Understanding mitochondrial DNA maintenance disorders at the single muscle fibre level.

Clonal expansion of mitochondrial DNA (mtDNA) deletions is an important pathological mechanism in adults with mtDNA maintenance disorders, leading to a mosaic mitochondrial respiratory chain deficiency in skeletal muscle. This study had two aims: (i) to determine if different Mendelian mtDNA maintenance disorders showed similar pattern of mtDNA deletions and respiratory chain deficiency and (ii) to investigate the correlation between the mitochondrial genetic defect and corresponding respiratory chain deficiency. We performed a quantitative analysis of respiratory chain deficiency, at a single cell level, in a cohort of patients with mutations in mtDNA maintenance genes. Using the same tissue section, we performed laser microdissection and single cell genetic analysis to investigate the relationship between mtDNA deletion characteristics and the respiratory chain deficiency. The pattern of respiratory chain deficiency is similar with different genetic defects. We demonstrate a clear correlation between the level of mtDNA deletion and extent of respiratory chain deficiency within a single cell. Long-range and single molecule PCR shows the presence of multiple mtDNA deletions in approximately one-third of all muscle fibres. We did not detect evidence of a replicative advantage for smaller mtDNA molecules in the majority of fibres, but further analysis is needed to provide conclusive evidence.

Subcellular origin of mitochondrial DNA deletions in human skeletal muscle.

OBJECTIVE: In patients with mitochondrial DNA (mtDNA) maintenance disorders and with aging, mtDNA deletions sporadically form and clonally expand within individual muscle fibers, causing respiratory chain deficiency. This study aimed to identify the sub-cellular origin and potential mechanisms underlying this process. METHODS: Serial skeletal muscle cryosections from patients with multiple mtDNA deletions were subjected to subcellular immunofluorescent, histochemical, and genetic analysis. RESULTS: We report respiratory chain-deficient perinuclear foci containing mtDNA deletions, which show local elevations of both mitochondrial mass and mtDNA copy number. These subcellular foci of respiratory chain deficiency are associated with a local increase in mitochondrial biogenesis and unfolded protein response signaling pathways. We also find that the commonly reported segmental pattern of mitochondrial deficiency is consistent with the three-dimensional organization of the human skeletal muscle mitochondrial network. INTERPRETATION: We propose that mtDNA deletions first exceed the biochemical threshold causing biochemical deficiency in focal regions adjacent to the myonuclei, and induce mitochondrial biogenesis before spreading across the muscle fiber. These subcellular resolution data provide new insights into the possible origin of mitochondrial respiratory chain deficiency in mitochondrial myopathy. Ann Neurol 2018;84:289-301.

Skeletal muscle mitochondrial oxidative phosphorylation function in idiopathic pulmonary arterial hypertension: in vivo and in vitro study.

Mitochondrial dysfunction within the pulmonary vessels has been shown to contribute to the pathology of idiopathic pulmonary arterial hypertension (IPAH). We investigated the hypothesis of whether impaired exercise capacity observed in IPAH patients is in part due to primary mitochondrial oxidative phosphorylation (OXPHOS) dysfunction in skeletal muscle. This could lead to potentially new avenues of treatment beyond targeting the pulmonary vessels. Nine clinically stable participants with IPAH underwent cardiopulmonary exercise testing, in vivo and in vitro assessment of mitochondrial function by 31P-magnetic resonance spectroscopy (31P-MRS) and laboratory muscle biopsy analysis. 31P-MRS showed abnormal skeletal muscle bioenergetics with prolonged recovery times of phosphocreatine and abnormal muscle pH handling. Histochemistry and quadruple immunofluorescence performed on muscle biopsies showed normal function and subunit protein abundance of the complexes within the OXPHOS system. Our findings suggest that there is no primary mitochondrial OXPHOS dysfunction but raises the possibility of impaired oxygen delivery to the mitochondria affecting skeletal muscle bioenergetics during exercise.

