Evaluating the involvement of metallothionein l in complex l deficiency : an in vitro study
Among the inborn errors of energy metabolism, complex I (CI) deficiency is the most frequently encountered and debilitating disorder of the oxidative phosphorylation system. Hallmarks include an early onset, progressive and heterogeneous course resulting in mortality, as well as a lack of curative treatment. On a cellular level, CI deficiency exhibits various biochemical consequences, of which the most destructive is the excessive production of reactive oxygen species (ROS). Previous studies at this institution have investigated the adaptive cellular responses associated with CI deficiency, with special focus on the increased expression of metallothioneins (MTs). MTs are small, non-enzymatic, endogenously expressed proteins, which have been shown to be involved in mitochondrial energy modulation and the detoxification of ROS. Consequently, these proteins may provide a novel therapeutic option against CI deficiency. However, a suitable experimental model to investigate this has been lacking. Therefore, this study aspired to realise two aims: Firstly, to produce and characterise (on a genetic and protein level) an in vitro model with which the effect of MTI overexpression on CI deficiency could be investigated; secondly, to perform an in vitro evaluation of the effect of MTI overexpression on the bioenergetics consequences of CI deficiency. To achieve the first, Ndufs4 knockout (a CI-deficient model) and TgMTI (an MTI overexpressing model) mice were used. In comparing their genetic backgrounds to C57BL6/J, a clear match was revealed. Consequently, the two mouse strains were crossbred to obtain four genotypes [wildtype, CI-deficient, MTI overexpressing and CI-deficient MTI overexpressing (experimental model)] from which primary skin fibroblasts — used for the remainder of the study — were successfully established. Upon characterisation, each cell line was found to correspond to its expected Ndufs4 and TgMTI genotypes, while TgMTI+/+ cell lines revealed MtI mRNA overexpression. CI deficiency could further be verified in the Ndufs4-/- cell lines, by the absence of the NDUFS4 [NADH dehydrogenase (ubiquinone) iron-sulphur protein 4] protein and instable and non-functional CI. Interestingly, these parameters were all increased in Ndufs4+/+:TgMTI+/+ cells. For the second aim, the effect of each genotype on cell viability, relative mitochondrial DNA copy number, cellular reduction-oxidation- and energy status, and ROS levels was determined. Finally, each genotype’s bioenergetics profile was evaluated, using the Seahorse XF Analyser. In this study, all objectives relating to the development and characterisation of primary mouse fibroblasts were thus successfully met. Since the four fibroblast models exhibited undeniable genetic and protein correspondence to the original mouse strains, they were regarded as suitable for further bioenergetics investigations. However, while the objectives of the second aim could be addressed using suitable methodologies, the results showed striking variation attributable to the process of cell culture, thereby making it impossible to accurately evaluate the effect of genotype on the bioenergetics consequences of CI deficiency. In conclusion, while the model produced corresponded exactly to genetic and protein expectations, primary skin fibroblasts from these mouse models are not suitable to investigate MTI overexpression on CI deficiency. It is therefore recommended that robust conclusions involving these models can only be reached via an in vivo approach.