Organization of the Respiratory Supercomplexes in Cells with Defective Complex III: Structural Features and Metabolic Consequences.

The mitochondrial respiratory chain encompasses four oligomeric enzymatic complexes (complex I, II, III and IV) which, together with the redox carrier ubiquinone and cytochrome c, catalyze electron transport coupled to proton extrusion from the inner membrane. The protonmotive force is utilized by complex V for ATP synthesis in the process of oxidative phosphorylation. Respiratory complexes are known to coexist in the membrane as single functional entities and as supramolecular aggregates or supercomplexes (SCs). Understanding the assembly features of SCs has relevant biomedical implications because defects in a single protein can derange the overall SC organization and compromise the energetic function, causing severe mitochondrial disorders. Here we describe in detail the main types of SCs, all characterized by the presence of complex III. We show that the genetic alterations that hinder the assembly of Complex III, not just the activity, cause a rearrangement of the architecture of the SC that can help to preserve a minimal energetic function. Finally, the major metabolic disturbances associated with severe SCs perturbation due to defective complex III are discussed along with interventions that may circumvent these deficiencies.

Coenzyme Q biosynthesis inhibition induces HIF-1α stabilization and metabolic switch toward glycolysis.

Coenzyme Q10 (CoQ, ubiquinone) is a redox-active lipid endogenously synthesized by the cells. The final stage of CoQ biosynthesis is performed at the mitochondrial level by the 'complex Q', where coq2 is responsible for the prenylation of the benzoquinone ring of the molecule. We report that the competitive coq2 inhibitor 4-nitrobenzoate (4-NB) decreased the cellular CoQ content and caused severe impairment of mitochondrial function in the T67 human glioma cell line. In parallel with the reduction in CoQ biosynthesis, the cholesterol level increased, leading to significant perturbation of the plasma membrane physicochemical properties. We show that 4-NB treatment did not significantly affect the cell viability, because of an adaptive metabolic rewiring toward glycolysis. Hypoxia-inducible factor 1α (HIF-1α) stabilization was detected in 4-NB-treated cells, possibly due to the contribution of both reduction in intracellular oxygen tension and ROS overproduction. Exogenous CoQ supplementation partially recovered cholesterol content, HIF-1α degradation, and ROS production, whereas only weakly improved the bioenergetic impairment induced by the CoQ depletion. Our data provide new insights on the effect of CoQ depletion and contribute to shed light on the pathogenic mechanisms of ubiquinone deficiency syndrome.

Drug repositioning as a therapeutic strategy for neurodegenerations associated with OPA1 mutations.

OPA1 mutations are the major cause of dominant optic atrophy (DOA) and the syndromic form DOA plus, pathologies for which there is no established cure. We used a 'drug repurposing' approach to identify FDA-approved molecules able to rescue the mitochondrial dysfunctions induced by OPA1 mutations. We screened two different chemical libraries by using two yeast strains carrying the mgm1I322M and the chim3P646L mutations, identifying 26 drugs able to rescue their oxidative growth phenotype. Six of them, able to reduce the mitochondrial DNA instability in yeast, have been then tested in Opa1 deleted mouse embryonic fibroblasts expressing the human OPA1 isoform 1 bearing the R445H and D603H mutations. Some of these molecules were able to ameliorate the energetic functions and/or the mitochondrial network morphology, depending on the type of OPA1 mutation. The final validation has been performed in patients' fibroblasts, allowing to select the most effective molecules. Our current results are instrumental to rapidly translating the findings of this drug repurposing approach into clinical trial for DOA and other neurodegenerations caused by OPA1 mutations.

Metabolomics hallmarks OPA1 variants correlating with their in vitro phenotype and predicting clinical severity.

