Urinary metabolomics investigation of Ndufs4 knockout mice

MSc (Biochemistry), North-West University, Potchefstroom Campus

Mitochondrial diseases (MDs) are the most common inborn errors of metabolism, with an estimated prevalence of approximately 1 in 5 000 live births, and are mainly caused by deficiencies of complex I (CI) of the oxidative phosphorylation (OXPHOS) system. Clinical presentations of CI deficiency are highly heterogeneous, with the most commonly reported, being Leigh syndrome (LS) – a devastating progressive, multi-systemic, neurodegenerative disorder. The Ndufs4 gene, which encodes for an 18 kDa subunit of CI, is a mutational hotspot in LS patients. To date, the efficacy of the limited available therapeutic interventions remains inconclusive, and can, in large, be attributed to our poor understanding of the pathological mechanisms behind these highly complex diseases. Fortunately, with a whole-body Ndufs4 knockout (KO) mouse model available, researchers have a great opportunity to gain a better understanding of this commonly reported MD. What remains lacking, however, is the incorporation of multi-platform metabolomics using urine. This biofluid shows promise in revealing global metabolic perturbations in MDs, and thus possesses the potential to elucidate disease mechanisms. The aim of this study, therefore, was to investigate the metabolic consequences of Ndufs4 deficiency by analysing the urine of the whole-body Ndufs4 KO mouse model. This was accomplished by implementing two main objectives: firstly, by validating the mouse model via genetic and phenotypic evaluation and the measurement of CI activity in the liver; and secondly, by comparing the urinary metabolome of Ndufs4 KO and wild-type mice, acquired via both untargeted and targeted analyses, in order to obtain a comprehensive view of the metabolic consequences. In this study, the mouse model was successfully validated on the genetic and phenotypic level, with Ndufs4 KO mice displaying well-reported phenotypic characteristics, including growth retardation, transient alopecia and hunched back posture. Biochemically, the mouse model was further confirmed with Ndufs4 KO mice exhibiting 15% residual CI activity in the liver. Urinary metabolomic analyses revealed multiple metabolic perturbations in the Ndufs4 KO mice. Most notably, were the markers classically observed in MDs and commonly believed to be the result of an altered redox status, namely elevated levels of pyruvate, lactate and alanine as well as some tricarboxylic acid cycle intermediates (2-ketoglutarate, fumarate and malate). A downregulation in protein/amino acid catabolism was observed, as indicated by decreased levels of numerous amino acids (e.g. glutamine, glutamate, leucine, isoleucine, valine and phenylalanine), 3-methylhistine (index of skeletal muscle breakdown) and metabolites associated with the urea cycle (arginine, citrulline and N-acetylglutamate). Similarly, lipid/fatty acid catabolism also appeared to be downregulated, as shown by lowered levels of glycerol as well as numerous carnitine- and glycine fatty acid conjugates (octanoyl- and decanoylcarntine; butyryl-, valeryl- and hexanoylglycine). Metabolites present in pathways associated with biosynthetic processes and/or ROS scavenging (including the pentose phosphate pathway, one-carbon metabolism and de novo pyrimidine synthesis) were also decreased. Taken together, the implementation of urinary metabolomics proved to be successful in revealing global metabolic perturbations in Ndufs4 KO mice.

Masters