Spectral components of detergent-solubilized bovine cytochrome oxidase.

Cytochrome oxidase is the terminal oxidase of the mitochondrial electron transport chain and pumps 4 protons per oxygen reduced to water. Spectral shifts in the α-band of heme a have been observed in multiple studies and these shifts have the potential to shed light on the proton pumping intermediates. Previously we found that heme a had two spectral components in the α-band during redox titrations in living RAW 264.7 mouse macrophage cells, the classical 605 nm form and a blue-shifted 602 nm form. To confirm these spectral changes were not an artifact due to the complex milieu of the living cell, redox titrations were performed in the isolated detergent-solubilized bovine enzyme from both the Soret- and α-band using precise multiwavelength spectroscopy. This data verified the presence of the 602 nm form in the α-band, revealed a similar shift of heme a in the Soret-band and ruled out the reversal of calcium binding as the origin of the blue shift. The 602 nm form was found to be stabilized at high pH or by binding of azide, which is known to blue shift the α-band of heme a. Azide also stabilized the 602 nm form in the living cells. It is concluded there is a form of cytochrome oxidase in which heme a undergoes a blue shift to a 602 nm form and that redox titrations can be successfully performed in living cells where the oxidase operates in its authentic environment and in the presence of a proton motive force.

Disclosing the functional changes of two genetic alterations in a patient with Chronic Progressive External Ophthalmoplegia: Report of the novel mtDNA m.7486G>A variant.

Chronic Progressive External Ophthalmoplegia (CPEO) is characterized by ptosis and ophthalmoplegia and is usually caused by mitochondrial DNA (mtDNA) deletions or mt-tRNA mutations. The aim of the present work was to clarify the genetic defect in a patient presenting with CPEO and elucidate the underlying pathogenic mechanism. This 62-year-old female first developed ptosis of the right eye at the age of 12 and subsequently the left eye at 45 years, and was found to have external ophthalmoplegia at the age of 55 years. Histopathological abnormalities were detected in the patient's muscle, including ragged-red fibres, a mosaic pattern of COX-deficient muscle fibres and combined deficiency of respiratory chain complexes I and IV. Genetic investigation revealed the "common deletion" in the patient's muscle and fibroblasts. Moreover, a novel, heteroplasmic mt-tRNASer(UCN) variant (m.7486G>A) in the anticodon loop was detected in muscle homogenate (50%), fibroblasts (11%) and blood (4%). Single-fibre analysis showed segregation with COX-deficient fibres for both genetic alterations. Assembly defects of mtDNA-encoded complexes were demonstrated in fibroblasts. Functional analyses showed significant bioenergetic dysfunction, reduction in respiration rate and ATP production and mitochondrial depolarization. Multilamellar bodies were detected by electron microscopy, suggesting disturbance in autophagy. In conclusion, we report a CPEO patient with two possible genetic origins, both segregating with biochemical and histochemical defect. The "common mtDNA deletion" is the most likely cause, yet the potential pathogenic effect of a novel mt-tRNASer(UCN) variant cannot be fully excluded.

Pathological mechanisms underlying single large-scale mitochondrial DNA deletions.

