Ultrastructural Changes of Mitochondria in the Cultivated Skin Fibroblasts of Patients with Point Mutations in Mitochondrial DNA

Mitochondrial disorders represent a heterogeneous group of multisystem diseases with extreme variability in clinical phenotype. The diagnosis of mitochondrial disorders relies heavily on extensive biochemical and molecular analyses combined with morphological studies including electron microscopy. Although muscle is the tissue of choice for electron microscopic studies, the authors investigated cultivated human skin fibroblasts (HSF) harboring 3 different pathologic mtDNA mutations: 3243A > G, 8344A > G, 8993T > G. They addressed to the possibility of whether mtDNA mutations influence mitochondrial morphology in HSF and if ultrastructural changes of mitochondria may be used for differential diagnostics of mitochondrial disorders caused by mtDNA mutations. Ultrastructural analysis of patients' HSF revealed a heterogeneous mixture of mainly abnormal, partially swelling mitochondria with unusual and sparse cristae. The most characteristic cristal abnormalities were heterogeneity in size and shapes or their absence. Typical filamentous and branched mitochondria with numerous cristae as appeared in control HSF were almost not observed. In all lines of cultured HSF with various mtDNA mutations, similar ultrastructural abnormalities and severely changed mitochondrial interior were found, although no alterations in function and amount of OXPHOS were detected by routinely used biochemical methods in two lines of cultured HSF. This highlights the importance of morphological analysis, even in cultured fibroblasts, in diagnostics of mitochondrial disorders.     

Mitochondrial DNA Mutations and Mitochondrial Abnormalities in Dilated Cardiomyopathy

Mitochondrial (mt)DNA defects, both deletions and tRNA point mutations, have been associated with cardiomyopathies. The aim of the study was to determine the prevalence of pathological mtDNA mutations and to assess associated defects of mitochondrial enzyme activity in dilated cardiomyopathy (DCM) patients with ultrastructural abnormalities of cardiac mitochondria. In a large cohort of 601 DCM patients we performed conventional light and electron microscopy on endomyocardial biopsy samples. Cases with giant organelles, angulated, tubular, and concentric cristae, and crystalloid or osmiophilic inclusion bodies were selected for mtDNA analysis. Mutation screening techniques, automated DNA sequencing, restriction enzyme digestion, and densitometric assays were performed to identify mtDNA mutations, assess heteroplasmy, and quantify the amount of mutant in myocardial and blood DNA. Of 601 patients (16 to 63 years; mean, 43.5 ± 12.7 years), 85 had ultrastructural evidence of giant organelles, with abnormal cristae and inclusion bodies; 19 of 85 (22.35%) had heteroplasmic mtDNA mutations (9 tRNA, 5 rRNA, and 4 missense, one in two patients) that were not found in 111 normal controls and in 32 DCM patients without the above ultrastructural mitochondrial abnormalities. In all cases, the amount of mutant was higher in heart than in blood. In hearts of patients that later underwent transplantation, cytochrome c oxidase (Cox) activity was significantly lower in cases with mutations than in those without or controls (P = 0.0008). NADH dehydrogenase activity was only slightly reduced in cases with mutations (P = 0.0388), whereas succinic dehydrogenase activity did not significantly differ between DCM patients with mtDNA mutations and those without or controls. The present study represents the first attempt to detect a morphological, easily identifiable marker to guide mtDNA mutation screening. Pathological mtDNA mutations are associated with ultrastructurally abnormal mitochondria, and reduced Cox activity in a small subgroup of non-otherwise-defined, idiopathic DCMs, in which mtDNA defects may constitute the basis for, or contribute to, the development of congestive heart failure.     

