The Blood-Brain Barrier Is Unaffected in the Ndufs4-/- Mouse Model of Leigh Syndrome.

Mitochondrial dysfunction plays a major role in physiological aging and in many pathological conditions. Yet, no study has explored the consequence of primary mitochondrial deficiency on the blood-brain barrier (BBB) structure and function. Addressing this question has major implications for pharmacological and genetic strategies aimed at ameliorating the neurological symptoms that are often predominant in patients suffering from these conditions. In this study, we examined the permeability of the BBB in the Ndufs4-/- mouse model of Leigh syndrome (LS). Our results indicated that the structural and functional integrity of the BBB was preserved in this severe model of mitochondrial disease. Our findings suggests that pharmacological or gene therapy strategies targeting the central nervous system in this mouse model and possibly other models of mitochondrial dysfunction require the use of specific tools to bypass the BBB. In addition, they raise the need for testing the integrity of the BBB in complementary in vivo models.

Sulfide catabolism in hibernation and neuroprotection.

The mammalian brain is exquisitely vulnerable to lack of oxygen. However, the mechanism underlying the brain's sensitivity to hypoxia is incompletely understood. In this narrative review, we present a case for sulfide catabolism as a key defense mechanism of the brain against acute oxygen shortage. We will examine literature on the role of sulfide in hypoxia/ischemia, deep hibernation, and leigh syndrome patients, and present our recent data that support the neuroprotective effects of sulfide catabolism and persulfide production. When oxygen levels become low, hydrogen sulfide (H2S) accumulates in brain cells and impairs the ability of these cells to use the remaining, available oxygen to produce energy. In recent studies, we found that hibernating ground squirrels, which can withstand very low levels of oxygen, have high levels of sulfide:quinone oxidoreductase (SQOR) and the capacity to catabolize hydrogen sulfide in the brain. Silencing SQOR increased the sensitivity of the brain of squirrels and mice to hypoxia, whereas neuron-specific SQOR expression prevented hypoxia-induced sulfide accumulation, bioenergetic failure, and ischemic brain injury in mice. Excluding SQOR from mitochondria increased sensitivity to hypoxia not only in the brain but also in heart and liver. Pharmacological agents that scavenge sulfide and/or increase persulfide maintained mitochondrial respiration in hypoxic neurons and made mice resistant to ischemic injury to the brain or spinal cord. Drugs that oxidize hydrogen sulfide and/or increase persulfide may prove to be an effective approach to the treatment of patients experiencing brain injury caused by oxygen deprivation or mitochondrial dysfunction.

Identification of a novel MT-ND3 variant and restoring mitochondrial function by allotopic expression of MT-ND3 gene.

Mitochondrial diseases are caused by nuclear, or mitochondrial DNA (mtDNA) variants and related co-factors. Here, we report a novel m.10197G > C variant in MT-ND3 in a patient, and two other patients with m.10191 T > C. MT-ND3 variants are known to cause Leigh syndrome or mitochondrial complex I deficiency. We performed the functional analyses of the novel m.10197G > C variant that significantly lowered MT-ND3 protein levels, causing complex I assembly and activity deficiency, and reduction of ATP synthesis. We adapted a previously described re-engineering technique of delivering mitochondrial genes into mitochondria through codon optimization for nuclear expression and translation by cytoplasmic ribosomes to rescue defects arising from the MT-ND3 variants. We constructed mitochondrial targeting sequences along with the codon-optimized MT-ND3 and imported them into the mitochondria. To achieve the goal, we imported codon-optimized MT-ND3 into mitochondria in three patients with m.10197G > C and m.10191 T > C missense variants in the MT-ND3. Nuclear expression of the MT-ND3 gene partially restored protein levels, complex I deficiency, and significant improvement of ATP production indicating a functional rescue of the mutant phenotype. The codon-optimized nuclear expression of mitochondrial protein and import inside the mitochondria can supplement the requirements for ATP in energy-deficient mitochondrial disease patients.

Lack of mitochondrial complex I assembly factor NDUFAF2 results in a distinctive infantile-onset brainstem neurodegenerative disease with early lethality.