Interpretation of variants of uncertain significance is an actual major challenge. We addressed this question on a set of OPA1 missense variants responsible for variable severity of neurological impairments. We used targeted metabolomics to explore the different signatures of OPA1 variants expressed in Opa1 deleted mouse embryonic fibroblasts (Opa1-/- MEFs), grown under selective conditions. Multivariate analyses of data discriminated Opa1+/+ from Opa1-/- MEFs metabolic signatures and classified OPA1 variants according to their in vitro severity. Indeed, the mild p.I382M hypomorphic variant was segregating close to the wild-type allele, while the most severe p.R445H variant was close to Opa1-/- MEFs, and the p.D603H and p.G439V alleles, responsible for isolated and syndromic presentations, respectively, were intermediary between the p.I382M and the p.R445H variants. The most discriminant metabolic features were hydroxyproline, the spermine/spermidine ratio, amino acid pool and several phospholipids, emphasizing proteostasis, endoplasmic reticulum (ER) stress and phospholipid remodeling as the main mechanisms ranking OPA1 allele impacts on metabolism. These results demonstrate the high resolving power of metabolomics in hierarchizing OPA1 missense mutations by their in vitro severity, fitting clinical expressivity. This suggests that our methodological approach can be used to discriminate the pathological significance of variants in genes responsible for other rare metabolic diseases and may be instrumental to select possible compounds eligible for supplementation treatment.

Fine-tuning of the respiratory complexes stability and supercomplexes assembly in cells defective of complex III.

The respiratory complexes are organized in supramolecular assemblies called supercomplexes thought to optimize cellular metabolism under physiological and pathological conditions. In this study, we used genetically and biochemically well characterized cells bearing the pathogenic microdeletion m.15,649-15,666 (ΔI300-P305) in MT-CYB gene, to investigate the effects of an assembly-hampered CIII on the re-organization of supercomplexes. First, we found that this mutation also affects the stability of both CI and CIV, and evidences the occurrence of a preferential structural interaction between CI and CIII2, yielding a small amount of active CI+CIII2 supercomplex. Indeed, a residual CI+CIII combined redox activity, and a low but detectable ATP synthesis driven by CI substrates are detectable, suggesting that the assembly of CIII into the CI+CIII2 supercomplex mitigates the detrimental effects of MT-CYB deletion. Second, measurements of oxygen consumption and ATP synthesis driven by NADH-linked and FADH2-linked substrates alone, or in combination, indicate a common ubiquinone pool for the two respiratory pathways. Finally, we report that prolonged incubation with rotenone enhances the amount of CI and CIII2, but reduces CIV assembly. Conversely, the antioxidant N-acetylcysteine increases CIII2 and CIV2 and partially restores respirasome formation. Accordingly, after NAC treatment, the rate of ATP synthesis increases by two-fold compared with untreated cell, while the succinate level, which is enhanced by the homoplasmic mutation, markedly decreases. Overall, our findings show that fine-tuning the supercomplexes stability improves the energetic efficiency of cells with the MT-CYB microdeletion.

Deciphering OPA1 mutations pathogenicity by combined analysis of human, mouse and yeast cell models.

OPA1 is the major gene responsible for Dominant Optic Atrophy (DOA) and the syndromic form DOA "plus". Over 370 OPA1 mutations have been identified so far, although their pathogenicity is not always clear. We have analyzed one novel and a set of known OPA1 mutations to investigate their impact on protein functions in primary skin fibroblasts and in two "ad hoc" generated cell systems: the MGM1/OPA1 chimera yeast model and the Opa1-/- MEFs model expressing the mutated human OPA1 isoform 1. The yeast model allowed us to confirm the deleterious effects of these mutations and to gain information on their dominance/recessivity. The MEFs model enhanced the phenotypic alteration caused by mutations, nicely correlating with the clinical severity observed in patients, and suggested that the DOA "plus" phenotype could be induced by the combinatorial effect of mitochondrial network fragmentation with variable degrees of mtDNA depletion. Overall, the two models proved to be valuable tools to functionally assess and define the deleterious mechanism and the pathogenicity of novel OPA1 mutations, and useful to testing new therapeutic interventions.

