Mutations in Cu/Zn superoxide dismutase (Sod1) have been reported in both familial and sporadic amyotrophic lateral sclerosis (ALS). In this study, we investigated the behavior of heteromeric combinations of wild-type (WT) and mutant Sod1 proteins A4V, L38V, G93A, and G93C in human cells. We showed that both WT and mutant Sod1 formed dimers and oligomers, but only mutant Sod1 accumulated in intracellular inclusions. Coexpression of WT and hSod1 mutants resulted in the formation of a larger number of intracellular inclusions per cell than that observed in cells coexpressing WT or mutant hSod1. The number of inclusions was greater in cells expressing A4V hSod1. To eliminate the contribution of endogenous Sod1, and better evaluate the effect of ALS-associated mutant Sod1 expression, we expressed human Sod1 WT and mutants in human cells knocked down for endogenous Sod1 (Sod1-KD), and in sod1Δ yeast cells. Using Sod1-KD cells we found that the WT–A4V heteromers formed higher molecular weight species compared with A4V and WT homomers. Using the yeast model, in conditions of chronological aging, we concluded that cells expressing Sod1 heterodimers showed decreased antioxidant activity, increased oxidative damage, reduced longevity, and oxidative stress-induced mutant Sod1 aggregation. In addition, we also found that ALS-associated Sod1 mutations reduced nuclear localization and, consequently, impaired the antioxidant response, suggesting this change in localization may contribute to disease in familial ALS. Overall, our study provides insight into the molecular underpinnings of ALS and may open avenues for the design of future therapeutic strategies. Significance Aggregation of the antioxidant enzyme Sod1 represents common factors of both familial (fALS) and sporadic cases of ALS, a fatal neurodegenerative disease. Although many ALS studies have focused on Sod1 homodimers/homomers, the investigation of Sod1 heterodimers/heteromers remains controversial and has mostly been performed with recombinant proteins in vitro, in the absence of a cellular environment. By using living cells, this study sheds light into a critical issue in the context of fALS, the high toxicity of the WT–mutant heteromeric inclusions, especially WT–A4V heteromers which accumulate both in human cells as well as in chronologically aged yeast cells. Besides the aggregation, we proposed that an inefficient heteromer response against oxidative conditions might contribute to fALS-linked mutant hSod1 toxicity.

Among the familial forms of amyotrophic lateral sclerosis (fALS), 20% are associated with the Cu,Zn-superoxide dismutase (Sod1). fALS is characterized by the accumulation of aggregated proteins and the increase in oxidative stress markers. Here, we used the non-invasive bimolecular fluorescence complementation (BiFC) assay in human H4 cells to investigate the kinetics of aggregation and subcellular localization of Sod1 mutants. We also studied the effect of the different Sod1 mutants to respond against oxidative stress by following the levels of reactive oxygen species (ROS) after treatment with hydrogen peroxide. Our results showed that only 30% of cells transfected with A4VSod1 showed no inclusions while for the other Sod1 mutants tested (L38V, G93A and G93C), this percentage was at least 70%. In addition, we found that 10% of cells transfected with A4VSod1 displayed more than five inclusions per cell and that A4V and G93A Sod1 formed inclusions more rapidly than L38V and G93C Sod1. Expression of WTSod1 significantly decreased the intracellular oxidation levels in comparison with expression of fALS Sod1 mutants, suggesting the mutations induce a functional impairment. All fALS mutations impaired nuclear localization of Sod1, which is important for maintaining genomic stability. Consistently, expression of WTSod1, but not of fALS Sod1 mutants, reduced DNA damage, as measured by the comet assay. Altogether, our study sheds light into the effects of fALS Sod1 mutations on inclusion formation, dynamics, and localization as well as on antioxidant response, opening novel avenues for investigating the role of fALS Sod1 mutations in pathogenesis.

Adverse experiences in the perinatal period have been associated with the methylation of the human glucocorticoid receptor gene (NR3C1) and long-term diseases. We conducted a systematic review on the association between adversities in the perinatal period and DNA methylation in the 1 _F region of the NR3C1 gene in newborns. We explored the MEDLINE, Web of Science, Scopus, Scielo, and Lilacs databases without time or language limitations. Two independent reviewers performed the selection of articles and data extraction. A third participated in the methodological quality assessment and consensus meetings at all stages. Finally, ten studies were selected. Methodological quality was considered moderate in six and low in four. Methylation changes were reported in 41 of the 47 CpG sites of exon 1 _F. Six studies addressed maternal conditions during pregnancy: two reported methylation changes at the same sites (CpG 10, 13, 20, 21 and 47), and four at one or more sites from CpG 35 to 39. Four studies addressed neonatal parameters and morbidities: methylation changes at the same sites 4, 8, 10, 16, 25, and 35 were reported in two. Hypermethylation associated with stressful conditions prevailed. Hypomethylation was more often associated with protective conditions (maternal-foetal attachment during pregnancy, breast milk intake, higher birth weight or Apgar). In conclusion, methylation changes in several sites of the 1 _F region of the NR3C1 gene in newborns and very young infants were associated with perinatal stress, but more robust and comparable results are needed to corroborate site-specific associations.