BACKGROUND: Congenital disorders of the mitochondrial respiratory chain are a heterogeneous group of inborn errors of metabolism. Among them, NADH:ubiquinone oxidoreductase (complex I, CI) deficiency is the most common. Biallelic pathogenic variants in NDUFAF2, encoding the nuclear assembly CI factor NDUFAF2, were initially reported to cause progressive encephalopathy beginning in infancy. Since the initial report in 2005, less than a dozen patients with NDUFAF2-related disease have been reported. METHODS: Clinical, biochemical, and neuroradiological features of four new patients residing in Northern Israel were collected during 2016-2022 at Emek Medical Center. Enzymatic activities of the five respiratory-chain complexes were determined in isolated fibroblast mitochondria by spectrophotometric methods. Western blot analyses were conducted with anti-human NDUFAF2 antibody; antibody against the mitochondrial marker VDAC1 was used as a loading control. Genetic studies were performed by chromosome microarray analysis using Affymetrix CytoScan 750 K arrays. RESULTS: All four patients presented with infantile-onset growth retardation, ophthalmological impairments with nystagmus, strabismus (starting between 5 and 9 months), and further progressed to life-threatening episodes of apnea usually triggered by trivial febrile illnesses (between 10 and 18 months) with gradual loss of acquired developmental milestones (3 of 4 patients). Serial magnetic-resonance imaging studies in two of the four patients showed a progressive pattern of abnormal T2-weighted hyperintense signals involving primarily the brainstem, the upper cervical cord, and later, the basal ganglia and thalami. Magnetic-resonance spectroscopy in one patient showed an increased lactate peak. Disease progression was marked by ventilatory dependency and early lethality. 3 of the 4 patients tested, harbored a homozygous 142-kb partial interstitial deletion that omits exons 2-4 of NDUFAF2. Mitochondrial CI activity was significantly decreased in the only patient tested. Western blot analysis disclosed the absence of NDUFAF2 protein compared to normal controls. In addition, we reviewed all 10 previously reported NDUFAF2-deficient cases to better characterize the disease. CONCLUSIONS: Biallelic loss-of-function mutations in NDUFAF2 result in a distinctive phenotype in the spectrum of Leigh syndrome with clinical and neuroradiological features that are primarily attributed to progressive brainstem damage.

Teamwork makes the dream work: functional collaborations between families, scientists, and healthcare providers to drive progress in the treatment of Leigh Syndrome.

BACKGROUND: Leigh syndrome, an inherited neurometabolic disorder, is estimated to be the most common pediatric manifestation of mitochondrial disease. No treatments are currently available for Leigh syndrome due to many hurdles in drug discovery efforts. Leigh syndrome causal variants span over 110 different genes and likely lead to both unique and shared biochemical alterations, often resulting in overlapping phenotypic features. The mechanisms by which pathogenic variants in mitochondrial genes alter cellular phenotype to promote disease remain poorly understood. The rarity of cases of specific causal variants creates barriers to drug discovery and adequately sized clinical trials. BODY: To address the current challenges in drug discovery and facilitate communication between researchers, healthcare providers, patients, and families, the Boston University integrative Cardiovascular Metabolism and Pathophysiology (iCAMP) Lab and Cure Mito Foundation hosted a Leigh Syndrome Symposium. This symposium brought together expert scientists and providers to highlight the current successes in drug discovery and novel models of mitochondrial disease, and to connect patients to providers and scientists to foster community and communication. CONCLUSION: In this symposium review, we describe the research presented, the hurdles ahead, and strategies to better connect the Leigh syndrome community members to advance treatments for Leigh syndrome.

N-acetylcysteine and cysteamine bitartrate prevent azide-induced neuromuscular decompensation by restoring glutathione balance in two novel surf1-/- zebrafish deletion models of Leigh syndrome.