Clonal expansion of mtDNA deletions: different disease models assessed by digital droplet PCR in single muscle cells.

Deletions in mitochondrial DNA (mtDNA) are an important cause of human disease and their accumulation has been implicated in the ageing process. As mtDNA is a high copy number genome, the coexistence of deleted and wild-type mtDNA molecules within a single cell defines heteroplasmy. When deleted mtDNA molecules, driven by intracellular clonal expansion, reach a sufficiently high level, a biochemical defect emerges, contributing to the appearance and progression of clinical pathology. Consequently, it is relevant to determine the heteroplasmy levels within individual cells to understand the mechanism of clonal expansion. Heteroplasmy is reflected in a mosaic distribution of cytochrome c oxidase (COX)-deficient muscle fibers. We applied droplet digital PCR (ddPCR) to single muscle fibers collected by laser-capture microdissection (LCM) from muscle biopsies of patients with different paradigms of mitochondrial disease, characterized by the accumulation of single or multiple mtDNA deletions. By combining these two sensitive approaches, ddPCR and LCM, we document different models of clonal expansion in patients with single and multiple mtDNA deletions, implicating different mechanisms and time points for the development of COX deficiency in these molecularly distinct mitochondrial cytopathies.

OPA1: How much do we know to approach therapy?

OPA1 is a GTPase that controls several functions, such as mitochondrial dynamics and energetics, mtDNA maintenance and cristae integrity. In the last years, there have been described other cellular pathways and mechanisms involving OPA1 directly or through its interaction. All this new information, by implementing our knowledge on OPA1 is instrumental to elucidating the pathogenic mechanisms of OPA1 mutations. Indeed, these are associated with dominant optic atrophy (DOA), one of the most common inherited optic neuropathies, and with an increasing number of heterogeneous neurodegenerative disorders. In this review, we overview all recent findings on OPA1 protein functions, on its dysfunction and related clinical phenotypes, focusing on the current therapeutic options and future perspectives to treat DOA and the other associated neurological disorders due to OPA1 mutations.

Eight human OPA1 isoforms, long and short: What are they for?

OPA1 is a dynamin-related GTPase that controls mitochondrial dynamics, cristae integrity, energetics and mtDNA maintenance. The exceptional complexity of this protein is determined by the presence, in humans, of eight different isoforms that, in turn, are proteolytically cleaved into combinations of membrane-anchored long forms and soluble short forms. Recent advances highlight how each OPA1 isoform is able to fulfill "essential" mitochondrial functions, whereas only some variants carry out "specialized" features. Long forms determine fusion, long or short forms alone build cristae, whereas long and short forms together tune mitochondrial morphology. These findings offer novel challenging therapeutic potential to gene therapy.

Cellular and mitochondrial determination of low molecular mass organic acids by LC-MS/MS.

A selective and sensitive method for the determination of low molecular mass organic acids (LMMOAs) in cell and mitochondrial extracts is presented. The analytical method consists in the separation by reversed phase liquid chromatography and detection with tandem mass spectrometry (LC-MS/MS) of the LMMOAs like malic, succinic, formic and citric acids. These acids are among the cellular intermediates of the tricarboxylic acid cycle (TCA), thus their quantitation can provide essential information about the catabolic and anabolic processes occurring in cells under physiological and pathological conditions. The analytical method was fully validated in terms of linearity, detection and quantification limits, recovery and precision. Detection limits (LOD) for malic, succinic and fumaric acids were in the range of 1-10nM, while 20nM was obtained for citric acid. Analytical recovery in cell and mitochondrial extracts was found between 88 and 105% (CV% ≤7.1) and matrix effect was estimated to be less than 108%. The LC-MS/MS method applied to the quantification of TCA cycle metabolites revealed a different distribution of the four acids in cells and mitochondria, and it could be used to monitoring metabolic alterations associated with TCA cycle and oxidative phosphorylation dysfunctions.

