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MSc (Biochemistry), North-West University, Potchefstroom Campus Mitochondrial disease is one of the most prevalent inherited paediatric disorders among children, with a prevalence of 1 in 5000 children. Leigh syndrome is the result of a Complex I (CI) deficiency and disrupts the redox balance needed for the function of various dehydrogenase enzymes. Due to the extensive heterogeneity of the disease, it remains a challenge to diagnose and treat mitochondrial diseases. One of the most overlooked areas for possible treatment is the solute carriers of the inner mitochondrial membrane (IMM). These solute carriers function together to form shuttle systems that transport electrons over the impermeable IMM, which can possibly aid in recovery of the disrupted redox balance. Targeted transcriptomic analysis was done on liver, heart and brain tissue collected from a CI deficient (Ndufs4 knockout) mice model to analyse the gene expression of the selected solute carriers part of the malate-aspartate shuttle and the citrate-pyruvate shuttle. The proteins of the glycerol-3-phosphate shuttle was also included due to its ability to also carry electrons over the IMM. This was done by making use of the Ion GeneStudioâ„¢ S5 Semiconductor Sequencer accompanied by the Ion Chefâ„¢ Instrument. Targeted metabolomics was also done on the selected tissue on the metabolites transported by the solute carriers, also to deduce if there are any changes in their abundance with diseases like Leigh syndrome. This was done via GC-TOF-MS and LC-MS/MS analysis. Transcriptomic analysis revealed for the liver tissue, Gpd1 (glycerol-3-phosphate dehydrogenase 1), Mdh1 (malate dehydrogenase 1), Me1 (malic enzyme 1) and Slc25a1 (solute carrier family 25 member 1, citrate carrier) were all down regulated in the knockout group. For the heart tissue, Slc25a22 (solute carrier family 25 member 22, glutamate carrier) and Me1 (malic enzyme 1) were also down regulated in the knockout group and for the brain tissue, Me3 (malic enzyme 3), Slc25a3 (solute carrier 25 member 3, phosphate carrier) and Slc25a22 (solute carrier family 25 member 22, glutamate carrier) were all up regulated in the knockout group. Changes in the expression of these genes all seem to be an effect of the redox imbalance due to complex I deficiency or revolve around an attempt to maintain energy production. Metabolomic analysis indicated an increase in abundance in metabolites associated with the down-regulated enzymes and shuttles. These changes all seem to be an effect of the complex I knockout and not an attempt to compensate for the disruption of the cells function. Considering this study involved an unconventional method for gene expression analysis, the overall success in the outcome of this study can be attributed to the combination of transcriptomics and metabolomics data to give a better understanding of the events at molecular level with abnormalities such as complex I deficiency. Masters

DSc (Science with Biochemistry), North-West University, Potchefstroom Campus 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. Doctoral

MSc (Biochemistry), North-West University, Potchefstroom Campus Mitochondria are involved in a plethora of cellular processes, the most prominent being the production of energy as adenosine triphosphate (ATP) through the oxidative phosphorylation (OXPHOS) system. Dysfunction of the OXPHOS system, especially complex I (CI) is one of the most common groups of inborn errors of metabolism that results in heterogeneous diseases like Leigh Syndrome, which are difficult to treat. To study mitochondrial disease in vivo a whole body Ndufs4 knockout mouse model has been developed and is now commonly used in research. Although mitochondrial disorders have been extensively studied, there are still a lot of gaps in the literature, for example, its effect on biotransformation. The limited ATP of individuals with mitochondrial disorders could affect the activity and capacity of the biotransformation pathway by dysregulating any of the factors that influence metabolism including gene expression through retrograde signalling, allosteric regulation, and the availability of cofactors. The aim of this study was to investigate differential expression of genes involved in phase two biotransformation in an Ndufs4 knockout mouse model. This was achieved by first investigating differential gene expression of glycine conjugation as a primary pathway of phase two biotransformation between Ndufs4 knockout (KO) and wild-type (WT) mice. This investigation was then expanded to explore if the changes observed in glycine conjugation were reflected in the gene expression of seven remaining phase two biotransformation pathways. Significant downregulation of the glycine conjugation pathway in KO mice compared to WT mice was indicated by gene expression analysis of Acsm2 and Glyat. These genes had fold changes of ~-3.6 and ~-1.6, respectively. Gene expression data was validated through enzyme activity analysis of GLYAT, which confirmed the downregulation in KO mice, by exhibiting ~50% residual GLYAT activity compared to WT mice. Significant differential expression was also revealed for 50 of the remaining phase two biotransformation pathway genes. Expression of most of these genes were significantly downregulated. As the exception, gene expression of sulfotransferases was significantly increased. Downstream effects of decreased ATP production and redox imbalance that contribute to retrograde signalling, is hypothesised to be the main contributors to the downregulation of genes investigated. Overall, the data strongly suggests that phase two biotransformation pathways are highly affected by mitochondrial dysfunction. Masters

