Oxidative stress mechanisms related to ribotoxic stress and endoplasmic reticulum stress

神经元缠结（NFT）和神经元细胞死亡是阿尔茨海默病（AD）的标志，异常的过度磷酸化tau蛋白是NFT的主要亚基。然而，tau蛋白磷酸化在AD神经退行性变过程中的作用机制尚不清楚。我们研究了肽基转移酶抑制剂（包括茴香霉素和T2-triol）处理SY 5 Y人神经母细胞瘤细胞后tau蛋白磷酸化和神经元细胞死亡的变化。Western blot分析显示茴香霉素和T2-triol诱导SAPK和p38 MAPK的活化以及tau蛋白在M4（Thr-231/Ser-235）、PHF-1（Ser-396/404）和S422（Ser-422）位点的过度磷酸化。p38的抑制剂SB 203580减弱了这种反应中tau蛋白的磷酸化，而GSK-3的抑制剂锂则没有。在这种反应中，Hoechst 33342观察到的凋亡细胞数小于Live/Dead试剂盒（分子探针）观察到的总死亡细胞数，表明可能涉及非凋亡细胞死亡机制。这种肽基转移酶抑制是一种核糖毒性胁迫。进一步用玫瑰红和光处理细胞，诱导单线态氧。在此条件下，核糖体RNA被氧化，tau蛋白被磷酸化，可能是通过核糖毒性应激。在AD脑tau蛋白的过度磷酸化和神经元细胞死亡可能解释这种核糖毒性应激机制。

Neurofibliraly tangles (NFTs) and neuronal cell death are hallmark of Alzheimer's disease (AD) and abnormal hyperphospholylated tau protein is the major subunit of NFT. However the mechanisms of phosphorylation of tau in neurodegenerative process of AD is unknown. We investigated the changes of phosphorylation of tau protein and neuronal cell death in SY5Y human neuroblastoma cells treated with peptidyltransferase inhibitors including anisomycin and T2-triol. Western blot analysis revealed that anisomycin and T2-triol induced activation of SAPK and p38 MAPK and hyperphosphorylation of tau protein at the M4 (Thr-231/Ser-235), the PHF-1 (Ser-396/404) and the S422 (Ser-422) site. The inhibitor of p38, SB203580, attenuated the phosphorylation of tau protein in this response, whereas an inhibition of GSK-3, lithium, did not. In this response the number of apoptotic cells visualized by Hoechst33342 was less than *he number of total dead cells visualized by Live/Dead kit (Molecular Probe), suggesting the non-apoptotoc cell death mechanism might be involved. This peptidyltransferase-inhibition is one kind of ribotoxic stresses. Further the cells were treated with rose bengal and light, and singlet oxygen was induced. Under this condition, ribosomal RNA was oxidized and tau protein was phosphorylated, probably through ribotoxic stress. In AD brain hyperphosphorylation of tau protein and neuronal cell death might be explained by this ribotoxic stress mechanism.