Investigating tissue-specificity in a mouse model of Leigh Syndrome using metabolomics

Abstract

Mitochondrial disease (MD) is one of the most challenging groups of inborn disorders featuring a tissue-specific vulnerability, governed by largely unknown mechanisms. The value of metabolomics in multi-systemic MD research has been increasingly recognised; however, minimally invasive biofluids are the sample type that is generally favoured and it remains unknown whether such systemic metabolomes clearly reflect tissue-specific metabolic alterations. Here, we mine and cross-compare metabolomes from Ndufs4 knockout (KO) mice with global complex I (CI) deficiency-related Leigh syndrome (LS) -- a complex neurometabolic MD defined by progressive focal lesions in specific brain regions. With this approach, the study aimed to identify and evaluate the extent of common and unique metabolic alterations associated with LS, on a brain regional and systemic level. Semi-targeted liquid chromatography-tandem mass spectrometry and untargeted gas chromatography time-of-flight mass spectrometry were performed on seven sample types from Ndufs4 KO (n≥19) vs wild type (WT, n≥20) mice; which include one lesion-resistant and three lesion-prone brain regions; two skeletal muscles of different fibre type composition; and urine. To gain mechanistic insight into regional neurodegeneration, metabolic alterations in the four brain regions were investigated alongside regional respiratory chain enzyme activity. Enzyme assays confirmed significantly decreased (60-80%) CI activity in all investigated Ndufs4 KO brain regions, with the lesion-resistant region displaying the highest residual CI activity (38% of WT). A higher residual CI activity and a less perturbed NADH/NAD+ ratio, correlate with less severe metabolic perturbations in Ndufs4 KO brain regions. Moreover, less perturbed BCAA oxidation and increased glutamate oxidation seem to distinguish lesionresistant from -prone KO brain regions, thereby identifying key areas of metabolism to target in future therapeutic intervention studies. The subsequent cross-comparison of all seven sample-type metabolomes further revealed widespread alterations in alanine, aspartate, glutamate, and arginine metabolism in Ndufs4 KO mice. An adaptive rewiring of glutamate and nitrogen metabolism, along with increased oxidation of alternative respiratory chain ubiquinone cycle fuels, α-hydroxyglutarate, succinate, and glycerol-3-phosphate seem to be a system-wide response to CI deficiency. Brain region-specific metabolic signatures include the accumulation of BCAAs, proline, and glycolytic intermediates, whereas skeletal muscles display evidence of hyper-catabolism. Furthermore, we describe a systemic dysregulation in one-carbon metabolism and the tricarboxylic acid cycle, which was not clearly reflected in the Ndufs4 KO brain. Altogether, our results provide novel insight into brain region-specific and systemic metabolic responses to LS, while confirming the value of urinary metabolomics when evaluating MD-associated metabolites but cautioning against mechanistic studies relying solely on systemic biofluids.