Oxidative stress mechanisms related to ribotoxic stress and endoplasmic reticulum stress

神经纤维缠结(NFT)和神经细胞死亡是阿尔茨海默病(AD)的特征，异常的高磷酸化tau蛋白是NFT的主要亚单位。然而，tau蛋白在阿尔茨海默病神经退变过程中的磷酸化机制尚不清楚。我们研究了氨基转移酶抑制剂(包括樟柳霉素和T2-Triol)对SY5Y人神经母细胞瘤细胞tau蛋白磷酸化和神经细胞死亡的影响。Western印迹分析显示，山奈素和T2-Triol可诱导SAPK和p38MAPK的激活以及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SB203580减弱了这种反应中tau蛋白的磷酸化，而GSK-3的抑制剂锂则不能。在这一反应中，Hoechst33342显示的凋亡细胞数少于Live/Dead Kit(分子探针)显示的死亡细胞总数，提示可能参与了非凋亡性细胞死亡机制。这种肽转移酶抑制是核毒应激的一种。进一步用孟加拉玫瑰和光处理细胞，并诱导单线态氧。在这种情况下，核糖体RNA被氧化，tau蛋白被磷酸化，可能是通过核毒应激。在阿尔茨海默病模型中，tau蛋白过度磷酸化和神经细胞死亡可能是这种核毒性应激机制的解释。

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.