JSW Life Sciences GmbH, Parkring 12, 8074 Grambach, Austria.
J Mol Neurosci. 2012 May;47(1):192-203. doi: 10.1007/s12031-012-9716-6. Epub 2012 Feb 19.
The role of hyperphosphorylation of the microtubule-associated protein tau in the pathological processes of several neurodegenerative diseases is becoming better understood. Consequently, development of new compounds capable of preventing tau hyperphosphorylation is an increasingly hot topic. For this reason, dependable in vitro and in vivo models that reflect tau hyperphosphorylation in human diseases are needed. In this study, we generated and validated an in vitro model appropriate to test potential curative and preventive compound effects on tau phosphorylation. For this purpose, a stably transfected SH-SY5Y cell line was constructed over-expressing mutant human tau441 (SH-SY5Y-TMHT441). Analyses of expression levels and tau phosphorylation status in untreated cells confirmed relevance to human diseases. Subsequently, the effect of different established kinase inhibitors on tau phosphorylation (e.g., residues Thr231, Thr181, and Ser396) was examined. It was shown with several methods including immunosorbent assays and mass spectrometry that the phosphorylation pattern of tau in SH-SY5Y-TMHT441 cells can be reliably modulated by these compounds, specifically targeting JNK, GSK-3, CDK1/5, and CK1. These four protein kinases are known to be involved in in vivo tau phosphorylation and are therefore authentic indicators for the suitability of this new cell culture model for tauopathies.
微管相关蛋白 tau 的过度磷酸化在几种神经退行性疾病的病理过程中的作用正逐渐被人们所理解。因此，开发能够预防 tau 过度磷酸化的新化合物成为一个日益热门的话题。出于这个原因，需要有可靠的体外和体内模型来反映人类疾病中的 tau 过度磷酸化。在这项研究中，我们构建了一种体外模型，用于测试潜在的治疗和预防化合物对 tau 磷酸化的影响。为此，构建了一个稳定转染的 SH-SY5Y 细胞系，过度表达突变型人 tau441（SH-SY5Y-TMHT441）。未经处理的细胞中 tau 表达水平和磷酸化状态的分析证实与人类疾病相关。随后，研究了不同已建立的激酶抑制剂对 tau 磷酸化（例如，残基 Thr231、Thr181 和 Ser396）的影响。包括免疫吸附测定和质谱在内的多种方法表明，这些化合物可以可靠地调节 SH-SY5Y-TMHT441 细胞中 tau 的磷酸化模式，特别是针对 JNK、GSK-3、CDK1/5 和 CK1。这四种蛋白激酶已知参与体内 tau 磷酸化，因此是这种新的细胞培养模型适合 tau 病的可靠指标。