OBJECTIVE: Single, large-scale deletions in mitochondrial DNA (mtDNA) are a common cause of mitochondrial disease. This study aimed to investigate the relationship between the genetic defect and molecular phenotype to improve understanding of pathogenic mechanisms associated with single, large-scale mtDNA deletions in skeletal muscle. METHODS: We investigated 23 muscle biopsies taken from adult patients (6 males/17 females with a mean age of 43 years) with characterized single, large-scale mtDNA deletions. Mitochondrial respiratory chain deficiency in skeletal muscle biopsies was quantified by immunoreactivity levels for complex I and complex IV proteins. Single muscle fibers with varying degrees of deficiency were selected from 6 patient biopsies for determination of mtDNA deletion level and copy number by quantitative polymerase chain reaction. RESULTS: We have defined 3 "classes" of single, large-scale deletion with distinct patterns of mitochondrial deficiency, determined by the size and location of the deletion. Single fiber analyses showed that fibers with greater respiratory chain deficiency harbored higher levels of mtDNA deletion with an increase in total mtDNA copy number. For the first time, we have demonstrated that threshold levels for complex I and complex IV deficiency differ based on deletion class. INTERPRETATION: Combining genetic and immunofluorescent assays, we conclude that thresholds for complex I and complex IV deficiency are modulated by the deletion of complex-specific protein-encoding genes. Furthermore, removal of mt-tRNA genes impacts specific complexes only at high deletion levels, when complex-specific protein-encoding genes remain. These novel findings provide valuable insight into the pathogenic mechanisms associated with these mutations. Ann Neurol 2018;83:115-130.

Using a quantitative quadruple immunofluorescent assay to diagnose isolated mitochondrial Complex I deficiency.

Isolated Complex I (CI) deficiency is the most commonly observed mitochondrial respiratory chain biochemical defect, affecting the largest OXPHOS component. CI is genetically heterogeneous; pathogenic variants affect one of 38 nuclear-encoded subunits, 7 mitochondrial DNA (mtDNA)-encoded subunits or 14 known CI assembly factors. The laboratory diagnosis relies on the spectrophotometric assay of enzyme activity in mitochondrially-enriched tissue homogenates, requiring at least 50 mg skeletal muscle, as there is no reliable histochemical method for assessing CI activity directly in tissue cryosections. We have assessed a validated quadruple immunofluorescent OXPHOS (IHC) assay to detect CI deficiency in the diagnostic setting, using 10 µm transverse muscle sections from 25 patients with genetically-proven pathogenic CI variants. We observed loss of NDUFB8 immunoreactivity in all patients with mutations affecting nuclear-encoding structural subunits and assembly factors, whilst only 3 of the 10 patients with mutations affecting mtDNA-encoded structural subunits showed loss of NDUFB8, confirmed by BN-PAGE analysis of CI assembly and IHC using an alternative, commercially-available CI (NDUFS3) antibody. The IHC assay has clear diagnostic potential to identify patients with a CI defect of Mendelian origins, whilst highlighting the necessity of complete mitochondrial genome sequencing in the diagnostic work-up of patients with suspected mitochondrial disease.

The genetics and pathology of mitochondrial disease.

Mitochondria are double-membrane-bound organelles that are present in all nucleated eukaryotic cells and are responsible for the production of cellular energy in the form of ATP. Mitochondrial function is under dual genetic control - the 16.6-kb mitochondrial genome, with only 37 genes, and the nuclear genome, which encodes the remaining ∼1300 proteins of the mitoproteome. Mitochondrial dysfunction can arise because of defects in either mitochondrial DNA or nuclear mitochondrial genes, and can present in childhood or adulthood in association with vast clinical heterogeneity, with symptoms affecting a single organ or tissue, or multisystem involvement. There is no cure for mitochondrial disease for the vast majority of mitochondrial disease patients, and a genetic diagnosis is therefore crucial for genetic counselling and recurrence risk calculation, and can impact on the clinical management of affected patients. Next-generation sequencing strategies are proving pivotal in the discovery of new disease genes and the diagnosis of clinically affected patients; mutations in >250 genes have now been shown to cause mitochondrial disease, and the biochemical, histochemical, immunocytochemical and neuropathological characterization of these patients has led to improved diagnostic testing strategies and novel diagnostic techniques. This review focuses on the current genetic landscape associated with mitochondrial disease, before focusing on advances in studying associated mitochondrial pathology in two, clinically relevant organs - skeletal muscle and brain. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Dysferlin mutations and mitochondrial dysfunction.

