Pathogenesis of mtDNA point mutation m.10191T>C affecting complex I function is a multifactorial process leading to metabolic remodeling of mitochondria.

Human mitochondrial complex I (NADH:ubiquinone oxidoreductase) of the oxidative phosphorylation system is a multiprotein assembly comprising both nuclear and mitochondrially encoded subunits. Deficiency of this complex is associated with numerous clinical syndromes ranging from highly progressive, often early lethal encephalopathies, of which Leigh disease is the most frequent, to neurodegenerative disorders in adult life, including Leber's hereditary optic neuropathy and Parkinson disease. We show here that the cytosolic Ca2+ signal in response to hormonal stimulation with bradykinin was impaired in skin fibroblasts from children between the ages of 0 and 5 years with an isolated complex I deficiency caused by mutations in nuclear encoded structural subunits of the complex. Inhibition of mitochondrial Na+-Ca2+ exchange by the benzothiazepine CGP37157 completely restored the aberrant cytosolic Ca2+ signal. This effect of the inhibitor was paralleled by complete restoration of the bradykinin-induced increases in mitochondrial Ca2+ concentration and ensuing ATP production. Thus, impaired mitochondrial Ca2+ accumulation during agonist stimulation is a major consequence of human complex I deficiency, a finding that may provide the basis for the development of new therapeutic approaches to this disorder.

Deleterious Mutation in the Mitochondrial Arginyl–Transfer RNA Synthetase Gene Is Associated with Pontocerebellar Hypoplasia

Leigh syndrome (LS) has emerged as the quintessential neuropathological signature of a mitochondrial encephalopathy. Did D. Leigh ever imagine this possibility in 1951 when he described the neuropathology of a young infant who died after 6 weeks of devastating neurological symptoms [9]? It is unlikely. But to his credit he recognized the histopathological overlap between his case and Wernicke encephalopathy; and he speculated about the role of an altered thiamine-dependent process as the basis for the similarity. Not far off the mark as we would learn over the next several decades. Leigh’s case showed sparing of the mamillary bodies and the putamen. The former observation has remained true but the latter appears now to be an exception. The difficulty in defining LS as a clinical entity followed subsequent observations by noted neuropathologists who reported the histopathology of LS in all age groups with a diversity of clinical prodromes and phenotypes. This debate was perhaps best epitomized by Crosby and Chou [2] when they described ragged-red fibers (RRF) in LS. At the time, this report engendered a vigorous debate as to whether the patient had LS or Kearns–Sayre syndrome (KSS). The current view of this historic controversy is the fact that both LS and KSS are mitochondrial diseases. RRF are rarely seen in LS, and are the rule in KSS. Montpetit et al. [11] described the clinical findings in a series of LS cases and concluded that the condition was mainly a subcortical brainstem syndrome. For the reasons stated earlier, the disease phenotype was not clearly defined. Insights into the biochemical basis for LS emerged in the 1960s. Worsley et al. [13] described two siblings with lactic acidosis. At autopsy, LS was confirmed. This report also represented the first description of familial lactic acidosis. In 1968, Hommes et al. described pyruvate carboxylase (PC) deficiency associated with LS. This association later was disputed by several authors. On the other hand, LS has been described with biotinidase deficiency, a form of multiple carboxylase deficiency. Willems et al. [12] described a young child with cytochrome c oxidase (COX) deficiency and LS, viewed by many at that time as an implausible correlate. In 1979, De Vivo et al. described an infant with LS and defective activation of the pyruvate dehydrogenase complex [3]. It was presumed that the basic defect involved the pyruvate dehydrogenase-specific phosphatase. Two other siblings, a male and female, also had LS suggesting autosomal recessive inheritance. The importance of COX deficiency in LS was emphasized by DiMauro et al. in 1987 when five patients were described with generalized partial COX deficiency and LS [4]. Numerous reports have since appeared linking COX deficiency and LS (Table 1). In 1990, Fujii et al. [7] described two children with complex I deficiency and LS, and Morris et al. [10] emphasized the deficiency of complex I as a common correlate to LS. In 1990, Holt et al. described a new mitochondrial disease associated with heteroplasmy and maternal inheritance [8]. This condition later was referred to as the NARP syndrome (neuropathy, ataxia and