Complex II phosphorylation is triggered by unbalanced redox homeostasis in cells lacking complex III.

A marked stimulation of complex II enzymatic activity was detected in cybrids bearing a homoplasmic MTCYB microdeletion causing disruption of both the activity and the assembly of complex III, but not in cybrids harbouring another MTCYB mutation affecting only the complex III activity. Moreover, complex II stimulation was associated with SDHA subunit tyrosine phosphorylation. Despite the lack of detectable hydrogen peroxide production, up-regulation of the levels of mitochondrial antioxidant defenses revealed a significant redox unbalance. This effect was also supported by the finding that treatment with N-acetylcysteine dampened the complex II stimulation, SDHA subunit tyrosine phosphorylation, and levels of antioxidant enzymes. In the absence of complex III, the cellular amount of succinate, but not fumarate, was markedly increased, indicating that enhanced activity of complex II is hampered due to the blockage of respiratory electron flow. Thus, we propose that complex II phosphorylation and stimulation of its activity represent a molecular mechanism triggered by perturbation of mitochondrial redox homeostasis due to severe dysfunction of respiratory complexes. Depending on the site and nature of the damage, complex II stimulation can either bypass the energetic deficit as an efficient compensatory mechanism, or be ineffectual, leaving cells to rely on glycolysis for survival.

OPA1 Isoforms in the Hierarchical Organization of Mitochondrial Functions.

OPA1 is a GTPase that controls mitochondrial fusion, cristae integrity, and mtDNA maintenance. In humans, eight isoforms are expressed as combinations of long and short forms, but it is unclear whether OPA1 functions are associated with specific isoforms and/or domains. To address this, we expressed each of the eight isoforms or different constructs of isoform 1 in Opa1-/- MEFs. We observed that any isoform could restore cristae structure, mtDNA abundance, and energetic efficiency independently of mitochondrial network morphology. Long forms supported mitochondrial fusion; short forms were better able to restore energetic efficiency. The complete rescue of mitochondrial network morphology required a balance of long and short forms of at least two isoforms, as shown by combinatorial isoform silencing and co-expression experiments. Thus, multiple OPA1 isoforms are required for mitochondrial dynamics, while any single isoform can support all other functions. These findings will be useful in designing gene therapies for patients with OPA1 haploinsufficiency.

Syndromic parkinsonism and dementia associated with OPA1 missense mutations.

OBJECTIVE: Mounting evidence links neurodegenerative disorders such as Parkinson disease and Alzheimer disease with mitochondrial dysfunction, and recent emphasis has focused on mitochondrial dynamics and quality control. Mitochondrial dynamics and mtDNA maintenance is another link recently emerged, implicating mutations in the mitochondrial fusion genes OPA1 and MFN2 in the pathogenesis of multisystem syndromes characterized by neurodegeneration and accumulation of mtDNA multiple deletions in postmitotic tissues. Here, we report 2 Italian families affected by dominant chronic progressive external ophthalmoplegia (CPEO) complicated by parkinsonism and dementia. METHODS: Patients were extensively studied by optical coherence tomography (OCT) to assess retinal nerve fibers, and underwent muscle and brain magnetic resonance spectroscopy (MRS), and muscle biopsy and fibroblasts were analyzed. Candidate genes were sequenced, and mtDNA was analyzed for rearrangements. RESULTS: Affected individuals displayed a slowly progressive syndrome characterized by CPEO, mitochondrial myopathy, sensorineural deafness, peripheral neuropathy, parkinsonism, and/or cognitive impairment, in most cases without visual complains, but with subclinical loss of retinal nerve fibers at OCT. Muscle biopsies showed cytochrome c oxidase-negative fibers and mtDNA multiple deletions, and MRS displayed defective oxidative metabolism in muscle and brain. We found 2 heterozygous OPA1 missense mutations affecting highly conserved amino acid positions (p.G488R, p.A495V) in the guanosine triphosphatase domain, each segregating with affected individuals. Fibroblast studies showed a reduced amount of OPA1 protein with normal mRNA expression, fragmented mitochondria, impaired bioenergetics, increased autophagy and mitophagy. INTERPRETATION: The association of CPEO and parkinsonism/dementia with subclinical optic neuropathy widens the phenotypic spectrum of OPA1 mutations, highlighting the association of defective mitochondrial dynamics, mtDNA multiple deletions, and altered mitophagy with parkinsonism.