Dysferlinopathies are caused by mutations in the DYSF gene and patients may present with proximal or distal myopathy. Dysferlin is responsible for membrane resealing, and mutations may result in a defect in membrane repair following mechanical or chemical stress, causing an influx of Ca2+. Since mitochondria are involved in Ca2+ buffering, we hypothesised that mitochondrial defects may be present in skeletal muscle biopsies from patients with mutations in this gene. The aim was to characterise mitochondrial defects in muscle from patients with dysferlinopathies. Here, we analysed skeletal muscle biopsies for eight patients by quadruple immunofluorescent assay to assess oxidative phosphorylation protein abundance. Long-range PCR in single muscle fibres was used to look for presence of clonally expanded large-scale mitochondrial DNA rearrangements in patients' skeletal muscle (n = 3). Immunofluorescence demonstrated that the percentage of complex I- and complex IV-deficient fibres was higher in patients with DYSF mutations than in age-matched controls. No clonally expanded mtDNA deletions were detected using long-range PCR in any of the analysed muscle fibres. We conclude that complex I and complex IV deficiency is higher in patients than age matched controls but patients do not have rearrangements of the mtDNA. We hypothesise that respiratory chain deficiency may be the results of an increased cytosolic Ca2+ concentration (due to a membrane resealing defect) causing mitochondrial aberrations.

Mitochondrial dysfunction in myofibrillar myopathy.

Myofibrillar myopathies (MFM) are characterised by focal myofibrillar destruction and accumulation of myofibrillar elements as protein aggregates. They are caused by mutations in the DES, MYOT, CRYAB, FLNC, BAG3, DNAJB6 and ZASP genes as well as other as yet unidentified genes. Previous studies have reported changes in mitochondrial morphology and cellular positioning, as well as clonally-expanded, large-scale mitochondrial DNA (mtDNA) deletions and focal respiratory chain deficiency in muscle of MFM patients. Here we examine skeletal muscle from patients with desmin (n = 6), ZASP (n = 1) and myotilin (n = 2) mutations and MFM protein aggregates, to understand how mitochondrial dysfunction may contribute to the underlying mechanisms causing disease pathology. We have used a validated quantitative immunofluorescent assay to study respiratory chain protein levels, together with oxidative enzyme histochemistry and single cell mitochondrial DNA analysis, to examine mitochondrial changes. Results demonstrate a small number of clonally-expanded mitochondrial DNA deletions, which we conclude are due to both ageing and disease pathology. Further to this we report higher levels of respiratory chain complex I and IV deficiency compared to age matched controls, although overall levels of respiratory deficient muscle fibres in patient biopsies are low. More strikingly, a significantly higher percentage of myofibrillar myopathy patient muscle fibres have a low mitochondrial mass compared to controls. We concluded this is mechanistically unrelated to desmin and myotilin protein aggregates; however, correlation between mitochondrial mass and muscle fibre area is found. We suggest this may be due to reduced mitochondrial biogenesis in combination with muscle fibre hypertrophy.

Unique quadruple immunofluorescence assay demonstrates mitochondrial respiratory chain dysfunction in osteoblasts of aged and PolgA(-/-) mice.

Fragility fractures caused by osteoporosis affect millions of people worldwide every year with significant levels of associated morbidity, mortality and costs to the healthcare economy. The pathogenesis of declining bone mineral density is poorly understood but it is inherently related to increasing age. Growing evidence in recent years, especially that provided by mouse models, suggest that accumulating somatic mitochondrial DNA mutations may cause the phenotypic changes associated with the ageing process including osteoporosis. Methods to study mitochondrial abnormalities in individual osteoblasts, osteoclasts and osteocytes are limited and impair our ability to assess the changes seen with age and in animal models of ageing. To enable the assessment of mitochondrial protein levels, we have developed a quadruple immunofluorescence method to accurately quantify the presence of mitochondrial respiratory chain components within individual bone cells. We have applied this technique to a well-established mouse model of ageing and osteoporosis and show respiratory chain deficiency.

Pathogenic mtDNA mutations causing mitochondrial myopathy: The need for muscle biopsy.