Deficiencies in the activity of cytochrome c oxidase (COX) are an important cause of autosomal recessive respiratory chain disorders. Patients with isolated COX deficiency are clinically and genetically heterogeneous, and mutations in several different assembly factors have been found to cause specific clinical phenotypes. Two of the most common clinical presentations, Leigh Syndrome and hypertrophic cardiomyopathy, have so far only been associated with mutations in SURF1 or SCO2 and COX15, respectively. Here we show that expression of COX10 from a retroviral vector complements the COX deficiency in a patient with anemia and Leigh Syndrome, and in a patient with anemia, sensorineural deafness and fatal infantile hypertrophic cardiomyopathy. A partial rescue was also obtained following microcell-mediated transfer of mouse chromosomes into patient fibroblasts. COX10 functions in the first step of the mitochondrial heme A biosynthetic pathway, catalyzing the conversion of protoheme (heme B) to heme O via the farnesylation of a vinyl group at position C2. Heme A content was reduced in mitochondria from patient muscle and fibroblasts in proportion to the reduction in COX enzyme activity and the amount of fully assembled enzyme. Mutation analysis of COX10 identified four different missense alleles, predicting amino acid substitutions at evolutionarily conserved residues. A topological model places these residues in regions of the protein shown to have important catalytic functions by mutation analysis of a prokaryotic ortholog. Mutations in COX10 have previously been reported in a single family with tubulopathy and leukodystrophy. This study shows that mutations in this gene can cause nearly the full range of clinical phenotypes associated with early onset isolated COX deficiency.

Obesity is associated with insulin resistance (IR) development, a risk factor for type 2 diabetes (T2D). How mitochondrial bioenergetics, in adipose tissue (AT), differs according to distinct metabolic profiles (i.e. insulin sensitive (IS), IR normoglycaemic (IR-NG), pre-diabetes (PD) and T2D) is still poorly understood. The purpose of this study was to evaluate and compare bioenergetics and energy substrate preference by omental AT (OAT) and subcutaneous AT (SAT) from subjects with obesity (OB, n = 40) at distinct metabolic stages. Furthermore, AT bioenergetics was also evaluated pre- and post-bariatric/metabolic surgery (BMS). High-resolution respirometry (HRR) was used to measure the real-time oxidative phosphorylation (OXPHOS) capacity and mitochondrial substrate preferences in both tissues. Substrate-uncoupler-inhibitor titration protocols were used: SUIT-P1 (complex I and II-linked mitochondrial respiration) and SUIT-P2 (fatty acid oxidation (FAO)-linked mitochondrial respiration). Flux control ratios (FCRs) were calculated. In SUIT-P1, lower OXPHOS capacity was observed in AT, particularly in SAT, during the establishment of IR (OB-IR-NG) and in the T2D group, due to alterations of mitochondrial coupling, evaluated by FCRs. In SUIT-P2, the OXPHOS coupling efficiency was highest in the OB-IR-NG group. AT from OB-IS, OB-IR-NG and OB-IR-PD preferred pyruvate, malate and glutamate oxidation and/or FAO during OXPHOS, whereas AT from T2D preferred succinate oxidation. BMS enhanced mitochondrial respiration in OAT, even under poor OXPHOS coupling efficiency. In conclusion, real-time OXPHOS analysis by HRR may be a sensitive biomarker of mitochondrial fitness, particularly in AT. Interventions based on modulating energetic substrate availability may become a good tool for obesity treatment stratification. KEY POINTS: Omental adipose tissue shows higher oxidative phosphorylation (OXPHOS) capacity compared to subcutaneous adipose tissue in paired explants from subjects with obesity. The OXPHOS capacity of adipose tissue differs through the progression of metabolic disease. Subjects with obesity and diabetes have the lowest OXPHOS capacity in paired explants of subcutaneous and omental adipose tissues. Bariatric surgery enhanced the OXPHOS capacity in omental adipose tissue, even under poor OXPHOS coupling efficiency. Assessment of the oxidative capacity in fresh adipose tissue explants could be a sensitive tool for early diagnosis of metabolic disease.

Decreased mitochondrial oxidative phosphorylation capacity in the human heart with left ventricular systolic dysfunction

To investigate whether ovariectomy affects mitochondrial respiratory function, gene expression of the biogenesis markers and mitochondrial dynamics of the vastus lateralis muscle, female Wistar rats divided into ovariectomized (OVX) and intact (INT) groups were kept sedentary (SED) or submitted to resistance training (RT) performed for thirteen weeks on a vertical ladder in which animals climbed with a workload apparatus. RT sessions were performed with four climbs with 65, 85, 95, and 100 % of the rat's previous maximum workload. Mitochondrial Respiratory Function data were obtained by High-resolution respirometry. Gene expression of FIS1, MFN1 and PGC1-α was evaluated by real-time PCR. There was a decrease on oxidative phosphorylation capacity in OVX-SED compared to other groups. Trained groups presented increase on oxidative phosphorylation capacity when compared to sedentary groups. For respiratory control ratio (RCR), OVX-SED presented lower values when compared to INT-SED and to trained groups. Trained groups presented RCR values higher compared to INT-SED. Exercise increased the values of FIS1, MFN1 and PGC1-α expression compared to OVX-SED. Our results demonstrated that in the absence of ovarian hormones, there is a great decrease in oxidative phosphorylation and electron transfer system capacities of sedentary animals. RT was able to increase the expression of genes related to mitochondrial dynamics markers, reversing the condition determined by ovariectomy.