Mitochondria: Biogenesis and mitophagy balance in segregation and clonal expansion of mitochondrial DNA mutations.

Mitochondria are cytoplasmic organelles containing their own multi-copy genome. They are organized in a highly dynamic network, resulting from balance between fission and fusion, which maintains homeostasis of mitochondrial mass through mitochondrial biogenesis and mitophagy. Mitochondrial DNA (mtDNA) mutates much faster than nuclear DNA. In particular, mtDNA point mutations and deletions may occur somatically and accumulate with aging, coexisting with the wild type, a condition known as heteroplasmy. Under specific circumstances, clonal expansion of mutant mtDNA may occur within single cells, causing a wide range of severe human diseases when mutant overcomes wild type. Furthermore, mtDNA deletions accumulate and clonally expand as a consequence of deleterious mutations in nuclear genes involved in mtDNA replication and maintenance, as well as in mitochondrial fusion genes (mitofusin-2 and OPA1), possibly implicating mtDNA nucleoids segregation. We here discuss how the intricacies of mitochondrial homeostasis impinge on the intracellular propagation of mutant mtDNA. This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.

A novel in-frame 18-bp microdeletion in MT-CYB causes a multisystem disorder with prominent exercise intolerance.

A novel heteroplasmic mitochondrial DNA (mtDNA) microdeletion affecting the cytochrome b gene (MT-CYB) was identified in an Italian female patient with a multisystem disease characterized by sensorineural deafness, cataracts, retinal pigmentary dystrophy, dysphagia, postural and gait instability, and myopathy with prominent exercise intolerance. The deletion is 18-base pair long and encompasses nucleotide positions 15,649-15,666, causing the loss of six amino acids (Ile-Leu-Ala-Met-Ile-Pro) in the protein, but leaving the remaining of the MT-CYB sequence in frame. The defective complex III function was cotransferred with mutant mtDNA in cybrids, thus unequivocally establishing its pathogenic role. Maternal relatives failed to show detectable levels of the deletion in blood and urinary epithelium, suggesting a de novo mutational event. This is the second report of an in-frame intragenic deletion in MT-CYB, which most likely occurred in early stages of embryonic development, associated with a severe multisystem disorder with prominent exercise intolerance.

Different mtDNA mutations modify tumor progression in dependence of the degree of respiratory complex I impairment.

Mitochondrial DNA mutations are currently investigated as modifying factors impinging on tumor growth and aggressiveness, having been found in virtually all cancer types and most commonly affecting genes encoding mitochondrial complex I (CI) subunits. However, it is still unclear whether they exert a pro- or anti-tumorigenic effect. We here analyzed the impact of three homoplasmic mtDNA mutations (m.3460G>A/MT-ND1, m.3571insC/MT-ND1 and m.3243A>G/MT-TL1) on osteosarcoma progression, chosen since they induce different degrees of oxidative phosphorylation impairment. In fact, the m.3460G>A/MT-ND1 mutation caused only a reduction in CI activity, whereas the m.3571insC/MT-ND1 and the m.3243A>G/MT-TL1 mutations induced a severe structural and functional CI alteration. As a consequence, this severe CI dysfunction determined an energetic defect associated with a compensatory increase in glycolytic metabolism and AMP-activated protein kinase activation. Osteosarcoma cells carrying such marked CI impairment displayed a reduced tumorigenic potential both in vitro and in vivo, when compared with cells with mild CI dysfunction, suggesting that mtDNA mutations may display diverse impact on tumorigenic potential depending on the type and severity of the resulting oxidative phosphorylation dysfunction. The modulation of tumor growth was independent from reactive oxygen species production but correlated with hypoxia-inducible factor 1α stabilization, indicating that structural and functional integrity of CI and oxidative phosphorylation are required for hypoxic adaptation and tumor progression.