Pathogenic mitochondrial tRNA (mt-tRNA) gene mutations represent a prominent cause of primary mitochondrial DNA (mtDNA)-related disease despite accounting for only 5%-10% of the mitochondrial genome.(1,2) Although some common mt-tRNA mutations, such as the m.3243A>G mutation, exist, the majority are rare and have been reported in only a small number of cases.(3) The MT-TP gene, encoding mt-tRNA(Pro), is one of the less polymorphic mt-tRNA genes, and only 5 MT-TP mutations have been reported as a cause of mitochondrial muscle disease to date (table e-1 at Neurology.org/ng, P6-10). We report 5 patients with myopathic phenotypes, each harboring different pathogenic mutations in the MT-TP gene, highlighting the importance of MT-TP mutations as a cause of mitochondrial muscle disease and the requirement to study clinically relevant tissue.

A novel immunofluorescent assay to investigate oxidative phosphorylation deficiency in mitochondrial myopathy: understanding mechanisms and improving diagnosis.

Oxidative phosphorylation defects in human tissues are often challenging to quantify due to a mosaic pattern of deficiency. Biochemical assays are difficult to interpret due to the varying enzyme deficiency levels found in individual cells. Histochemical analysis allows semi-quantitative assessment of complex II and complex IV activities, but there is no validated histochemical assay to assess complex I activity which is frequently affected in mitochondrial pathology. To help improve the diagnosis of mitochondrial disease and to study the mechanisms underlying mitochondrial abnormalities in disease, we have developed a quadruple immunofluorescent technique enabling the quantification of key respiratory chain subunits of complexes I and IV, together with an indicator of mitochondrial mass and a cell membrane marker. This assay gives precise and objective quantification of protein abundance in large numbers of individual muscle fibres. By assessing muscle biopsies from subjects with a range of different mitochondrial genetic defects we have demonstrated that specific genotypes exhibit distinct biochemical signatures in muscle, providing evidence for the diagnostic use of the technique, as well as insight into the underlying molecular pathology. Stringent testing for reproducibility and sensitivity confirms the potential value of the technique for mechanistic studies of disease and in the evaluation of therapeutic approaches.

Mitochondrial and inflammatory changes in sporadic inclusion body myositis.

AIMS: Sporadic inclusion body myositis (sIBM) is the most common late onset muscle disease causing progressive weakness. In light of the lack of effective treatment, we investigated potential causes underlying muscle wasting. We hypothesized that accumulation of mitochondrial respiratory deficiency in muscle fibres may lead to fibre atrophy and degeneration, contributing to muscle mass reduction. METHODS: Histochemical and immunohistochemical analyses were performed on muscle biopsies from 16 sIBM patients to detect activity of mitochondrial enzymes and expression of mitochondrial respiratory chain proteins along with inflammatory markers respectively. Mitochondrial DNA mutations were assessed in single muscle fibres using real-time PCR. RESULTS: We identified respiratory-deficient fibres at different stages of mitochondrial dysfunction, with downregulated expression of complex I of mitochondrial respiratory chain being the initial feature. We detected mitochondrial DNA rearrangements in the majority of individual respiratory-deficient muscle fibres. There was a strong correlation between number of T lymphocytes and macrophages residing in muscle tissue and the abundance of respiratory-deficient fibres. Moreover, we found that respiratory-deficient muscle fibres were more likely to be atrophic compared with respiratory-normal counterparts. CONCLUSIONS: Our findings suggest that mitochondrial dysfunction has a role in sIBM progression. A strong correlation between the severity of inflammation, degree of mitochondrial changes and atrophy implicated existence of a mechanistic link between these three parameters. We propose a role for inflammatory cells in the initiation of mitochondrial DNA damage, which when accumulated, causes respiratory dysfunction, fibre atrophy and ultimately degeneration of muscle fibres.

Quantitative quadruple-label immunofluorescence of mitochondrial and cytoplasmic proteins in single neurons from human midbrain tissue.

