Department of Medical Biology, Cerrahpasa Faculty of Medicine, Istanbul University, Istanbul, Turkey.
Department of Medical Biology, Cerrahpasa Faculty of Medicine, Istanbul University, Istanbul, Turkey.
J Chem Neuroanat. 2018 Oct;92:41-47. doi: 10.1016/j.jchemneu.2018.05.006. Epub 2018 May 31.
Hyperphosphorylation of tau leading to neurofibrillary tangles (NFT) is one of the key pathological hallmarks in neurodegenerative disorders such as Alzheimer disease (AD). Peptidyl-prolyl cis-trans isomerase (Pin1) regulates the phosphorylation of Ser/Thr sites of tau protein, and promotes microtubule assembly. In this study, we aimed to determine the effect of tau hyperphosphorylation on Pin1 expression in primary cortical neurons in order to investigate the results of the pathological process on Pin1, an important enzyme involved in various cellular mechanisms. Primary cortical neurons were prepared from embryonic day 16 -Sprague Dawley rat embryos. The cultures were treated with 25 nM okadaic acid (OKA) on day 7 in order to promote tau hyperphosphorylation. The cytotoxicity was determined with LDH release and measured by ELISA. Tau phosphorylation was confirmed by western blot using anti-tau antibodies Thr231 and Tau-1. Pin1 mRNA expression level was determined by qRT-PCR at 8 and 24 h. Pin1 protein expression was analyzed with immunofluorescent labeling at 8 and 24 h. Tau phosphorylation on Thr231 was increased and non-phosphorylated Tau-1 was decreased in OKA treated group compared with the untreated control at 8 h of treatment. While Pin1 mRNA expression levels at 8 h post-OKA treatment were lower than that of control groups, there were no differences between OKA-treated group and control groups in Pin1 protein expression. Whereas no significant differences for Pin1 mRNA expression, protein expression levels were decreased OKA-treated group compared to control groups at 24 h of treatment. The LDH release of OKA-treated group was significantly increased at 24 h. Our study indicates that although OKA treatment suppressed Pin1 mRNA expression and induced tau phosphorylation at 8 h of treatment, its influence on Pin1 protein expression has 16 h phase delay. Given the important role of Pin1 in many cellular mechanisms these results might indicate that tau hyperphosphorylation involved in many neurodegenerative disorders may cause some alterations in brain microenvironment via Pin1.This is the first demonstration of the alteration of the Pin1 mRNA and protein expression in OKA induced model in primary cortical neurons.
tau 的过度磷酸化导致神经原纤维缠结 (NFT) 是阿尔茨海默病 (AD) 等神经退行性疾病的关键病理学特征之一。肽基脯氨酰顺反异构酶 (Pin1) 调节 tau 蛋白丝氨酸/苏氨酸位点的磷酸化，并促进微管组装。在这项研究中，我们旨在确定 tau 过度磷酸化对原代皮质神经元中 Pin1 表达的影响，以研究病理过程对参与各种细胞机制的重要酶 Pin1 的影响。原代皮质神经元从胚胎第 16 天的 Sprague Dawley 大鼠胚胎中分离得到。第 7 天用 25 nM 岗田酸 (OKA) 处理培养物以促进 tau 过度磷酸化。通过 LDH 释放和 ELISA 测量来确定细胞毒性。用抗 tau 抗体 Thr231 和 Tau-1 通过 Western blot 确认 tau 磷酸化。用 qRT-PCR 在 8 和 24 h 时测定 Pin1 mRNA 表达水平。用免疫荧光标记在 8 和 24 h 时分析 Pin1 蛋白表达。与未处理对照组相比，OKA 处理组在处理 8 h 时 Thr231 上的 tau 磷酸化增加，而非磷酸化的 Tau-1 减少。虽然 OKA 处理后 8 h Pin1 mRNA 表达水平低于对照组，但 OKA 处理组与对照组之间的 Pin1 蛋白表达无差异。而 OKA 处理组的 Pin1 mRNA 表达无显著差异，蛋白表达水平在处理 24 h 时低于对照组。OKA 处理组的 LDH 释放明显增加。我们的研究表明，尽管 OKA 处理在 8 h 时抑制了 Pin1 mRNA 表达并诱导了 tau 磷酸化，但它对 Pin1 蛋白表达的影响有 16 h 的时滞。鉴于 Pin1 在许多细胞机制中的重要作用，这些结果可能表明，许多神经退行性疾病中 tau 的过度磷酸化可能通过 Pin1 导致大脑微环境发生一些改变。这是首次在原代皮质神经元的 OKA 诱导模型中证明 Pin1 mRNA 和蛋白表达的改变。