SURF1 deficiency (OMIM # 220110) causes Leigh syndrome (LS, OMIM # 256000), a mitochondrial disorder typified by stress-induced metabolic strokes, neurodevelopmental regression and progressive multisystem dysfunction. Here, we describe two novel surf1-/- zebrafish knockout models generated by CRISPR/Cas9 technology. While gross larval morphology, fertility, and survival into adulthood appeared unaffected, surf1-/- mutants manifested adult-onset ocular anomalies and decreased swimming activity, as well as classical biochemical hallmarks of human SURF1 disease, including reduced complex IV expression and enzymatic activity and increased tissue lactate. surf1-/- larvae also demonstrated oxidative stress and stressor hypersensitivity to the complex IV inhibitor, azide, which exacerbated their complex IV deficiency, reduced supercomplex formation, and induced acute neurodegeneration typical of LS including brain death, impaired neuromuscular responses, reduced swimming activity, and absent heartrate. Remarkably, prophylactic treatment of surf1-/- larvae with either cysteamine bitartrate or N-acetylcysteine, but not other antioxidants, significantly improved animal resiliency to stressor-induced brain death, swimming and neuromuscular dysfunction, and loss of heartbeat. Mechanistic analyses demonstrated cysteamine bitartrate pretreatment did not improve complex IV deficiency, ATP deficiency, or increased tissue lactate but did reduce oxidative stress and restore glutathione balance in surf1-/- animals. Overall, two novel surf1-/- zebrafish models recapitulate the gross neurodegenerative and biochemical hallmarks of LS, including azide stressor hypersensitivity that was associated with glutathione deficiency and ameliorated by cysteamine bitartrate or N-acetylcysteine therapy.

The immune system as a driver of mitochondrial disease pathogenesis: a review of evidence.

BACKGROUND: Genetic mitochondrial diseases represent a significant challenge to human health. These diseases are extraordinarily heterogeneous in clinical presentation and genetic origin, and often involve multi-system disease with severe progressive symptoms. Mitochondrial diseases represent the most common cause of inherited metabolic disorders and one of the most common causes of inherited neurologic diseases, yet no proven therapeutic strategies yet exist. The basic cell and molecular mechanisms underlying the pathogenesis of mitochondrial diseases have not been resolved, hampering efforts to develop therapeutic agents. MAIN BODY: In recent pre-clinical work, we have shown that pharmacologic agents targeting the immune system can prevent disease in the Ndufs4(KO) model of Leigh syndrome, indicating that the immune system plays a causal role in the pathogenesis of at least this form of mitochondrial disease. Intriguingly, a number of case reports have indicated that immune-targeting therapeutics may be beneficial in the setting of genetic mitochondrial disease. Here, we summarize clinical and pre-clinical evidence suggesting a key role for the immune system in mediating the pathogenesis of at least some forms of genetic mitochondrial disease. CONCLUSIONS: Significant clinical and pre-clinical evidence indicates a key role for the immune system as a significant in the pathogenesis of at least some forms of genetic mitochondrial disease.

Metabolic rescue ameliorates mitochondrial encephalo-cardiomyopathy in murine and human iPSC models of Leigh syndrome.

BACKGROUND: Mice with deletion of complex I subunit Ndufs4 develop mitochondrial encephalomyopathy resembling Leigh syndrome (LS). The metabolic derangement and underlying mechanisms of cardio-encephalomyopathy in LS remains incompletely understood. METHODS: We performed echocardiography, electrophysiology, confocal microscopy, metabolic and molecular/morphometric analysis of the mice lacking Ndufs4. HEK293 cells, human iPS cells-derived cardiomyocytes and neurons were used to determine the mechanistic role of mitochondrial complex I deficiency. RESULTS: LS mice develop severe cardiac bradyarrhythmia and diastolic dysfunction. Human-induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) with Ndufs4 deletion recapitulate LS cardiomyopathy. Mechanistically, we demonstrate a direct link between complex I deficiency, decreased intracellular (nicotinamide adenine dinucleotide) NAD+ /NADH and bradyarrhythmia, mediated by hyperacetylation of the cardiac sodium channel NaV 1.5, particularly at K1479 site. Neuronal apoptosis in the cerebellar and midbrain regions in LS mice was associated with hyperacetylation of p53 and activation of microglia. Targeted metabolomics revealed increases in several amino acids and citric acid cycle intermediates, likely due to impairment of NAD+ -dependent dehydrogenases, and a substantial decrease in reduced Glutathione (GSH). Metabolic rescue by nicotinamide riboside (NR) supplementation increased intracellular NAD+ / NADH, restored metabolic derangement, reversed protein hyperacetylation through NAD+ -dependent Sirtuin deacetylase, and ameliorated cardiomyopathic phenotypes, concomitant with improvement of NaV 1.5 current and SERCA2a function measured by Ca2+ -transients. NR also attenuated neuronal apoptosis and microglial activation in the LS brain and human iPS-derived neurons with Ndufs4 deletion. CONCLUSIONS: Our study reveals direct mechanistic explanations of the observed cardiac bradyarrhythmia, diastolic dysfunction and neuronal apoptosis in mouse and human induced pluripotent stem cells (iPSC) models of LS.

