The effect of metallothionein overexpression on the transcription of selected genes involved in one-carbon metabolism and oxidative stress in NDUFS4-deficient mouse tissues

Abstract

Respiratory complex I (CI) defects are the most common mitochondrial disease, for which treatment is limited. Additionally, there is a lack of understanding of the impact of a CI defect upon the gene expression regulation of important metabolic pathways. In this study, the impact of a CI defect upon the gene expression of enzymes involved in one-carbon metabolism and oxidative stress was investigated. These two networks are vital for amino acid and purine production, and for the physiological antioxidant defence systems, respectively. The effect of metallothionein (MT1) over expression was also investigated in the context of a CI defect, since metallothionein is an endogenous antioxidant with therapeutic potential. An NADH:Ubiquinone Oxidoreductase Subunit S4 (NDUFS4)-deficient, and an MT1 over expressing mouse model and crossbred mice were used. Metallothionein (MT1), which is an endogenous antioxidant protein with therapeutic potential, was also investigated in the context of a CI defect. After the mouse models were successfully characterised on DNA, RNA and protein levels, real-time quantitative reverse transcriptase PCR (RT-qPCR) was applied to evaluate the expression of selected genes. The quadriceps and brain tissues of 24 mice (four genotypes, namely wildtype, NDUFS4 KO, MT1 over expressing and NDUFS4 KO:MT1 over expressing) were used. It was concluded that Mthfd2, Bhmt, Tyms and Mtrr showed the greatest down regulating change in expression in the brain tissue, whereas Mthfd2 and Gpx1 showed the greatest down regulating change in gene expression in quadriceps tissues. Mt1 mitigated the down regulation only of Tyms in the brain tissue. It is argued that the inhibition of 1-C metabolism by the 5' adenosine monophosphate-activated protein kinase (AMPK) pathway and alternative cellular adaptive mechanisms in the case of a CI defect, might be linked to the results. Factors that impacted the results were the differences in RNA concentration and transcription factors between the brain and quadriceps tissues, the tissue-types used and the number of genes investigated. Nevertheless, strengths of this study included the evidence-based selection of the investigated genes, the mouse models used and the in-depth evaluation of the processing of RT-qPCR data. The importance of considering the bigger gene expression regulation system of an organism, as well as the possible adverse effects of the use of antioxidants in mitochondrial myopathies during research, were also recognised. Taken together, this study has successfully applied empirical methods to investigate the expression – and its regulation – of genes involved in 1-C metabolism and oxidative stress, in the context of a CI defect and transgenic over expression of Mt1.