Ultrastructural Changes of Mitochondria in the Skeletal Muscle of Patients with Amyotrophic Lateral Sclerosis

Functional defects and morphological changes of mitochondria have been reported to be in the skeletal muscle of patients with amyotrophic lateral sclerosis (ALS). Recent studies suggested that mitochondrial abnormalities are related to the pathogenesis of ALS. The purpose of this study is to evaluate the ultrastructural changes of muscle mitochondria in ALS patients. The authors examined 49 cases of diagnostic muscle biopsy samples with definite or probable ALS by electron microscopy. Of the 49 cases, 5 (10%) had ultrastructural abnormalities of muscle mitochondria, including giant mitochondria, paracrystalline inclusions, and abnormal cristae. These abnormal mitochondria were mainly observed among subsarcolemmal mitochondrial aggregates.     

Ultrastructural changes of mitochondria in the skeletal muscle of patients with amyotrophic lateral sclerosis

Functional defects and morphological changes of mitochondria have been reported to be in the skeletal muscle of patients with amyotrophic lateral sclerosis (ALS). Recent studies suggested that mitochondrial abnormalities are related to the pathogenesis of ALS. The purpose of this study is to evaluate the ultrastructural changes of muscle mitochondria in ALS patients. The authors examined 49 cases of diagnostic muscle biopsy samples with definite or probable ALS by electron microscopy. Of the 49 cases, 5 (10%) had ultrastructural abnormalities of muscle mitochondria, including giant mitochondria, paracrystalline inclusions, and abnormal cristae. These abnormal mitochondria were mainly observed among subsarcolemmal mitochondrial aggregates.     

Morphological Methods in the Diagnosis of Mitochondrial Encephalomyopathies: The Role of Electron Microscopy

Mitochondrial encephalomyopathies (MEs) encompass a heterogeneous group of disorders that frequently present a diagnostic challenge to clinicians. Historically, MEs were diagnosed by finding ragged red fibers in the muscle biopsy and confirmatory evidence was provided by the presence of numerical and/or ultrastructural abnormalities in mitochondria. In most centers diagnosis involves clinical evaluation and the morphological, histochemical, and biochemical investigation of a skeletal muscle biopsy. However, with the availability of mitochondrial DNA analysis, the necessity and role of morphological methods and, in particular, electron microscopy has been questioned. The aim of this study was to delineate the role of electron microscopy in the diagnosis of MEs.     

Morphological Methods in the Diagnosis of Mitochondrial Encephalomyopathies: The Role of Electron Microscopy

Mitochondrial encephalomyopathies (MEs) encompass a heterogeneous group of disorders that frequently present a diagnostic challenge to clinicians. Historically, MEs were diagnosed by finding ragged red fibers in the muscle biopsy and confirmatory evidence was provided by the presence of numerical and/or ultrastructural abnormalities in mitochondria. In most centers diagnosis involves clinical evaluation and the morphological, histochemical, and biochemical investigation of a skeletal muscle biopsy. However, with the availability of mitochondrial DNA analysis, the necessity and role of morphological methods and, in particular, electron microscopy has been questioned. The aim of this study was to delineate the role of electron microscopy in the diagnosis of MEs.     

Morphological methods in the diagnosis of mitochondrial encephalomyopathies: the role of electron microscopy

Mitochondrial encephalomyopathies (MEs) encompass a heterogeneous group of disorders that frequently present a diagnostic challenge to clinicians. Historically, MEs were diagnosed by finding ragged red fibers in the muscle biopsy and confirmatory evidence was provided by the presence of numerical and/or ultrastructural abnormalities in mitochondria. In most centers diagnosis involves clinical evaluation and the morphological, histochemical, and biochemical investigation of a skeletal muscle biopsy. However, with the availability of mitochondrial DNA analysis, the necessity and role of morphological methods and, in particular, electron microscopy has been questioned. The aim of this study was to delineate the role of electron microscopy in the diagnosis of MEs.     

A novel system for assigning the mode of inheritance in mitochondrial disorders using cybrids and rhodamine 6G.