Patients suffering from chronic fatigue syndrome (CFS) or post-COVID syndrome (PCS) exhibit a reduced physiological performance capability. Impaired mitochondrial function and morphology may play a pivotal role. Thus, we aimed to measure the muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity and assess mitochondrial morphology in CFS and PCS patients in comparison to healthy controls (HCs). Mitochondrial OXPHOS capacity was measured in permeabilized muscle fibers using high-resolution respirometry. Mitochondrial morphology (subsarcolemmal/intermyofibrillar mitochondrial form/cristae/diameter/circumference/area) and content (number and proportion/cell) were assessed via electron microscopy. Analyses included differences in OXPHOS between HC, CFS, and PCS, whereas comparisons in morphology/content were made for CFS vs. PCS. OXPHOS capacity of complex I, which was reduced in PCS compared to HC. While the subsarcolemmal area, volume/cell, diameter, and perimeter were higher in PCS vs. CFS, no difference was observed for these variables in intermyofibrillar mitochondria. Both the intermyofibrillar and subsarcolemmal cristae integrity was higher in PCS compared to CFS. Both CFS and PCS exhibit increased fatigue and impaired mitochondrial function, but the progressed pathological morphological changes in CFS suggest structural changes due to prolonged inactivity or unknown molecular causes. Instead, the significantly lower complex I activity in PCS suggests probably direct virus-induced alterations.

The genetic causes of Leigh syndrome are heterogeneous, with a poor genotype-phenotype correlation. To date, more than 50 nuclear genes cause nuclear gene-encoded Leigh syndrome. NDUFS6 encodes a 13 kiloDaltons subunit, which is part of the peripheral arm of complex I and is localized in the iron-sulfur fraction. Only a few patients were reported with proven NDUFS6 pathogenic variants and all presented with severe neonatal lactic acidemia and complex I deficiency, leading to death in the first days of life. Here, we present a patient harboring two NDUFS6 variants with a phenotype compatible with Leigh syndrome. Although most of previous reports suggested that NDUFS6 pathogenic variants invariably lead to early neonatal death, this report shows that the clinical spectrum could be larger. We found a severe decrease of NDUFS6 protein level in patient's fibroblasts associated with a complex I assembly defect in patient's muscle and fibroblasts. These data confirm the importance of NDUFS6 and the Zn-finger domain for a correct assembly of complex I.

Leigh syndrome, a severe neurological disorder is commonly caused by homozygous or bi-allelic pathogenic variants in the SURF1 gene. SURF1 deficiency leads to dysfunction of Cytochrome C Oxidase (COX) activity, which is crucial for mitochondrial oxidative phosphorylation. Understanding COX activity's correlation with disease severity is essential for developing SURF1 Leigh Syndrome biomarkers. This study assesses the disease burden in SURF1 Leigh Syndrome and evaluates COX activity as a treatment biomarker. We reviewed records and questionnaires from 17 individuals, classifying them into phenotypic and genotypic groups. We compared COX activity assays in patient fibroblasts to age-matched controls, clinical data, and neuroimaging findings. Patient COX activity was at most 50% of controls, averaging 32% (p < 0.001). Common clinical features included brainstem abnormalities (93.3%), motor regression (92.3%), bi-allelic heterozygous SURF1 variants (88.2%), and delayed growth/development (35.7%). Homozygous and heterozygous nonsense/frameshift variants showed more severe phenotypes (p = 0.008) and more MRI abnormalities (p = 0.005). Significant COX activity reduction is linked to SURF1 Leigh Syndrome, with genotype influencing disease severity. Clinical and neuroimaging correlations show potential for prognostic indicators. This study lays the groundwork for future research and clinical application of COX activity as a SURF1 Leigh Syndrome biomarker.

Metabolic flexibility and carnitine flux: The role of carnitine acyltransferase in glucose homeostasis

Leigh Syndrome with Nephropathy and CoQ10 Deficiency Due to decaprenyl diphosphate synthase subunit 2 (PDSS2) Mutations

Chapter 14 - Nuclear Genetic Causes of Leigh and Leigh-Like Syndrome

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.

We recently reported an autosomal dominant form of renal Fanconi syndrome caused by a missense mutation in the third codon of the peroxisomal protein EHHADH. The mutation mistargets EHHADH to mitochondria, thereby impairing mitochondrial energy production and, consequently, reabsorption of electrolytes and low-molecular-weight nutrients in the proximal tubule. Here, we further elucidate the molecular mechanism underlying this pathology. We find that mutated EHHADH is incorporated into mitochondrial trifunctional protein (MTP), thereby disturbing β-oxidation of long-chain fatty acids. The resulting MTP deficiency leads to a characteristic accumulation of hydroxyacyl- and acylcarnitines. Mutated EHHADH also limits respiratory complex I and corresponding supercomplex formation, leading to decreases in oxidative phosphorylation capacity,mitochondrial membrane potential maintenance, and ATP generation. Activity of the Na(+)/K(+)-ATPase is thereby diminished, ultimately decreasing the transport activity of the proximal tubule cells.