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

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

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

MSc (Biochemistry), North-West University, Potchefstroom Campus The dysfunction of mitochondrial complex I (CI) impedes the most efficient mechanism feeding electrons into the respiratory chain (RC) — a system that, together with ATP synthase (CV), is responsible for the majority of cellular energy production via oxidative phosphorylation (OXPHOS). Although CI deficiency is the most common defect in mitochondrial energy metabolism, it is among the most complex, multifactorial, poorly understood disorders that currently lack an effective treatment. One of the most promising tools used to gain insight into this form of mitochondrial disease (MD), is the whole-body Ndufs4 knockout (Ndufs4/) mouse model. Although the neurological phenotype of these animals has been widely studied, the effect of CI deficiency on Ndufs4/skeletal muscle metabolism remains elusive. The lack of research on this tissue is largely owed to the view that these animals do not display strong muscle involvement. However, neuromuscular involvement is a hallmark feature of MD and considering skeletal muscle’s high energetic demand, metabolic activity, and integration with the nervous system, this tissue needs to be investigated. The aim of this study was, therefore, to combine hypothesis-generating metabolic profiling and biochemical strategies to gain insight into the energy metabolism of both glycolytic (white quadriceps) and oxidative (soleus) skeletal muscles from Ndufs4/mice. Profiling methods that utilise various high-content analytical platforms were employed in order to generate a broad metabolic phenotype of CI deficient muscles. This multi-platform metabolomics approach comprised of targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS), untargeted gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) and proton nuclear magnetic resonance (1H-NMR) spectroscopy. However, extensive multi-platform metabolomics of mouse tissues presents a challenge due to the limited-quantity samples obtained — especially in the case of NMR spectroscopy, which is inherently insensitive. Therefore, one of the chief objectives of the study was to develop a 1H-NMR method that enables the analysis of small-quantity biological samples. In the first part of the study, we present a novel miniaturised 1H-NMR method utilising 2 mm NMR tubes, which enables metabolic profiling on a tenth of the sample quantity required by a well-established standard operating protocol (SOP). We demonstrate the miniaturised method’s acceptability regarding precision (CV < 15%), relative accuracy (80–120 %), linearity (R2 > 0.95) and statistical equivalence (p < 0.05) to the SOP when analysing spiked synthetic urine as well as mouse muscle extracts. In addition, we exhibit the novel method’s advantages for large-scale metabolomics when; i) adequate sample quantities are available by analysing increasingly concentrated versions (up to 10×) of samples to expand metabolome coverage; or ii) sample quantities are limited by performing a pilot metabolomics study on minute Ndufs4/(n = 3) and wild-type (WT; n = 3) solei, which identified five metabolites (previously linked to MD) strongly discriminating the two genotypes. In the second part of the study, we investigate the effects of CI deficiency in Ndufs4/skeletal muscles. Through multi-platform metabolic profiling of Ndufs4/(n = 19), and WT (n = 20) white quadriceps and soleus muscles, we provide the first empirical evidence of adaptive responses to CI dysfunction involving non-classical pathways that fuel the ubiquinone (Q)-cycle. By restoring the electron flux to CIII via the Q-cycle, these adaptive mechanisms could maintain adequate oxidative ATP production, despite CI deficiency — providing a possible explanation for the lack of muscle involvement in the Ndufs4/phenotype. We report a respective 48 and 34 discriminatory metabolites between Ndufs4/, and WT white quadriceps and soleus muscles, among which the most prominent alterations indicate the involvement of the glycerol-3-phosphate shuttle, the electron transfer flavoprotein system, respiratory chain CII, and the proline cycle in fuelling the Q-cycle. Enzyme (CI-CIV and CS) activity assays confirmed severely reduced (80 %) CI activity in both Ndufs4/(n = 12) muscle types, compared to WTs (n = 10), along with moderate reductions in CS (12 %) and CIII (18 %) activities in Ndufs4/solei. When comparing muscle fibre types, glycolytic fibres seemed to be more vulnerable to CI deficiency, as greater disturbances in metabolic profiles were evident, along with much lower residual CI activity (4.58 ± 1.67 nmol/min/mg) compared to oxidative fibres (14.74 ± 6.67 nmol/min/mg). Taken together, this study contributes to the natural science field in two ways. Firstly, through our novel miniaturised 1H-NMR method, we provide a cost-effective alternative solution for the current restrictions of NMR spectroscopy in the analysis of limited-quantity biological samples. Traditionally, cryoprobe technology is required for such studies; however, we show that a typical NMR spectrometer with a standard probe head can be used to analyse small sample quantities with adequate analytical efficiency. Secondly, through discover-phase multi-platform metabolic profiling, we provide novel mechanistic insight into CI deficiency — thereby highlighting the value of metabolomics in MD research. We report skeletal muscle-specific changes in several metabolic pathways that result from whole-body mitochondrial dysfunction, which upon further investigation could provide novel targets for therapeutic intervention in CI deficiency and potentially lead to the development of new treatment strategies. Masters