When normal human cultured skin fibroblasts were treated with the fluorescent dye rhodamine 6G (R6G), there was a drastic reduction in numbers of intact mitochondria and electron transport chain enzyme activities, despite the fact that mitochondrial DNA (mtDNA) was still present in treated cells. We used this observation to develop a novel system for generating cybrids. When cultured skin fibroblast cells from a patient with the mitochondrial encephalopathy and ragged-red fibers (MERRF) syndrome harboring the A8344G mtDNA mutation and which showed a severe reduction in cytochrome c oxidase activity were treated with R6G and fused to enucleated HeLaCOT cells, the resulting cybrid clones showed recovery of cytochrome c oxidase activity, and were shown to have mtDNA derived solely from the HeLaCOT cell line. R6G has significant advantages over ethidium bromide in removing the mitochondrial elements from cultured cells, and the results reported here demonstrate that this strategy can be used to determine the origin of the genetic defect in patients with electron transport chain abnormalities.     

Organismal effects of mitochondrial dysfunction

Mitochondrial disease can lead to clinical abnormalities in any organ system. Both inherited and spontaneous disorders are known. The spontaneous forms can occur as a mitochondrial DNA (mtDNA) mutation early in embryogenesis or, later in life, as somatic mutations that accumulate with age. The inherited forms may arise from any of >100 characterized mutations in mtDNA or from >200 nuclear gene defects that affect proteins required for mitochondrial function. Most dividing cells survive and interact normally despite their mitochondrial defects. Thus post-mitotic, terminally differentiated cells are preferentially affected in mitochondrial disease. This review emphasizes cellular metabolic co-operation and the structural and biochemical diversity of mitochondria as the framework for understanding the clinical spectrum of mitochondrial disease. The principles of the mitochondrial clinical assessment scale I (MCAS-I) are presented to assist in the development of diagnostic spectra of mitochondrial disease.     

Morphological correlates of mitochondrial dysfunction in children

Morphological studies have traditionally played a major role in the study of adults with suspected mitochondrial diseases. Here we review their role in the investigation of paediatric patients. The morphological changes may be macroscopic, such as developmental abnormalities of the brain in pyruvate dehydrogenase deficiency, including ectopic inferior olives and the absence of corpus callosum and pyramids. Other changes are histological, such as rarefaction of the neuropil and endothelial prominence in Leigh syndrome, and spongiosis with neuronal loss and gliosis in Alpers disease. The ragged-red fibres typical of mitochondrial disease in adults are only rarely seen in skeletal muscle biopsies from children. On the other hand, dramatic ultrastructural changes involving the mitochondria may be seen in many organs, including the liver, heart and intestine. In Alpers and lethal infantile mitochondrial diseases, the hepatocytes show marked accumulation of small droplets of lipid alternating with densely packed mitochondria with pale matrix and loss of granules. These changes are associated with a marked decrease in respiratory chain enzyme activity in the liver, often without similar decrease in the skeletal muscle or fibroblasts. Enlarged mitochondria with concentric cristae are prominent in the cardiac myocytes in Barth syndrome. For the assessment of children with a suspected disorder of mitochondrial dysfunction, detailed morphological studies of the brain (at autopsy) and of biopsies (especially of the liver), including ultrastructural assessment of the mitochondria, can be a very useful preliminary investigation. The findings should then be correlated with the clinical features and used as a guide for further biochemical and molecular studies, preferably on multiple tissues.     

