Barbosa Livea F, Cerqueira Fernanda M, Macedo Antero F A, Garcia Camila C M, Angeli José Pedro F, Schumacher Robert I, Sogayar Mari Cleide, Augusto Ohara, Carrì Maria Teresa, Di Mascio Paolo, Medeiros Marisa H G
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil.
Biochim Biophys Acta. 2010 May;1802(5):462-71. doi: 10.1016/j.bbadis.2010.01.011. Epub 2010 Jan 25.
Mutations in the gene encoding cytosolic Cu,Zn-superoxide dismutase (SOD1) have been linked to familial amyotrophic lateral sclerosis (FALS). However the molecular mechanisms of motor neuron death are multi-factorial and remain unclear. Here we examined DNA damage, p53 activity and apoptosis in SH-SY5Y human neuroblastoma cells transfected to achieve low-level expression of either wild-type or mutant Gly(93)-->Ala (G93A) SOD1, typical of FALS. DNA damage was investigated by evaluating the levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and DNA strand breaks. Significantly higher levels of DNA damage, increased p53 activity, and a greater percentage of apoptotic cells were observed in SH-SY5Y cells transfected with G93A SOD1 when compared to cells overexpressing wild-type SOD1 and untransfected cells. Western blot, FACS, and confocal microscopy analysis demonstrated that G93A SOD1 is present in the nucleus in association with DNA. Nuclear G93A SOD1 has identical superoxide dismutase activity but displays increased peroxidase activity when compared to wild-type SOD1. These results indicate that the G93A mutant SOD1 association with DNA might induce DNA damage and trigger the apoptotic response by activating p53. This toxic activity of mutant SOD1 in the nucleus may play an important role in the complex mechanisms associated with motor neuron death observed in ALS pathogenesis.
编码胞质铜锌超氧化物歧化酶（SOD1）的基因突变与家族性肌萎缩侧索硬化症（FALS）有关。然而，运动神经元死亡的分子机制是多因素的，目前仍不清楚。在此，我们检测了转染后实现野生型或突变型甘氨酸（93）→丙氨酸（G93A）SOD1低水平表达的SH-SY5Y人神经母细胞瘤细胞中的DNA损伤、p53活性和细胞凋亡情况，G93A SOD1是FALS的典型特征。通过评估8-氧代-7,8-二氢-2'-脱氧鸟苷（8-氧代脱氧鸟苷）水平和DNA链断裂情况来研究DNA损伤。与过表达野生型SOD1的细胞和未转染细胞相比，在转染G93A SOD1的SH-SY5Y细胞中观察到DNA损伤水平显著更高、p53活性增加以及凋亡细胞百分比更高。蛋白质印迹、流式细胞术和共聚焦显微镜分析表明，G93A SOD1与DNA结合存在于细胞核中。与野生型SOD1相比，核内G93A SOD1具有相同的超氧化物歧化酶活性，但过氧化物酶活性增加。这些结果表明，G93A突变型SOD1与DNA的结合可能诱导DNA损伤并通过激活p53触发凋亡反应。突变型SOD1在细胞核中的这种毒性活性可能在肌萎缩侧索硬化症发病机制中观察到的与运动神经元死亡相关的复杂机制中起重要作用。
