Decreased Cortical Nrf2 Gene Expression in Autism Spectrum Disorder and its Relationship to Thiol and Cobalamin status

Abstract

Background The etiology of Autism spectrum disorder (ASD) is suspected to involve redox imbalances. In accordance with this, we previously published that sulfur metabolites in the methionine cycle and transsulfuration pathway were dysregulated in ASD frontal cortex. This was accompanied by disturbed vitamin B12 or cobalamin status. Cobalamin abundance and levels of the bioactive species methylcobalamin (MeCbl) were decreased, while levels of oxidized inert hydroxocobalamin (OHCbl) were increased in those tissues. Cellular cobalamin status is dependent on antioxidant availability. Reports have described decreased expression or activity of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in blood cells in ASD. Nrf2 is a transcription factor that promotes transcription of genes involved in maintaining antioxidant and detoxification status. We followed up on our postmortem study by performing real-time quantitative polymerase chain reaction (RT-qPCR) to investigate expression redox-linked genes. Genes included Nrf2 (NFE2L2), cystathionine beta synthase (CBS), cystathionine gamma-lyase (CTH), solute carrier family 1 member 1 (SLC1A1), glutathione synthetase (GSS), and glutathione reductase (GSR). We then searched for correlations among gene expression, thiols, and cobalamins to understand what factors may contribute to deranged sulfur and cobalamin metabolism in the brain in ASD. Methods RNA was isolated from control and ASD frontal cortex tissue using RNAqueous-4PCR kit manufactured by Ambion. Complementary DNA (cDNA) was synthesized with a Roche First Strand cDNA Synthesis kit. RT-qPCR was performed with a Roche LightCycler 480 model. Gene expression was normalized to glyceraldehyde 3-phosphate dehydrogenase. Bioinformatics searches were conducted with data downloaded from the consensus RNA dataset compiled by The Human Protein Atlas. Data were analyzed with Microsoft Excel and GraphPad Prism. Results NFE2L2, CBS, CTH, SLC1A1, and GSS RNA were decreased in ASD frontal cortex. Correlation analyses of pooled control and ASD data revealed strong associations between NFE2L2, cobalamin abundance, methionine, S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH). An association was also found with the transsulfuration metabolite cystathionine. Homocysteine (HCY) and OHCbl were inversely correlated with NFE2L2 expression. We decided to use a bioinformatics approach to further probe a general connection between Nrf2 and cobalamin status, utilizing cobalamin levels in various tissues published by Hsu et al. in 1966. Data obtained from The Human Protein Atlas revealed impressive associations between the expression of Nrf2-regulated genes, NFE2L2 expression, and tissue cobalamin abundance as described by Hsu et al. Conclusion Our RT-qPCR data support a role for decreased Nrf2 activity as an important feature of ASD. We further find that NFE2L2 expression is related to the metabolic patterns we characterized in ASD frontal cortex. Results of our bioinformatics inquiry suggest a potential causal role for Nrf2 that requires further investigation.