Human iPSC-derived cerebral organoids model features of Leigh syndrome and reveal abnormal corticogenesis.

Leigh syndrome (LS) is a rare, inherited neurometabolic disorder that presents with bilateral brain lesions caused by defects in the mitochondrial respiratory chain and associated nuclear-encoded proteins. We generated human induced pluripotent stem cells (iPSCs) from three LS patient-derived fibroblast lines. Using whole-exome and mitochondrial sequencing, we identified unreported mutations in pyruvate dehydrogenase (GM0372, PDH; GM13411, MT-ATP6/PDH) and dihydrolipoyl dehydrogenase (GM01503, DLD). These LS patient-derived iPSC lines were viable and capable of differentiating into progenitor populations, but we identified several abnormalities in three-dimensional differentiation models of brain development. LS patient-derived cerebral organoids showed defects in neural epithelial bud generation, size and cortical architecture at 100 days. The double mutant MT-ATP6/PDH line produced organoid neural precursor cells with abnormal mitochondrial morphology, characterized by fragmentation and disorganization, and showed an increased generation of astrocytes. These studies aim to provide a comprehensive phenotypic characterization of available patient-derived cell lines that can be used to study Leigh syndrome.

Microglial response promotes neurodegeneration in the Ndufs4 KO mouse model of Leigh syndrome.

Leigh syndrome is a mitochondrial disease characterized by neurodegeneration, neuroinflammation, and early death. Mice lacking NDUFS4, a mitochondrial complex I subunit (Ndufs4 KO mice), have been established as a good animal model for studying human pathology associated with Leigh syndrome. As the disease progresses, there is an increase in neurodegeneration and neuroinflammation, thereby leading to deteriorating neurological symptoms, including motor deficits, breathing alterations, and eventually, death of the animal. However, despite the magnitude of neuroinflammation associated with brain lesions, the role of neuroinflammatory pathways and their main cellular components have not been addressed directly as relevant players in the disease pathology. Here, we investigate the role of microglial cells, the main immune cells of the CNS, in Leigh-like syndrome pathology, by pharmacologically depleting them using the colony-stimulating factor 1 receptor antagonist PLX3397. Microglial depletion extended lifespan and delayed motor symptoms in Ndufs4 KO mice, likely by preventing neuronal loss. Next, we investigated the role of the major cytokine interleukin-6 (IL-6) in the disease progression. IL-6 deficiency partially rescued breathing abnormalities and modulated gliosis but did not extend the lifespan or rescue motor decline in Ndufs4 KO mice. The present results show that microglial accumulation is pathogenic, in a process independent of IL-6, and hints toward a contributing role of neuroinflammation in the disease of Ndufs4 KO mice and potentially in patients with Leigh syndrome.

A homozygous splice variant in ATP5PO, disrupts mitochondrial complex V function and causes Leigh syndrome in two unrelated families.

Mitochondrial complex V plays an important role in oxidative phosphorylation by catalyzing the generation of ATP. Most complex V subunits are nuclear encoded and not yet associated with recognized Mendelian disorders. Using exome sequencing, we identified a rare homozygous splice variant (c.87+3A>G) in ATP5PO, the complex V subunit which encodes the oligomycin sensitivity conferring protein, in three individuals from two unrelated families, with clinical suspicion of a mitochondrial disorder. These individuals had a similar, severe infantile and often lethal multi-systemic disorder that included hypotonia, developmental delay, hypertrophic cardiomyopathy, progressive epileptic encephalopathy, progressive cerebral atrophy, and white matter abnormalities on brain MRI consistent with Leigh syndrome. cDNA studies showed a predominant shortened transcript with skipping of exon 2 and low levels of the normal full-length transcript. Fibroblasts from the affected individuals demonstrated decreased ATP5PO protein, defective assembly of complex V with markedly reduced amounts of peripheral stalk proteins, and complex V hydrolytic activity. Further, expression of human ATP5PO cDNA without exon 2 (hATP5PO-∆ex2) in yeast cells deleted for yATP5 (ATP5PO homolog) was unable to rescue growth on media which requires oxidative phosphorylation when compared to the wild type construct (hATP5PO-WT), indicating that exon 2 deletion leads to a non-functional protein. Collectively, our findings support the pathogenicity of the ATP5PO c.87+3A>G variant, which significantly reduces but does not eliminate complex V activity. These data along with the recent report of an affected individual with ATP5PO variants, add to the evidence that rare biallelic variants in ATP5PO result in defective complex V assembly, function and are associated with Leigh syndrome.

