Variant load of mitochondrial DNA in single human mesenchymal stem cells.

Heteroplasmic mitochondrial DNA (mtDNA) variants accumulate as humans age, particularly in the stem-cell compartments, and are an important contributor to age-related disease. Mitochondrial dysfunction has been observed in osteoporosis and somatic mtDNA pathogenic variants have been observed in animal models of osteoporosis. However, this has never been assessed in the relevant human tissue. Mesenchymal stem cells (MSCs) are the progenitors to many cells of the musculoskeletal system and are critical to skeletal tissues and bone vitality. Investigating mtDNA in MSCs could provide novel insights into the role of mitochondrial dysfunction in osteoporosis. To determine if this is possible, we investigated the landscape of somatic mtDNA variation in MSCs through a combination of fluorescence-activated cell sorting and single-cell next-generation sequencing. Our data show that somatic heteroplasmic variants are present in individual patient-derived MSCs, can reach high heteroplasmic fractions and have the potential to be pathogenic. The identification of somatic heteroplasmic variants in MSCs of patients highlights the potential for mitochondrial dysfunction to contribute to the pathogenesis of osteoporosis.

Automated quantitative high-throughput multiplex immunofluorescence pipeline to evaluate OXPHOS defects in formalin-fixed human prostate tissue.

Advances in multiplex immunofluorescence (mIF) and digital image analysis has enabled simultaneous assessment of protein defects in electron transport chain components. However, current manual methodology is time consuming and labour intensive. Therefore, we developed an automated high-throughput mIF workflow for quantitative single-cell level assessment of formalin fixed paraffin embedded tissue (FFPE), leveraging tyramide signal amplification on a Ventana Ultra platform coupled with automated multispectral imaging on a Vectra 3 platform. Utilising this protocol, we assessed the mitochondrial oxidative phosphorylation (OXPHOS) protein alterations in a cohort of benign and malignant prostate samples. Mitochondrial OXPHOS plays a critical role in cell metabolism, and OXPHOS perturbation is implicated in carcinogenesis. Marked inter-patient, intra-patient and spatial cellular heterogeneity in OXPHOS protein abundance was observed. We noted frequent Complex IV loss in benign prostate tissue and Complex I loss in age matched prostate cancer tissues. Malignant regions within prostate cancer samples more frequently contained cells with low Complex I & IV and high mitochondrial mass in comparison to benign-adjacent regions. This methodology can now be applied more widely to study the frequency and distribution of OXPHOS alterations in formalin-fixed tissues, and their impact on long-term clinical outcomes.

A subcellular cookie cutter for spatial genomics in human tissue.

Intracellular heterogeneity contributes significantly to cellular physiology and, in a number of debilitating diseases, cellular pathophysiology. This is greatly influenced by distinct organelle populations and to understand the aetiology of disease, it is important to have tools able to isolate and differentially analyse organelles from precise location within tissues. Here, we report the development of a subcellular biopsy technology that facilitates the isolation of organelles, such as mitochondria, from human tissue. We compared the subcellular biopsy technology to laser capture microdissection (LCM) that is the state-of-the-art technique for the isolation of cells from their surrounding tissues. We demonstrate an operational limit of  >20 µm for LCM and then, for the first time in human tissue, show that subcellular biopsy can be used to isolate mitochondria beyond this limit.

The Role of Mitochondria-Linked Fatty-Acid Uptake-Driven Adipogenesis in Graves Orbitopathy.

CONTEXT: Depot-specific expansion of orbital adipose tissue (OAT) in Graves orbitopathy (GO; an autoimmune condition producing proptosis, visual impairment and reduced quality of life) is associated with fatty acid (FA)-uptake-driven adipogenesis in preadipocytes/fibroblasts (PFs). OBJECTIVE: This work sought a role for mitochondria in OAT adipogenesis in GO. METHODS: Confluent PFs from healthy OAT (OAT-H), OAT from GO (OAT-GO) and white adipose tissue in culture medium compared with culture medium containing a mixed hormonal cocktail as adipogenic medium (ADM), or culture-medium containing FA-supplementation, oleate:palmitate:linoleate (45:30:25%) with/without different concentration of mitochondrial biosubstrate adenosine 5'-diphosphate/guanosine 5'-diphosphate (ADP/GDP), AICAR (adenosine analogue), or inhibitor oligomycin-A for 17 days. Main outcome measures included oil-red-O staining and foci count of differentiated adipocytes for in vitro adipogenesis, flow cytometry, relative quantitative polymerase chain reaction, MTS-assay/106 cells, total cellular-ATP detection kit, and Seahorse-XFe96-Analyzer for mitochondria and oxidative-phosphorylation (OXPHOS)/glycolysis-ATP production analysis. RESULTS: During early adipogenesis before adipocyte formation (days 0, 4, and7), we observed OAT-specific cellular ATP production via mitochondrial OXPHOS in PFs both from OAT-H and OAT-GO, and substantially disrupted OXPHOS-ATP/glycolysis-ATP production in PFs from OAT-GO, for example, a 40% reduction in OXPHOS-ATP and trend-increased glycolysis-ATP production on days 4 and 7 compared with day 0, which contrasted with the stable levels in OAT-H. FA supplementation in culture-medium triggered adipogenesis in PFs both from OAT-H and OAT-GO, which was substantially enhanced by 1-mM GDP reaching 7% to 18% of ADM adipogenesis. The FA-uptake-driven adipogenesis was diminished by oligomycin-A but unaffected by treatment with ADP or AICAR. Furthermore, we observed a significant positive correlation between FA-uptake-driven adipogenesis by GDP and the ratios of OXPHOS-ATP/glycolysis-ATP through adipogenesis of PFs from OAT-GO. CONCLUSION: Our study confirmed that FA uptake can drive OAT adipogenesis and revealed a fundamental role for mitochondria-OXPHOS in GO development, which provides potential for therapeutic interventions.