Altered Mitochondrial Anatomy of Trigeminal Ganglia Neurons in Diabetes

Neurons from sensory ganglia are exposed to oxidative attack in diabetes. Altered mitochondrial morphologies are due to impaired dynamics (fusion, fission) and to cristae remodeling. This study aimed to evaluate using transmission electron microscopy mitochondrial changes in diabetic trigeminal ganglia suggestive for ignition of apoptosis, in absence of “classical” morphological signs of apoptosis. We used samples of trigeminal ganglia (from six type 2 diabetes human donors and five streptozotocin (STZ)‐induced diabetic rats). In human diabetic samples we found three main distributions of mitochondria: (a) small “dark” normal mitochondria, seemingly resulted from fission processes; (b) small “dark” damaged mitochondria, with side‐vesiculations (single‐ and double‐coated), large matrix vesicles and cytosolic leakage of reactive species, mixed with larger “light” mitochondria, swollen, and with crystolysis; (c) prevailing “light” mitochondria. In STZ‐treated rats a type (c) distribution prevailed, except for nociceptive neurons where we found a different distribution: large and giant mitochondria, suggestive for impaired mitochondrial fission, mitochondrial fenestrations, matrix vesicles interconnected by lamellar cristae, and mitochondrial leakage into the cytosol. Thus, the ultrastructural pattern of mitochondria damage in diabetic samples of sensory neurons may provide clues on the initiation of intrinsic apoptosis, even if the classical morphological signs of apoptosis are not present. Further studies, combining use of biochemical and ultrastructural techniques, may allow a better quantification of the degree in which mitochondrial damage, with membrane alterations and cytosolic leaks, may be used as morphological signs suggesting the point‐of‐no return for apoptosis. Anat Rec, 299:1561–1570, 2016. © 2016 Wiley Periodicals, Inc.     

Analysis of Mitochondrial Network Morphology in Cultured Myoblasts from Patients with Mitochondrial Disorders

Mitochondrial morphology was studied in cultivated myoblasts obtained from patients with mitochondrial disorders, including CPEO, MELAS and TMEM70 deficiency. Mitochondrial networks and ultrastructure were visualized by fluorescence microscopy and transmission electron microscopy, respectively. A heterogeneous picture of abnormally sized and shaped mitochondria with fragmentation, shortening, and aberrant cristae, lower density of mitochondria and an increased number of “megamitochondria” were found in patient myoblasts. Morphometric Fiji analyses revealed different mitochondrial network properties in myoblasts from patients and controls. The small number of cultivated myoblasts required for semiautomatic morphometric image analysis makes this tool useful for estimating mitochondrial disturbances in patients with mitochondrial disorders.     

Comparison of lymphoblast mitochondria from normal subjects and patients with Barth syndrome using electron microscopic tomography

Barth syndrome (BTHS) is a mitochondrial disorder that is caused by mutations in the tafazzin gene, which affects phospholipid composition. To determine whether this defect leads to alterations in the internal three-dimensional organization of mitochondrial membranes, we applied electron microscopic tomography to lymphoblast mitochondria from BTHS patients and controls. Tomograms were formed from 50 and 150 nm sections of chemically fixed lymphoblasts and the data were used to manually segment volumes of relevant structural details. Normal lymphoblast mitochondria contained well-aligned, lamellar cristae with slot-like junctions to the inner boundary membrane. In BTHS, mitochondrial size was more variable and the total mitochondrial volume per cell increased mainly due to clusters of fragmented mitochondria inside nuclear invaginations. However, mitochondria showed reduced cristae density, less cristae alignment, and inhomogeneous cristae distribution. Three-dimensional reconstruction of BTHS mitochondria revealed zones of adhesion of the opposing inner membranes, causing obliteration of the intracrista space. We found small isolated patches of adhesion as well as extended adhesion zones, resulting in sheets of collapsed cristae packaged in multiple concentric layers. We also found large tubular structures (diameter 30-150 nm) that appeared to be derivatives of the adhesion zones. The data suggest that mitochondrial abnormalities of BTHS involve adhesions of inner mitochondrial membranes with subsequent collapse of the intracristae space.     