Cross-comparison of systemic and tissue-specific metabolomes in a mouse model of Leigh syndrome.

INTRODUCTION: The value of metabolomics in multi-systemic mitochondrial disease research has been increasingly recognized, with the ability to investigate a variety of biofluids and tissues considered a particular advantage. Although minimally invasive biofluids are the generally favored sample type, it remains unknown whether systemic metabolomes provide a clear reflection of tissue-specific metabolic alterations. OBJECTIVES: Here we cross-compare urine and tissue-specific metabolomes in the Ndufs4 knockout mouse model of Leigh syndrome-a complex neurometabolic MD defined by progressive focal lesions in specific brain regions-to identify and evaluate the extent of common and unique metabolic alterations on a systemic and brain regional level. METHODS: Untargeted and semi-targeted multi-platform metabolomics were performed on urine, four brain regions, and two muscle types of Ndufs4 KO (n≥19) vs wildtype (n≥20) mice. RESULTS: Widespread alterations were evident in alanine, aspartate, glutamate, and arginine metabolism in Ndufs4 KO mice; while brain-region specific metabolic signatures include the accumulation of branched-chain amino acids, proline, and glycolytic intermediates. 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. CONCLUSION: Our results confirm the value of urinary metabolomics when evaluating MD-associated metabolites, while cautioning against mechanistic studies relying solely on systemic biofluids.

Evaluating the Bioenergetics Health Index Ratio in Leigh Syndrome Fibroblasts to Understand Disease Severity.

Several pediatric mitochondrial disorders, including Leigh syndrome (LS), impact mitochondrial (mt) genetics, development, and metabolism, leading to complex pathologies and energy failure. The extent to which pathogenic mtDNA variants regulate disease severity in LS is currently not well understood. To better understand this relationship, we computed a glycolytic bioenergetics health index (BHI) for measuring mitochondrial dysfunction in LS patient fibroblast cells harboring varying percentages of pathogenic mutant mtDNA (T8993G, T9185C) exhibiting deficiency in complex V or complex I (T10158C, T12706C). A high percentage (>90%) of pathogenic mtDNA in cells affecting complex V and a low percentage (<39%) of pathogenic mtDNA in cells affecting complex I was quantified. Levels of defective enzyme activities of the electron transport chain correlated with the percentage of pathogenic mtDNA. Subsequent bioenergetics assays showed cell lines relied on both OXPHOS and glycolysis for meeting energy requirements. Results suggest that whereas the precise mechanism of LS has not been elucidated, a multi-pronged approach taking into consideration the specific pathogenic mtDNA variant, glycolytic BHI, and the composite BHI (average ratio of oxphos to glycolysis) can aid in better understanding the factors influencing disease severity in LS.

Cell-Permeable Succinate Increases Mitochondrial Membrane Potential and Glycolysis in Leigh Syndrome Patient Fibroblasts.

Mitochondrial disorders represent a large group of severe genetic disorders mainly impacting organ systems with high energy requirements. Leigh syndrome (LS) is a classic example of a mitochondrial disorder resulting from pathogenic mutations that disrupt oxidative phosphorylation capacities. Currently, evidence-based therapy directed towards treating LS is sparse. Recently, the cell-permeant substrates responsible for regulating the electron transport chain have gained attention as therapeutic agents for mitochondrial diseases. We explored the therapeutic effects of introducing tricarboxylic acid cycle (TCA) intermediate substrate, succinate, as a cell-permeable prodrug NV118, to alleviate some of the mitochondrial dysfunction in LS. The results suggest that a 24-hour treatment with prodrug NV118 elicited an upregulation of glycolysis and mitochondrial membrane potential while inhibiting intracellular reactive oxygen species in LS cells. The results from this study suggest an important role for TCA intermediates for treating mitochondrial dysfunction in LS. We show, here, that NV118 could serve as a therapeutic agent for LS resulting from mutations in mtDNA in complex I and complex V dysfunctions.

