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Background Aging in postmitotic tissues is associated with clonal expansion of somatic mitochondrial deletions, the origin of which is not well understood. Such deletions are often flanked by direct nucleotide repeats, but this alone does not fully explain their distribution. Here, we hypothesized that the close proximity of direct repeats on single-stranded mitochondrial DNA (mtDNA) might play a role in the formation of deletions. Results By analyzing human mtDNA deletions in the major arc of mtDNA, which is single-stranded during replication and is characterized by a high number of deletions, we found a non-uniform distribution with a “hot spot” where one deletion breakpoint occurred within the region of 6–9 kb and another within 13–16 kb of the mtDNA. This distribution was not explained by the presence of direct repeats, suggesting that other factors, such as the spatial proximity of these two regions, can be the cause. In silico analyses revealed that the single-stranded major arc may be organized as a large-scale hairpin-like loop with a center close to 11 kb and contacting regions between 6–9 kb and 13–16 kb, which would explain the high deletion activity in this contact zone. The direct repeats located within the contact zone, such as the well-known common repeat with a first arm at 8470–8482 bp (base pair) and a second arm at 13,447–13,459 bp, are three times more likely to cause deletions compared to direct repeats located outside of the contact zone. A comparison of age- and disease-associated deletions demonstrated that the contact zone plays a crucial role in explaining the age-associated deletions, emphasizing its importance in the rate of healthy aging. Conclusions Overall, we provide topological insights into the mechanism of age-associated deletion formation in human mtDNA, which could be used to predict somatic deletion burden and maximum lifespan in different human haplogroups and mammalian species.

To report the retinal phenotype and the associated genetic and systemic findings in patients with mitochondrial disease.Retrospective case series.Twenty-three patients with retinopathy and mitochondrial disease, including chronic progressive external ophthalmoplegia (CPEO), maternally inherited diabetes and deafness (MIDD), mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), Kearns-Sayre syndrome, neuropathy, ataxia, and retinitis pigmentosa (NARP) syndrome, and other systemic manifestations.Review of case notes, retinal imaging, electrophysiologic assessment, molecular genetic testing including protein modeling, and histologic analysis of muscle biopsy.Phenotypic characteristics of mitochondrial retinopathy.Genetic testing identified sporadic large-scale mitochondrial DNA deletions and variants in MT-TL1, MT-ATP6, MT-TK, MT-RNR1, or RRM2B. Based on retinal imaging, 3 phenotypes could be differentiated: type 1 with mild, focal pigmentary abnormalities; type 2 characterized by multifocal white-yellowish subretinal deposits and pigment changes limited to the posterior pole; and type 3 with widespread granular pigment alterations. Advanced type 2 and 3 retinopathy presented with chorioretinal atrophy that typically started in the peripapillary and paracentral areas with foveal sparing. Two patients exhibited a different phenotype: 1 revealed an occult retinopathy, and the patient with RRM2B-associated retinopathy showed no foveal sparing, no severe peripapillary involvement, and substantial photoreceptor atrophy before loss of the retinal pigment epithelium. Two patients with type 1 disease showed additional characteristics of mild macular telangiectasia type 2. Patients with type 1 and mild type 2 or 3 disease demonstrated good visual acuity and no symptoms associated with the retinopathy. In contrast, patients with advanced type 2 or 3 disease often reported vision problems in dim light conditions, reduced visual acuity, or both. Short-wavelength autofluorescence usually revealed a distinct pattern, and near-infrared autofluorescence may be severely reduced in type 3 disease. The retinal phenotype was key to suspecting mitochondrial disease in 11 patients, whereas 12 patients were diagnosed before retinal examination.Different types of mitochondrial retinopathy show characteristic features. Even in absence of visual symptoms, their recognition may facilitate the often challenging and delayed diagnosis of mitochondrial disease, in particular in patients with mild or nebulous multisystem disease.

Additional file 1. Distribution of the perfect direct repeatsand deletions from MitoBreakin the major arc.

Additional file 2. The third principal component scores, associated with aging-related deletions of healthy samples from a paper [36]. The contact, marked by the pink square, is characterized by the increased scores.

Additional file 4. Hi-C contact matrix of mtDNA obtained from the human olfactory epithelium autopsies. The top row represents two contact matrixes from covid patients, middle and bottom rows represent the contact matrices from controls. Solid white squares mark the potential contact zone. Dotted white rectangles mark the contacts, emphasizing the circularity of AQmtDNA.

Additional file 3. Hi-C contact matrix of mtDNA obtained from the human lymphoblastoid cells. Dotted squares mark the potential contact zones. Ovals mark the contacts, emphasizing the circularity of mtDNA.