Late onset Leigh syndrome and ataxia due to a T to C mutation at bp 9,185 of mitochondrial DNA

A T-to-C missense mutation at nucleotide position 9,185 in the protein-coding ATP6 gene of the mitochondrial genome was present at high heteroplasmy in members of a Canadian family with Leigh syndrome with predominant ataxia and peripheral neuropathy. This mutation results in the substitution of a proline residue for an evolutionary-conserved leucine at position of amino acid 220 near the carboxyl terminus of the mitochondrial protein. The index patient and brother, who had an identical clinical presentation, had >90% mutant mtDNA in cultured skin fibroblasts, lymphocytes, and whole blood. Their mother and a maternal uncle, symptomatic with a peripheral neuropathy alone, had 86% and 85% heteroplasmy, respectively. Symptomatic maternal cousins with early onset revealed 90% and 91% mutant mtDNA in all tissues analyzed. Studies of lymphoblasts from the asymptomatic maternal grandmother and eldest brother of the proband were heteroplasmic for mutant mtDNA with 56% and 17%, respectively. Biochemical analysis demonstrated normal respiratory chain enzyme activity in muscle and fibroblasts, normal ATP synthesis, but reduced oligomycin-sensitive H(+)ATPase in cultured lymphoblast mitochondria. We propose that the 9,185T > C mtDNA mutation is pathogenic even though the initial phenotype is mild and the biochemical phenotype not easily detectable.     

The mitochondrial genome and psychiatric illness

Psychiatric disorders are a leading cause of morbidity and mortality, yet their underlying pathophysiology remains unclear. Searches for a genetic cause of bipolar disorder, schizophrenia, and major depressive disorder have yielded inconclusive results. There is increasing interest in the possibility that defects in the mitochondrial genome may play an important role in psychiatric illness. We undertook a review of the literature investigating mitochondria and adult psychiatric disorders. MEDLINE, PsycINFO, and EMBASE were searched from their inception through September 2011, and the reference lists of identified articles were reviewed for additional studies. While multiple lines of evidence, including clinical, genetic, ultrastructural, and biochemical studies, support the involvement of mitochondria in the pathophysiology of psychiatric illness, many studies have methodological limitations and their findings have not been replicated. Clinical studies suggest that psychiatric features can be prominent, and the presenting features of mitochondrial disorders. There is limited but inconsistent evidence for the involvement of mitochondrial DNA haplogroups and mitochondria-related nuclear gene polymorphisms, and for mitochondrial ultrastructural and biochemical abnormalities in psychiatric illness. The current literature suggests that mitochondrial dysfunction and mitochondrial genetic variations may play an important role in psychiatric disorders, but additional methodologically rigorous and adequately powered studies are needed before definitive conclusions can be drawn. ? 2012 Wiley Periodicals, Inc.     

Mutations in TIMM50 cause severe mitochondrial dysfunction by targeting key aspects of mitochondrial physiology

3‐Methylglutaconic aciduria (3‐MGA‐uria) syndromes comprise a heterogeneous group of diseases associated with mitochondrial membrane defects. Whole‐exome sequencing identified compound heterozygous mutations in TIMM50 (c.[341 G>A];[805 G>A]) in a boy with West syndrome, optic atrophy, neutropenia, cardiomyopathy, Leigh syndrome, and persistent 3‐MGA‐uria. A comprehensive analysis of the mitochondrial function was performed in fibroblasts of the patient to elucidate the molecular basis of the disease. TIMM50 protein was severely reduced in the patient fibroblasts, regardless of the normal mRNA levels, suggesting that the mutated residues might be important for TIMM50 protein stability. Severe morphological defects and ultrastructural abnormalities with aberrant mitochondrial cristae organization in muscle and fibroblasts were found. The levels of fully assembled OXPHOS complexes and supercomplexes were strongly reduced in fibroblasts from this patient. High‐resolution respirometry demonstrated a significant reduction of the maximum respiratory capacity. A TIMM50‐deficient HEK293T cell line that we generated using CRISPR/Cas9 mimicked the respiratory defect observed in the patient fibroblasts; notably, this defect was rescued by transfection with a plasmid encoding the TIMM50 wild‐type protein. In summary, we demonstrated that TIMM50 deficiency causes a severe mitochondrial dysfunction by targeting key aspects of mitochondrial physiology, such as the maintenance of proper mitochondrial morphology, OXPHOS assembly, and mitochondrial respiratory capacity.     

