Althausen S, Paschen W
Department of Experimental Neurology, Max-Planck-Institute for Neurological Research, Gleuelerstr. 50, 50931 Köln, Germany.
Brain Res Mol Brain Res. 2000 Dec 8;84(1-2):32-40. doi: 10.1016/s0169-328x(00)00208-4.
Elevated homocysteine levels have been suggested to contribute to various pathological states of the brain. However, the basic mechanisms underlying homocysteine-induced neurotoxicity have not yet been fully elucidated. In the present series of experiments, we investigated the effect of homocysteine on mRNA levels of genes coding for cytoplasmic- or endoplasmic reticulum-resident stress proteins. Primary neuronal cell cultures were exposed to different homocysteine levels for 1-24 h. Cell injury was evaluated using the MTT assay, protein synthesis was studied by measuring the incorporation of L-[4,5-3H]leucine into proteins, mRNA levels of hsp70, gadd153, grp78, and grp94 were evaluated by quantitative PCR, and changes in protein levels of hsp70, grp78 and grp94 were analyzed by immunoblotting. Exposure of cells to 5 or 10 mM homocysteine for 24 h induced marked cell injury (decrease of viability to 58 or 45% of control respectively). After 6 h treatment, gadd153, grp78 and grp94 mRNA levels increased markedly, but only when cells were exposed to levels of homocysteine high enough to induce cell injury. In addition, hsp70 mRNA levels and protein synthesis were significantly reduced. At earlier (1 or 3 h) or later (12 or 24 h) time intervals, homocysteine exposure induced a marked increase in mRNA levels of all genes studied. GRP78 and GRP94 protein levels were increased in cells exposed to 5 mM homocysteine for 24 h but not in cells exposed to 10 mM homocysteine. HSP70 protein levels, in contrast, were decreased in cells exposed to homocysteine for different periods. The expression of genes coding for ER-resident stress proteins is specifically activated under conditions of ER stress. The close relationship between the extent of cell injury and increase in grp78 mRNA levels suggests that ER dysfunction may contribute to the pathological process. The results imply that the ER is an intracellular target of homocysteine toxicity.
同型半胱氨酸水平升高被认为与大脑的各种病理状态有关。然而，同型半胱氨酸诱导神经毒性的基本机制尚未完全阐明。在本系列实验中，我们研究了同型半胱氨酸对编码细胞质或内质网驻留应激蛋白的基因mRNA水平的影响。将原代神经元细胞培养物暴露于不同水平的同型半胱氨酸中1 - 24小时。使用MTT法评估细胞损伤，通过测量L-[4,5-³H]亮氨酸掺入蛋白质来研究蛋白质合成，通过定量PCR评估hsp70、gadd153、grp78和grp94的mRNA水平，并通过免疫印迹分析hsp70、grp78和grp94蛋白质水平的变化。将细胞暴露于5或10 mM同型半胱氨酸24小时会诱导明显的细胞损伤（活力分别降至对照的58%或45%）。处理6小时后，gadd153、grp78和grp94的mRNA水平显著升高，但仅当细胞暴露于足以诱导细胞损伤的高同型半胱氨酸水平时才会如此。此外，hsp70的mRNA水平和蛋白质合成显著降低。在更早（1或3小时）或更晚（12或24小时）的时间间隔，同型半胱氨酸暴露会导致所有研究基因的mRNA水平显著增加。暴露于5 mM同型半胱氨酸24小时的细胞中GRP78和GRP94的蛋白质水平升高，但暴露于10 mM同型半胱氨酸的细胞中则没有。相反，在暴露于同型半胱氨酸不同时间段的细胞中，HSP70的蛋白质水平降低。在内质网应激条件下，编码内质网驻留应激蛋白的基因表达会被特异性激活。细胞损伤程度与grp78 mRNA水平升高之间的密切关系表明内质网功能障碍可能参与了病理过程。结果表明内质网是同型半胱氨酸毒性的细胞内靶点。