OBJECTIVE: Levetiracetam (LEV) is an effective antiseizure medicine, but 10%-20% of people treated with LEV report psychiatric side-effects, and up to 1% may have psychotic episodes. Pharmacogenomic predictors of these adverse drug reactions (ADRs) have yet to be identified. We sought to determine the contribution of both common and rare genetic variation to psychiatric and behavioral ADRs associated with LEV. METHODS: This case-control study compared cases of LEV-associated behavioral disorder (n = 149) or psychotic reaction (n = 37) to LEV-exposed people with no history of psychiatric ADRs (n = 920). All samples were of European ancestry. We performed genome-wide association study (GWAS) analysis comparing those with LEV ADRs to controls. We estimated the polygenic risk scores (PRS) for schizophrenia and compared cases with LEV-associated psychotic reaction to controls. Rare variant burden analysis was performed using exome sequence data of cases with psychotic reactions (n = 18) and controls (n = 122). RESULTS: Univariate GWAS found no significant associations with either LEV-associated behavioural disorder or LEV-psychotic reaction. PRS analysis showed that cases of LEV-associated psychotic reaction had an increased PRS for schizophrenia relative to contr ols (p = .0097, estimate = .4886). The rare-variant analysis found no evidence of an increased burden of rare genetic variants in people who had experienced LEV-associated psychotic reaction relative to controls. SIGNIFICANCE: The polygenic burden for schizophrenia is a risk factor for LEV-associated psychotic reaction. To assess the clinical utility of PRS as a predictor, it should be tested in an independent and ideally prospective cohort. Larger sample sizes are required for the identification of significant univariate common genetic signals or rare genetic signals associated with psychiatric LEV ADRs.

In past decades, the identification of genes involved in epileptic disorders has grown exponentially. The pace of gene identification in epileptic disorders began to accelerate in the late 2000s, driven by new technologies such as molecular cytogenetics and next-generation sequencing (NGS). These technologies have also been applied to genetic diagnostics, with different configurations, such as gene panels, whole-exome sequencing and whole-genome sequencing. The clinician must be aware that any technology has its limitations and complementary techniques must still be used to establish a diagnosis for specific diseases. In addition, increasing the amount of genetic information available in a larger patient sample also increases the need for rigorous interpretation steps, when taking into account the clinical, electroclinical, and when available, functional data. Local, multidisciplinary discussions have proven valuable in difficult diagnostic situations, especially in cases where precision medicine is being considered. They also serve to improve genetic counseling in complex situations. In this article, we will briefly review the genetic basis of rare and common epilepsies, the current strategies used for molecular diagnosis, including their limitations, and some pitfalls for data interpretation, in the context of etiological diagnosis and genetic counseling.

Here, we report a consanguineous family harboring a novel homozygous frame-shift mutation in

Background and ObjectivesWe report the pathogenic sequence variant m.5789T>C in the anticodon stem of the mitochondrial tRNA for cysteine as a novel cause of neuropathy, ataxia, and retinitis pigmentosa (NARP), which is usually associated with pathogenic variants in the MT-ATP6 gene.MethodsTo address the correlation of oxidative phosphorylation deficiency with mutation loads, we performed genotyping on single laser-dissected skeletal muscle fibers. Stability of the mitochondrial tRNACys was investigated by Northern blotting. Accompanying deletions of the mitochondrial genome were detected by long-range PCR and their breakpoints were determined by sequencing of single-molecule amplicons.ResultsThe sequence variant m.5789T>C, originating from the patient's mother, decreases the stability of the mitochondrial tRNA for cysteine by disrupting the anticodon stem, which subsequently leads to a combined oxidative phosphorylation deficiency. In parallel, we observed a prominent cluster of low-abundance somatic deletions with breakpoints in the immediate vicinity of the m.5789T>C variant. Strikingly, all deletion-carrying mitochondrial DNA (mtDNA) species, in which the corresponding nucleotide position was not removed, harbored the mutant allele, and none carried the wild-type allele.DiscussionIn addition to providing evidence for the novel association of a tRNA sequence alteration with NARP syndrome, our observations support the hypothesis that single nucleotide changes can lead to increased occurrence of site-specific mtDNA deletions through the formation of an imperfect repeat. This finding might be relevant for understanding mechanisms of deletion generation in the human mitochondrial genome.

Brain : a journal of neurology (2022). doi:10.1093/brain/awac119, Published by Oxford Univ. Press, Oxford

Chronic progressive external ophthalmoplegia (CPEO) is a frequent clinical manifestation of disorders caused by pathogenic mitochondrial DNA mutations. However, for diagnostic purposes skeletal muscle tissue is used, since extraocular muscle tissue is usually not available for work-up. In the present study we aimed to identify causative factors that are responsible for extraocular muscle to be primarily affected in CPEO. We performed comparative histochemical and molecular genetic analyses of extraocular muscle and skeletal muscle single fibers in a case of isolated CPEO caused by the heteroplasmic m.5667G>A mutation in the mitochondrial tRNAAsn gene (MT-TN). Histochemical analyses revealed higher proportion of cytochrome c oxidase deficient fibers in extraocular muscle (41%) compared to skeletal muscle (10%). However, genetic analyses of single fibers revealed no significant difference either in the mutation loads between extraocular muscle and skeletal muscle cytochrome c oxidase deficient single fibers (extraocular muscle 86% ± 4.6%; skeletal muscle 87.8 %± 5.7%, p = 0.246) nor in the mutation threshold (extraocular muscle 74% ± 3%; skeletal muscle 74% ± 4%). We hypothesize that higher proportion of cytochrome c oxidase deficient fibers in extraocular muscle compared to skeletal muscle might be due to facilitated segregation of the m.5667G>A mutation into extraocular muscle, which may explain the preferential ocular manifestation and clinically isolated CPEO.