Role of mitochondrial mutations in cancer

A role for mitochondria in cancer causation has been implicated through identification of mutations in the mitochondrial DNA (mtDNA) and in nuclear-encoded mitochondrial genes. Although many mtDNA mutations were detected in common tumors, an unequivocal causal link between heritable mitochondrial abnormalities and cancer is provided only by the germ line mutations in the nuclear-encoded genes for succinate dehydrogenase (mitochondrial complex II) and fumarate hydratase (fumarase). The absence of evidence for highly penetrant tumors caused by inherited mtDNA mutations contrasts with the frequent occurrence of mtDNA mutations in many different tumor types. Thus, either the majority of diverse mtDNA mutations observed in tumors are not important for the process of carcinogenesis or that they play a common oncogenic role.     

Respiratory chain supercomplexes set the threshold for respiration defects in human mtDNA mutant cybrids

Mitochondrial DNA (mtDNA) mutations cause heterogeneous disorders in humans. MtDNA exists in multiple copies per cell, and mutations need to accumulate beyond a critical threshold to cause disease, because coexisting wild-type mtDNA can complement the genetic defect. A better understanding of the molecular determinants of functional complementation among mtDNA molecules could help us shedding some light on the mechanisms modulating the phenotypic expression of mtDNA mutations in mitochondrial diseases. We studied mtDNA complementation in human cells by fusing two cell lines, one containing a homoplasmic mutation in a subunit of respiratory chain complex IV, COX I, and the other a distinct homoplasmic mutation in a subunit of complex III, cytochrome b. Upon cell fusion, respiration is recovered in hybrids cells, indicating that mitochondria fuse and exchange genetic and protein materials. Mitochondrial functional complementation occurs frequently, but with variable efficiency. We have investigated by native gel electrophoresis the molecular organization of the mitochondrial respiratory chain in complementing hybrid cells. We show that the recovery of mitochondrial respiration correlates with the presence of supramolecular structures (supercomplexes) containing complexes I, III and IV. We suggest that critical amounts of complexes III or IV are required in order for supercomplexes to form and provide mitochondrial functional complementation. From these findings, supercomplex assembly emerges as a necessary step for respiration, and its defect sets the threshold for respiratory impairment in mtDNA mutant cells.     

Enhanced ROS production and antioxidant defenses in cybrids harbouring mutations in mtDNA

It has been suggested that mutations in mitochondrial DNA (mtDNA) can produce an increase in reactive oxygen species (ROS) and that this can play a major role in the pathogenic mechanisms of mitochondrial encephalomyopathies. Many studies exist using electron transport chain (ETC) inhibitors, however there are only a few studies that examine ROS production associated with mutations in the mtDNA. To investigate this issue, we have studied ROS production, antioxidant defences and oxidative damage to lipids and proteins in transmitochondrial cybrids carrying different mtDNA mutations. Here, we report that two different mutant cell lines carrying mutations in their mitochondrial tRNA genes (A3243G in tRNA LeuUUR and A8344G in tRNA Lys) showed an increased ROS production with a parallel increase in the antioxidant enzyme activities, which may protect cells from oxidative damage in our experimental conditions (no overt oxidative damage to lipids and proteins has been observed). In contrast, cytochrome c oxidase (COX) mutant cybrids (carrying the stop-codon mutation G6930A in the COXI gene) showed neither an increase in ROS production nor elevation of antioxidant enzyme activities or oxidative damage. These results suggest that the specific location of mutations in mtDNA has a strong influence on the phenotype of the antioxidant response. Therefore, this issue should be carefully considered when antioxidant therapies are investigated in patients with mitochondrial disorders.