Respiratory complex I is essential to induce a Warburg profile in mitochondria-defective tumor cells.

BACKGROUND: Aerobic glycolysis, namely the Warburg effect, is the main hallmark of cancer cells. Mitochondrial respiratory dysfunction has been proposed to be one of the major causes for such glycolytic shift. This hypothesis has been revisited as tumors appear to undergo waves of gene regulation during progression, some of which rely on functional mitochondria. In this framework, the role of mitochondrial complex I is still debated, in particular with respect to the effect of mitochondrial DNA mutations in cancer metabolism. The aim of this work is to provide the proof of concept that functional complex I is necessary to sustain tumor progression. METHODS: Complex I-null osteosarcoma cells were complemented with allotopically expressed complex I subunit 1 (MT-ND1). Complex I re-assembly and function recovery, also in terms of NADH consumption, were assessed. Clones were tested for their ability to grow in soft agar and to generate tumor masses in nude mice. Hypoxia levels were evaluated via pimonidazole staining and hypoxia-inducible factor-1α (HIF-1α) immunoblotting and histochemical staining. 454-pyrosequencing was implemented to obtain global transcriptomic profiling of allotopic and non-allotopic xenografts. RESULTS: Complementation of a truncative mutation in the gene encoding MT-ND1, showed that a functional enzyme was required to perform the glycolytic shift during the hypoxia response and to induce a Warburg profile in vitro and in vivo, fostering cancer progression. Such trigger was mediated by HIF-1α, whose stabilization was regulated after recovery of the balance between α-ketoglutarate and succinate due to a recuperation of NADH consumption that followed complex I rescue. CONCLUSION: Respiratory complex I is essential for the induction of Warburg effect and adaptation to hypoxia of cancer cells, allowing them to sustain tumor growth. Differently from other mitochondrial tumor suppressor genes, therefore, a complex I severe mutation such as the one here reported may confer anti-tumorigenic properties, highlighting the prognostic values of such genetic markers in cancer.

Molecular mechanisms of superoxide production by complex III: a bacterial versus human mitochondrial comparative case study.

In this mini review, we briefly survey the molecular processes that lead to reactive oxygen species (ROS) production by the respiratory complex III (CIII or cytochrome bc1). In particular, we discuss the "forward" and "reverse" electron transfer pathways that lead to superoxide generation at the quinol oxidation (Qo) site of CIII, and the components that affect these reactions. We then describe and compare the properties of a bacterial (Rhodobacter capsulatus) mutant enzyme producing ROS with its mitochondrial (human cybrids) counterpart associated with a disease. The mutation under study is located at a highly conserved tyrosine residue of cytochrome b (Y302C in R. capsulatus and Y278C in human mitochondria) that is at the heart of the quinol oxidation (Qo) site of CIII. Similarities of the major findings of bacterial and human mitochondrial cases, including decreased catalytic activity of CIII, enhanced ROS production and ensuing cellular responses and damages, are remarkable. This case illustrates the usefulness of undertaking parallel and complementary studies using biologically different yet evolutionarily related systems, such as α-proteobacteria and human mitochondria. It progresses our understanding of CIII mechanism of function and ROS production, and underlines the possible importance of supra-molecular organization of bacterial and mitochondrial respiratory chains (i.e., respirasomes) and their potential disease-associated protective roles. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.