Age-associated mitochondrial DNA mutations cause metabolic remodelling that contributes to accelerated intestinal tumorigenesis.

Oxidative phosphorylation (OXPHOS) defects caused by somatic mitochondrial DNA (mtDNA) mutations increase with age in human colorectal epithelium and are prevalent in colorectal tumours, but whether they actively contribute to tumorigenesis remains unknown. Here we demonstrate that mtDNA mutations causing OXPHOS defects are enriched during the human adenoma/carcinoma sequence, suggesting they may confer a metabolic advantage. To test this we deleted the tumour suppressor Apc in OXPHOS deficient intestinal stem cells in mice. The resulting tumours were larger than in control mice due to accelerated cell proliferation and reduced apoptosis. We show that both normal crypts and tumours undergo metabolic remodelling in response to OXPHOS deficiency by upregulating the de novo serine synthesis pathway (SSP). Moreover, normal human colonic crypts upregulate the SSP in response to OXPHOS deficiency prior to tumorigenesis. Our data show that age-associated OXPHOS deficiency causes metabolic remodelling that can functionally contribute to accelerated intestinal cancer development.

The feasibility of muscle mitochondrial respiratory chain phenotyping across the cognitive spectrum in Parkinson's disease.

INTRODUCTION: There has been little work on the relationship between sarcopenia, a progressive skeletal muscle disorder, and age-related neurodegenerative diseases such as Parkinson's disease (PD). OBJECTIVES: We aimed to determine: 1) the feasibility of characterizing skeletal muscle across a range of cognitive function in PD; 2) if muscle mitochondrial respiratory chain (MRC) function and content are preserved in older adults with PD. METHODS: Sarcopenia was defined using handgrip strength, chair rise and bioimpedance analysis. MRC function was assessed using phosphorous magnetic resonance spectroscopy (MRS) by estimating τ1/2 PCr (s) (phosphocreatine half-time recovery) in the calf muscles following a bout of aerobic exercise. Biopsy of the vastus lateralis muscle was performed, and MRC content assessed by fluorescent immunohistochemistry for porin and components of MRC Complexes I and IV. RESULTS: Nine participants (78% male; mean age 79.9; PD duration 3.3 years) were recruited. Four had cognitive impairment. Six participants had probable sarcopenia. Eight participants completed MRS and had mean (SD) τ1/2 PCr of 37.8 (7.6) seconds, suggesting preserved mitochondrial function. Muscle biopsies were obtained in all and the procedure was well tolerated. Porin Z-score, a proxy for mitochondrial mass, was lower than expected compared to controls (0-89% of fibres with low porin). There was a small amount of Complex I (0.16-4.59%) and Complex IV (0-3.79%) deficiency. CONCLUSIONS: Detailed phenotyping, muscle biopsy and imaging was feasible and acceptable across a spectrum of cognitive function in PD. Sarcopenia was relatively common and may be associated with lower mitochondrial mass and low levels of MRC deficiency.

The rise and rise of mitochondrial DNA mutations.

How mitochondrial DNA mutations clonally expand in an individual cell is a question that has perplexed mitochondrial biologists for decades. A growing body of literature indicates that mitochondrial DNA mutations play a major role in ageing, metabolic diseases, neurodegenerative diseases, neuromuscular disorders and cancers. Importantly, this process of clonal expansion occurs for both inherited and somatic mitochondrial DNA mutations. To complicate matters further there are fundamental differences between mitochondrial DNA point mutations and deletions, and between mitotic and post-mitotic cells, that impact this pathogenic process. These differences, along with the challenges of investigating a longitudinal process occurring over decades in humans, have so far hindered progress towards understanding clonal expansion. Here we summarize our current understanding of the clonal expansion of mitochondrial DNA mutations in different tissues and highlight key unanswered questions. We then discuss the various existing biological models, along with their advantages and disadvantages. Finally, we explore what has been achieved with mathematical modelling so far and suggest future work to advance this important area of research.

