A metabolomics and biochemical investigation of selected brain regions from Ndufs4 knockout mice
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Mitochondria, the organelles found throughout the cytoplasm of most eukaryotic cells, have essential functions which have been implicated in the etiology of numerous metabolic and degenerative diseases. The mitochondrial oxidative phosphorylation (OXPHOS) system produces up to 90% of cellular energy. It comprises the respiratory chain (RC) of four enzyme complexes and the ATP synthase complex. Genetic mutations that affect the OXPHOS system cause a clinically heterogenous group of disorders which fall under the umbrella term, primary mitochondrial disease (MD). Collectively, MDs are the most common among the inborn errors of metabolism in humans. These diseases generally present with severe, detrimental clinical phenotypes and primarily affect tissues with a high energy demand. An isolated OXPHOS complex I (CI) deficiency is the most commonly observed childhood-onset MD. It is often caused by a mutation in the nuclear coded NADH dehydrogenase (ubiquinone) iron-sulfur protein 4 (Ndufs4) gene. The resulting phenotype, known as Leigh syndrome, is characterised by progressive neurodegeneration in specific brain regions that drives disease progression and premature death. Currently, the mechanisms governing the brain’s regional susceptibility to a CI deficiency are unclear and therapeutic strategies are lacking. Using the Ndufs4 knockout (KO) mouse, an accurate model of Leigh syndrome, this study aimed to determine whether brain regional differences in RC enzyme activities or metabolic profiles could be correlated with neurodegeneration. A combination of spectrophotometric enzyme activity assays and multi-platform metabolomics techniques were applied to investigate four selected brain regions: three neurodegeneration-prone regions (brainstem, cerebellum and olfactory bulbs) and a neurodegeneration-resilient region (anterior cortex). These were obtained from male Ndufs4 KO and wild-type mice. The enzyme assays (biochemical investigation) confirmed that CI activity was significantly reduced (60% to 80%) in the KO brain regions. Additionally, the findings suggested that lower residual CI activity, as well as higher OXPHOS requirements, or differential OXPHOS organisation, could underlie region-specific neurodegeneration. In accordance, a global disturbance in cellular metabolism distinguished the metabolic profiles (metabolomics investigation) of the KO brain regions. These global disturbances seemed to reflect a compensatory response in classic and non-classic metabolic pathways to alleviate the consequences of a CI deficiency. However, these adaptative responses seemed sub-optimal since they are susceptible to the detrimental effects of a CI deficiency and entail maladaptive features. Furthermore, the global metabolic perturbations had a gradient of severity across the brain regions which correlated with neurodegeneration and lower residual CI activity. It therefore seemed that the neurodegeneration-prone brain regions had greater requirements of the sub-optimal compensatory pathways which ultimately reached a detrimental threshold. This then triggered neurodegenerative processes. The impairment of various redox-sensitive reactions also suggested that a lower cellular NAD+/NADH ratio in the neurodegeneration-prone brain regions might augment neurodegenerative processes. In addition, a few discriminatory metabolites unique to the anterior cortex suggested that inherent regional differences in metabolism might play a role in regional neurodegeneration. Conclusively, the results enabled a better understanding of the regional neurodegeneration in Ndufs4 KO mice. The potential metabolic targets for treatment and for monitoring disease progression or therapeutic interventions revealed in this study, warrant further investigation.