A high mutation load of m.14597A>G in MT-ND6 causes Leigh syndrome.

Leigh syndrome (LS) is an early-onset progressive neurodegenerative disorder associated with mitochondrial deficiency. m.14597A>G (p.Ile26Thr) in the MT-ND6 gene was reported to cause Leber's hereditary optic neuropathy (LHON) or dementia/dysarthria. In previous reports, less than 90% heteroplasmy was shown to result in adult-onset disease. Here, by whole mitochondrial sequencing, we identified m.14597A>G mutation of a patient with LS. PCR-RFLP analysis on fibroblasts from the patient revealed a high mutation load (> 90% heteroplasmy). We performed functional assays using cybrid cell models generated by fusing mtDNA-less rho0 HeLa cells with enucleated cells from patient fibroblasts carrying the m.14597A>G variant. Cybrid cell lines bearing the m.14597A>G variant exhibited severe effects on mitochondrial complex I activity. Additionally, impairment of cell proliferation, decreased ATP production and reduced oxygen consumption rate were observed in the cybrid cell lines bearing the m.14597A>G variant when the cells were metabolically stressed in medium containing galactose, indicating mitochondrial respiratory chain defects. These results suggest that a high mutation load of m.14597A>G leads to LS via a mitochondrial complex I defect, rather than LHON or dementia/dysarthria.

Defective metabolic programming impairs early neuronal morphogenesis in neural cultures and an organoid model of Leigh syndrome.

Leigh syndrome (LS) is a severe manifestation of mitochondrial disease in children and is currently incurable. The lack of effective models hampers our understanding of the mechanisms underlying the neuronal pathology of LS. Using patient-derived induced pluripotent stem cells and CRISPR/Cas9 engineering, we developed a human model of LS caused by mutations in the complex IV assembly gene SURF1. Single-cell RNA-sequencing and multi-omics analysis revealed compromised neuronal morphogenesis in mutant neural cultures and brain organoids. The defects emerged at the level of neural progenitor cells (NPCs), which retained a glycolytic proliferative state that failed to instruct neuronal morphogenesis. LS NPCs carrying mutations in the complex I gene NDUFS4 recapitulated morphogenesis defects. SURF1 gene augmentation and PGC1A induction via bezafibrate treatment supported the metabolic programming of LS NPCs, leading to restored neuronal morphogenesis. Our findings provide mechanistic insights and suggest potential interventional strategies for a rare mitochondrial disease.

Hypoxia ameliorates brain hyperoxia and NAD+ deficiency in a murine model of Leigh syndrome.

Leigh syndrome is a severe mitochondrial neurodegenerative disease with no effective treatment. In the Ndufs4-/- mouse model of Leigh syndrome, continuously breathing 11% O2 (hypoxia) prevents neurodegeneration and leads to a dramatic extension (~5-fold) in lifespan. We investigated the effect of hypoxia on the brain metabolism of Ndufs4-/- mice by studying blood gas tensions and metabolite levels in simultaneously sampled arterial and cerebral internal jugular venous (IJV) blood. Relatively healthy Ndufs4-/- and wildtype (WT) mice breathing air until postnatal age ~38 d were compared to Ndufs4-/- and WT mice breathing air until ~38 days old followed by 4-weeks of breathing 11% O2. Compared to WT control mice, Ndufs4-/- mice breathing air have reduced brain O2 consumption as evidenced by an elevated partial pressure of O2 in IJV blood (PijvO2) despite a normal PO2 in arterial blood, and higher lactate/pyruvate (L/P) ratios in IJV plasma revealed by metabolic profiling. In Ndufs4-/- mice, hypoxia treatment normalized the cerebral venous PijvO2 and L/P ratios, and decreased levels of nicotinate in IJV plasma. Brain concentrations of nicotinamide adenine dinucleotide (NAD+) were lower in Ndufs4-/- mice breathing air than in WT mice, but preserved at WT levels with hypoxia treatment. Although mild hypoxia (17% O2) has been shown to be an ineffective therapy for Ndufs4-/- mice, we find that when combined with nicotinic acid supplementation it provides a modest improvement in neurodegeneration and lifespan. Therapies targeting both brain hyperoxia and NAD+ deficiency may hold promise for treating Leigh syndrome.