Diagnosis and Treatment of Mitochondrial Myopathies.

Mitochondrial myopathies are progressive muscle conditions caused primarily by the impairment of oxidative phosphorylation (OXPHOS) in the mitochondria. This causes a deficit in energy production in the form of adenosine triphosphate (ATP), particularly in skeletal muscle. The diagnosis of mitochondrial myopathy is reliant on the combination of numerous techniques including traditional histochemical, immunohistochemical, and biochemical testing combined with the fast-emerging molecular genetic techniques, namely next-generation sequencing (NGS). This has allowed for the diagnosis to become more effective in terms of determining causative or novel genes. However, there are currently no effective or disease-modifying treatments available for the vast majority of patients with mitochondrial myopathies. Existing therapeutic options focus on the symptomatic management of disease manifestations. An increasing number of clinical trials have investigated the therapeutic effects of various vitamins, cofactors, and small molecules, though these trials have failed to show definitive outcome measures for clinical practice thus far. In addition, new molecular strategies, specifically mtZFNs and mtTALENs, that cause beneficial heteroplasmic shifts in cell lines harboring varying pathogenic mtDNA mutations offer hope for the future. Moreover, recent developments in the reproductive options for patients with mitochondrial myopathies mean that for some families, the possibility of preventing transmission of the mutation to the next generation is now possible.

Inherited pathogenic mitochondrial DNA mutations and gastrointestinal stem cell populations.

Inherited mitochondrial DNA (mtDNA) mutations cause mitochondrial disease, but mtDNA mutations also occur somatically and accumulate during ageing. Studies have shown that the mutation load of some inherited mtDNA mutations decreases over time in blood, suggesting selection against the mutation. However, it is unknown whether such selection occurs in other mitotic tissues, and where it occurs within the tissue. Gastrointestinal epithelium is a canonical mitotic tissue rapidly renewed by stem cells. Intestinal crypts (epithelium) undergo monoclonal conversion with a single stem cell taking over the niche and producing progeny. We show: (1) that there is a significantly lower mtDNA mutation load in the mitotic epithelium of the gastrointestinal tract when compared to the smooth muscle in the same tissue in patients with the pathogenic m.3243A>G and m.8344A>G mutations; (2) that there is considerable variation seen in individual crypts, suggesting changes in the stem cell population; (3) that this lower mutation load is reflected in the absence of a defect in oxidative phosphorylation in the epithelium. This suggests that there is selection against inherited mtDNA mutations in the gastrointestinal stem cells that is in marked contrast to the somatic mtDNA mutations that accumulate with age in epithelial stem cells leading to a biochemical defect. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Predominant Asymmetrical Stem Cell Fate Outcome Limits the Rate of Niche Succession in Human Colonic Crypts.

Stem cell (SC) dynamics within the human colorectal crypt SC niche remain poorly understood, with previous studies proposing divergent hypotheses on the predominant mode of SC self-renewal and the rate of SC replacement. Here we use age-related mitochondrial oxidative phosphorylation (OXPHOS) defects to trace clonal lineages within human colorectal crypts across the adult life-course. By resolving the frequency and size distribution of OXPHOS-deficient clones, quantitative analysis shows that, in common with mouse, long-term maintenance of the colonic epithelial crypt relies on stochastic SC loss and replacement mediated by competition for limited niche access. We find that the colonic crypt is maintained by ~5 effective SCs. However, with a SC loss/replacement rate estimated to be slower than once per year, our results indicate that the vast majority of individual SC divisions result in asymmetric fate outcome. These findings provide a quantitative platform to detect and study deviations from human colorectal crypt SC niche homeostasis during the process of colorectal carcinogenesis.

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.

Preferential amplification of a human mitochondrial DNA deletion in vitro and in vivo.

We generated induced pluripotent stem cells (iPSCs) from patient fibroblasts to yield cell lines containing varying degrees of heteroplasmy for a m.13514 A > G mtDNA point mutation (2 lines) and for a ~6 kb single, large scale mtDNA deletion (3 lines). Long term culture of the iPSCs containing a single, large-scale mtDNA deletion showed consistent increase in mtDNA deletion levels with time. Higher levels of mtDNA heteroplasmy correlated with increased respiratory deficiency. To determine what changes occurred in deletion level during differentiation, teratomas comprising all three embryonic germ layers were generated from low (20%) and intermediate heteroplasmy (55%) mtDNA deletion clones. Regardless of whether iPSCs harbouring low or intermediate mtDNA heteroplasmy were used, the final levels of heteroplasmy in all teratoma germ layers increased to a similar high level (>60%). Thus, during human stem cell division, cells not only tolerate high mtDNA deletion loads but seem to preferentially replicate deleted mtDNA genomes. This has implications for the involvement of mtDNA deletions in both disease and ageing.

