Evaluating the effect of a ketogenic diet in a Leigh syndrome mouse model

Abstract

Mitochondrial diseases (MDs) are a heterogeneous group of metabolic disorders with clinical manifestations resulting from impaired energy production and tissue-specific vulnerabilities. Leigh syndrome (LS), a mitochondrial disorder, is a severe paediatric neurodegenerative disorder primarily associated with complex I (CI) deficiency. CI dysfunction in LS is typically linked to mutations in nuclear DNA-encoded subunits of CI like NDUFS4, which plays a critical role in CI assembly and stability. Defects in this subunit leads to mitochondrial dysfunction and neurodegeneration. With current MD treatments typically focusing on symptom management, there is an urgent need for innovative therapeutic strategies that target the underlying metabolic and mitochondrial dysfunctions. This thesis investigates the therapeutic potential of dietary interventions to alleviate mitochondrial dysfunction in LS, using the preclinical Ndufs4 knockout (KO) mouse model, which recapitulates hallmark features of LS.

In Phase A of the study, a preliminary investigation was conducted in Ndufs4 wild-type (WT) and heterozygous (HET) mice to evaluate the impact of a ketogenic diet (KD) on mTOR activity, redox balance, and systemic metabolism. Given the known association between alcohol consumption and redox dysregulation, chronic alcohol administration was used to simulate the redox and metabolic alterations observed in MD. Western blot analyses of liver samples revealed that the KD significantly suppressed mTOR complex 1 (mTORC1) activity, a key regulator of cellular metabolism. Metabolomic profiling using multi-platform approaches, such as LC-MS/MS, ¹H-NMR, and GC-TOF-MS, demonstrated that the KD induced significant alterations in metabolic pathways, particularly affecting amino acid metabolism and the tricarboxylic acid (TCA) cycle. Flow cytometry assessments further showed a reduction in oxidative stress markers, although prolonged alcohol exposure appeared to blunt some of the KD benefits. These findings underscored the complex interaction between dietary composition and redox homeostasis. Building upon Phase A of the study, the dietary intervention was modified in Phase B to a highfat diet (HFD) enriched with omega-3 (ω-3) polyunsaturated fatty acids (PUFAs) derived from fish oil. In this phase, both phenotypic and biochemical parameters were assessed in a Ndufs4 KO mouse after dietary intervention. Phenotypic evaluations, included locomotor activity tests, clasping tests, grip strength measurements, and survival assessments. These evaluations revealed that mice fed the ω-3 PUFA-enriched HFD exhibited significant improvements in clasping symptoms and an extended lifespan compared to those on a normal diet. Biochemical analyses of whole brain tissues indicated that signalling pathways such as mTOR, PGC1-α, SIRT1 and TNF-α was unaffected by the diet. However, systemic inflammation in serum was reduced by the diet intervention as indicated by a decrease in IL-6 and TNF cytokines Comprehensive metabolomic analyses further indicated that the HFD reprogrammed metabolicpathways involving the TCA cycle, branched-chain amino acid metabolism, and lipid metabolism.

In conclusion, the findings of this thesis imply that dietary interventions using a PUFA-enriched HFD offers a promising, safer, and more effective strategy for mitigating LS pathology than a standard KD. The HFD may alleviate some of the metabolic impairments underlying LS through the attenuation of systemic inflammation and alteration of metabolic pathways. These results provide a compelling rationale for further investigation into dietary interventions as complementary therapeutic strategies for MDs. Future studies are needed to investigate the precise molecular mechanisms underlying this PUFA enriched HFD and to evaluate the potential for clinical translation.