TRMU deficiency: A broad clinical spectrum responsive to cysteine supplementation.

TRMU is a nuclear gene crucial for mitochondrial DNA translation by encoding tRNA 5-methylaminomethyl-2-thiouridylate methyltransferase, which thiolates mitochondrial tRNA. Biallelic pathogenic variants in TRMU are associated with transient infantile liver failure. Other less common presentations such as Leigh syndrome, myopathy, and cardiomyopathy have been reported. Recent studies suggested that provision of exogenous L-cysteine or N-acetylcysteine may ameliorate the effects of disease-causing variants and improve the natural history of the disease. Here, we report six infants with biallelic TRMU variants, including four previously unpublished patients, all treated with exogenous cysteine. We highlight the first report of an affected patient undergoing orthotopic liver transplantation, the long-term effects of cysteine supplementation, and the ability of the initial presentation to mimic multiple inborn errors of metabolism. We propose that TRMU deficiency should be suspected in all children presenting with persistent lactic acidosis and hypoglycemia, and that combined N-acetylcysteine and L-cysteine supplementation should be considered prior to molecular diagnosis, as this is a low-risk approach that may increase survival and mitigate the severity of the disease course.

Aberrant BCAA and glutamate metabolism linked to regional neurodegeneration in a mouse model of Leigh syndrome.

The dysfunction of respiratory chain complex I (CI) is the most common form of mitochondrial disease that most often presents as Leigh syndrome (LS) in children - a severe neurometabolic disorder defined by progressive focal lesions in specific brain regions. The mechanisms underlying this region-specific vulnerability to CI deficiency, however, remain elusive. Here, we examined brain regional respiratory chain enzyme activities and metabolic profiles in a mouse model of LS with global CI deficiency to gain insight into regional vulnerability to neurodegeneration. One lesion-resistant and three lesion-prone brain regions were investigated in Ndufs4 knockout (KO) mice at the late stage of LS. Enzyme assays confirmed significantly decreased (60-80%) CI activity in all investigated KO brain regions, with the lesion-resistant region displaying the highest residual CI activity (38% of wild type). A higher residual CI activity, and a less perturbed NADH/NAD+ ratio, correlate with less severe metabolic perturbations in KO brain regions. Moreover, less perturbed BCAA oxidation and increased glutamate oxidation seem to distinguish lesion-resistant from -prone KO brain regions, thereby identifying key areas of metabolism to target in future therapeutic intervention studies.

Delineating the neurological phenotype in children with defects in the ECHS1 or HIBCH gene.

The neurological phenotype of 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) and short-chain enoyl-CoA hydratase (SCEH) defects is expanding and natural history studies are necessary to improve clinical management. From 42 patients with Leigh syndrome studied by massive parallel sequencing, we identified five patients with SCEH and HIBCH deficiency. Fourteen additional patients were recruited through collaborations with other centres. In total, we analysed the neurological features and mutation spectrum in 19 new SCEH/HIBCH patients. For natural history studies and phenotype to genotype associations we also included 70 previously reported patients. The 19 newly identified cases presented with Leigh syndrome (SCEH, n = 11; HIBCH, n = 6) and paroxysmal dystonia (SCEH, n = 2). Basal ganglia lesions (18 patients) were associated with small cysts in the putamen/pallidum in half of the cases, a characteristic hallmark for diagnosis. Eighteen pathogenic variants were identified, 11 were novel. Among all 89 cases, we observed a longer survival in HIBCH compared to SCEH patients, and in HIBCH patients carrying homozygous mutations on the protein surface compared to those with variants inside/near the catalytic region. The SCEH p.(Ala173Val) change was associated with a milder form of paroxysmal dystonia triggered by increased energy demands. In a child harbouring SCEH p.(Ala173Val) and the novel p.(Leu123Phe) change, an 83.6% reduction of the protein was observed in fibroblasts. The SCEH and HIBCH defects in the catabolic valine pathway were a frequent cause of Leigh syndrome in our cohort. We identified phenotype and genotype associations that may help predict outcome and improve clinical management.