Topoisomerase 3α Is Required for Decatenation and Segregation of Human mtDNA.

How mtDNA replication is terminated and the newly formed genomes are separated remain unknown. We here demonstrate that the mitochondrial isoform of topoisomerase 3α (Top3α) fulfills this function, acting independently of its nuclear role as a component of the Holliday junction-resolving BLM-Top3α-RMI1-RMI2 (BTR) complex. Our data indicate that mtDNA replication termination occurs via a hemicatenane formed at the origin of H-strand replication and that Top3α is essential for resolving this structure. Decatenation is a prerequisite for separation of the segregating unit of mtDNA, the nucleoid, within the mitochondrial network. The importance of this process is highlighted in a patient with mitochondrial disease caused by biallelic pathogenic variants in TOP3A, characterized by muscle-restricted mtDNA deletions and chronic progressive external ophthalmoplegia (CPEO) plus syndrome. Our work establishes Top3α as an essential component of the mtDNA replication machinery and as the first component of the mtDNA separation machinery.

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.

Multipotent Basal Stem Cells, Maintained in Localized Proximal Niches, Support Directed Long-Ranging Epithelial Flows in Human Prostates.

Sporadic mitochondrial DNA mutations serve as clonal marks providing access to the identity and lineage potential of stem cells within human tissues. By combining quantitative clonal mapping with 3D reconstruction of adult human prostates, we show that multipotent basal stem cells, confined to discrete niches in juxta-urethral ducts, generate bipotent basal progenitors in directed epithelial migration streams. Basal progenitors are then dispersed throughout the entire glandular network, dividing and differentiating to replenish the loss of apoptotic luminal cells. Rare lineage-restricted luminal stem cells, and their progeny, are confined to proximal ducts and provide only minor contribution to epithelial homeostasis. In situ cell capture from clonal maps identified delta homolog 1 (DLK1) enrichment of basal stem cells, which was validated in functional spheroid assays. This study establishes significant insights into niche organization and function of prostate stem and progenitor cells, with implications for disease.

The Spectrum of Mitochondrial Ultrastructural Defects in Mitochondrial Myopathy.

Mitochondrial functions are intrinsically linked to their morphology and membrane ultrastructure. Characterizing abnormal mitochondrial structural features may thus provide insight into the underlying pathogenesis of inherited and acquired mitochondrial diseases. Following a systematic literature review on ultrastructural defects in mitochondrial myopathy, we investigated skeletal muscle biopsies from seven subjects with genetically defined mtDNA mutations. Mitochondrial ultrastructure and morphology were characterized using two complimentary approaches: transmission electron microscopy (TEM) and serial block face scanning EM (SBF-SEM) with 3D reconstruction. Six ultrastructural abnormalities were identified including i) paracrystalline inclusions, ii) linearization of cristae and abnormal angular features, iii) concentric layering of cristae membranes, iv) matrix compartmentalization, v) nanotunelling, and vi) donut-shaped mitochondria. In light of recent molecular advances in mitochondrial biology, these findings reveal novel aspects of mitochondrial ultrastructure and morphology in human tissues with implications for understanding the mechanisms linking mitochondrial dysfunction to disease.

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.

Complex mitochondrial DNA rearrangements in individual cells from patients with sporadic inclusion body myositis.

Mitochondrial DNA (mtDNA) rearrangements are an important cause of mitochondrial disease and age related mitochondrial dysfunction in tissues including brain and skeletal muscle. It is known that different mtDNA deletions accumulate in single cells, but the detailed nature of these rearrangements is still unknown. To evaluate this we used a complementary set of sensitive assays to explore the mtDNA rearrangements in individual cells from patients with sporadic inclusion body myositis, a late-onset inflammatory myopathy with prominent mitochondrial changes. We identified large-scale mtDNA deletions in individual muscle fibres with 20% of cytochrome c oxidase-deficient myofibres accumulating two or more mtDNA deletions. The majority of deletions removed only the major arc but ∼10% of all deletions extended into the minor arc removing the origin of light strand replication (OL) and a variable number of genes. Some mtDNA molecules contained two deletion sites. Additionally, we found evidence of mitochondrial genome duplications allowing replication and clonal expansion of these complex rearranged molecules. The extended spectrum of mtDNA rearrangements in single cells provides insight into the process of clonal expansion which is fundamental to our understanding of the role of mtDNA mutations in ageing and disease.