BACKGROUND: Respiratory chain (RC) deficiencies are found in primary mtDNA diseases. Focal RC defects are also associated with ageing and neurodegenerative disorders, e.g. in substantia nigra (SN) neurons from Parkinson's disease patients. In mitochondrial disease and ageing, mtDNA mutational loads vary considerably between neurons necessitating single cell-based assessment of RC deficiencies. Evaluating the full extent of RC deficiency within SN neurons is challenging because their size precludes investigations in serial sections. We developed an assay to measure RC abnormalities in individual SN neurons using quadruple immunofluorescence. NEW METHOD: Using antibodies against subunits of complex I (CI) and IV, porin and tyrosine hydroxylase together with IgG subtype-specific fluorescent labelled secondary antibodies, we quantified the expression of CI and CIV compared to mitochondrial mass in dopaminergic neurons. CI:porin and CIV:porin ratios were determined relative to a standard control. RESULTS: Quantification of expression of complex subunits in midbrain sections from patients with mtDNA disease and known RC deficiencies consistently showed reduced CI:porin and/or CIV:porin ratios. COMPARISON WITH EXISTING METHOD(S): The standard histochemical method to investigate mitochondrial dysfunction, the cytochrome c oxidase/succinate dehydrogenase assay, measures CIV and CII activities. To also study CI in a patient, immunohistology in additional sections, i.e. in different neurons, is required. Our method allows correlation of the expression of CI, CIV and mitochondrial mass at a single cell level. CONCLUSION: Quantitative quadruple-label immunofluorescence is a reliable tool to measure RC deficiencies in individual neurons that will enable new insights in the molecular mechanisms underlying inherited and acquired mitochondrial dysfunction.

Early changes of neuromuscular transmission in the SOD1(G93A) mice model of ALS start long before motor symptoms onset.

Amyotrophic lateral sclerosis is characterized by a progressive degeneration of the corticospinal tract motor neurons. Growing evidence suggests that degeneration may begin at the distal axon proceeding in a dying-back pattern. It seemed therefore of interest to investigate synaptic transmission at the neuromuscular junction (NMJ) in pre- and symptomatic phases of the disease. Endplate potentials (EPPs), miniatures endplate potentials (MEPPs) and giant MEPPs (GMEPPs) were recorded from innervated diaphragm muscle fibers from 4-6 and 12-15 weeks-old SOD1(G93A) mice and non-transgenic aged-matched littermates (WT). In the pre-symptomatic phase, SOD1(G93A) mice exhibited a significant increase in the mean amplitude of EPPs together with an increase in the mean quantal content of EPPs, suggesting that more acetylcholine is being released into the synaptic cleft. SOD1(G93A) mice presented a higher frequency of GMEPPs, suggestive of intracellular Ca(2+) deregulation in nerve terminals. The increase in the mean amplitude of MEPPs and the decreased mean rise-time of MEPPs in SOD1(G93A) mice point to post-synaptic related changes. In the symptomatic phase, electrophysiological data showed evidence for two NMJ groups in SOD1(G93A) mice: SOD1a and SOD1b. SOD1a group presented reduced mean amplitude of both EPPs and MEPPs. The mean rise-time of MEPPs was increased, when compared to WT and to SOD1b group, indicating impairments in the neuromuscular transmission. In contrast, the neuromuscular transmission of SOD1b group was not different from age-matched WT nor pre-symptomatic SOD1(G93A) mice, being somehow in between both groups. Altogether these results show that the neuromuscular transmission of SOD1(G93A) mice is enhanced in the pre-symptomatic phase. In the symptomatic phase our results are consistent with the hypothesis that the diaphragm of SOD1(G93A) mice is undergoing cycles of denervation/re-innervation supported by mixed neuromuscular junction populations. These early changes in the neuromuscular transmission of SOD1(G93A) mice suggest that the ALS associated events start long before symptoms onset.