independent mechanism. Bioactive natural products reported to have therapeutic potential for AD such as curcumin and demethoxycurcumin were screened in an murine neuroblastoma (N2A) cell model. Western blot analyses for the expression of APP C-terminal protein, human tau-1, and phosphorylated tau at Serine 262 (p(262)) and Serine 396 (pS(396)) were done after treatment of N2A cells with the test compounds.
The bi-cistronic reporter assay revealed that curcumin was more effective than demethoxycurcumin, a structural analog of curcumin, in inhibiting both APP and tau IRES-dependent translation initiation. This result was further confirmed by Western blot analysis for the expression of APP C-terminal protein, human tau-1, pS(262) and pS(396) suggesting that curcumin may play a role in AD pathology alleviation through the inhibition of the APP and tau IRES-mediated translation mechanism. On the other hand, demethoxycurcumin was observed to inhibit the phosphorylation of both tau pS(262) and pS(396).
A novel assay system using the bi-cistronic reporter constructs for the identification of compounds with activity against the translation directed by APP and tau IRES was developed. The results provide novel suggestive insights for the potential use of the mentioned compounds as prophylactic and therapeutic anti-AD agents.
本研究旨在通过双顺反子报告基因检测，筛选出姜黄素和脱甲氧基姜黄素对淀粉样前体蛋白（APP）和 tau 蛋白内部核糖体进入位点（IRES）的影响，从而寻找治疗阿尔茨海默病（AD）的药物。
采用双顺反子检测，第一顺反子为受巨细胞病毒启动子控制的β-半乳糖苷酶基因，代表经典的帽依赖性翻译起始机制；第二顺反子涉及 APP 或 tau IRES 元件的利用，在无帽依赖机制下驱动分泌型碱性磷酸酶（SEAP）的表达。筛选了报道具有治疗 AD 潜力的生物活性天然产物，如姜黄素和脱甲氧基姜黄素，在小鼠神经母细胞瘤（N2A）细胞模型中进行筛选。用测试化合物处理 N2A 细胞后，通过 Western blot 分析检测 APP C 端蛋白、人 tau-1 和磷酸化 tau 在丝氨酸 262（p(262)）和丝氨酸 396（pS(396)）的表达。
双顺反子报告基因检测结果表明，姜黄素比脱甲氧基姜黄素（姜黄素的结构类似物）更能有效抑制 APP 和 tau IRES 依赖性翻译起始。这一结果进一步通过 Western blot 分析 APP C 端蛋白、人 tau-1、pS(262)和 pS(396)的表达得到证实，表明姜黄素可能通过抑制 APP 和 tau IRES 介导的翻译机制，在缓解 AD 病理方面发挥作用。另一方面，脱甲氧基姜黄素被观察到抑制 tau pS(262)和 pS(396)的磷酸化。
建立了一种新的检测系统，使用双顺反子报告载体鉴定对 APP 和 tau IRES 指导的翻译有活性的化合物。研究结果为这些化合物作为预防和治疗 AD 药物的潜在用途提供了新的见解。