Individuals withWe identified novelThese findings implicate

BackgroundKCNC2 encodes a member of the shaw-related voltage-gated potassium channel family (KV3.2), which are important for sustained high-frequency firing and optimized energy efficiency of action potentials in the brain.MethodsIndividuals with KCNC2 variants detected by exome sequencing were selected for clinical, further genetic and functional analysis. The cases were referred through clinical and research collaborations in our study. Four de novo variants were examined electrophysiologically in Xenopus laevis oocytes.ResultsWe identified novel KCNC2 variants in 27 patients with various forms of epilepsy. Functional analysis demonstrated gain-of-function in severe and loss-of-function in milder phenotypes as the underlying pathomechanisms with specific response to valproic acid.ConclusionThese findings implicate KCNC2 as a novel causative gene for epilepsy emphasizing the critical role of KV3.2 in the regulation of brain excitability with an interesting genotype-phenotype correlation and a potential concept for precision medicine.

Leber's hereditary optic neuropathy (LHON) is the most frequent mitochondrial disease and was the first to be genetically defined by a point mutation in mitochondrial DNA (mtDNA). A molecular diagnosis is achieved in up to 95% of cases, the vast majority of which are accounted for by 3 mutations within mitochondrial complex I subunit-encoding genes in the mtDNA (mtLHON). Here, we resolve the enigma of LHON in the absence of pathogenic mtDNA mutations. We describe biallelic mutations in a nuclear encoded gene, DNAJC30, in 33 unsolved patients from 29 families and establish an autosomal recessive mode of inheritance for LHON (arLHON), which to date has been a prime example of a maternally inherited disorder. Remarkably, all hallmarks of mtLHON were recapitulated, including incomplete penetrance, male predominance, and significant idebenone responsivity. Moreover, by tracking protein turnover in patient-derived cell lines and a DNAJC30-knockout cellular model, we measured reduced turnover of specific complex I N-module subunits and a resultant impairment of complex I function. These results demonstrate that DNAJC30 is a chaperone protein needed for the efficient exchange of complex I subunits exposed to reactive oxygen species and integral to a mitochondrial complex I repair mechanism, thereby providing the first example to our knowledge of a disease resulting from impaired exchange of assembled respiratory chain subunits.

In this work we studied molecular and functional effects of the loss of the smallest nuclear encoded subunit of cytochrome c oxidase COX8A in fibroblasts from a patient with a homozygous splice site mutation and in CRISPR/Cas9 genome-edited HEK293T cells. In both cellular model systems, between 20 to 30% of the residual enzymatic activity of cytochrome c oxidase (COX) was detectable. In immunoblots of BN-PAGE separated mitochondria from both cellular models almost no monomers and dimers of the fully assembled COX could be visualized. Interestingly, supercomplexes of COX formed with complex III and also with complexes I and III retained considerable immunoreactivity, while nearly no immunoreactivity attributable to subassemblies was found. That indicates that COX lacking subunit 8A is stabilized in supercomplexes, while monomers and dimers are rapidly degraded. With transcriptome analysis by 3'-RNA sequencing we failed to detect in our cellular models of COX8A deficiency transcriptional changes of genes involved in the mitochondrial unfolded protein response (mtUPR) and the integrated stress response (ISR). Thus, our data strongly suggest that the smallest subunit of cytochrome c oxidase COX8A is required for maintenance of the structural stability of COX monomers and dimers.

The variation in the mutational spectrum of the mitochondrial genome (mtDNA) among species is not well understood. Recently, we demonstrated an increase in A>G substitutions on a heavy chain (hereafter AH>GH) of mtDNA in aged mammals, interpreting it as a hallmark of age-related oxidative damage. In this study, we hypothesized that the occurrence of AH>GHsubstitutions may depend on the level of aerobic metabolism, which can be inferred from an organism’s body temperature. To test this hypothesis, we used body temperature in endotherms and environmental temperature in ectotherms as proxies for metabolic rate and reconstructed mtDNA mutational spectra for 1350 vertebrate species. Our results showed that temperature was associated with increased rates of AH>GHand asymmetry of AH>GHin different species of ray-finned fishes and within geographically distinct clades of European anchovy. Analysis of nucleotide composition in the most neutral synonymous sites of fishes revealed that warm-water species were expectedly more A-poor and G-rich compared to cold-water species. Finally, we extended our analyses to all vertebrates and observed higher AH>GHand increased asymmetry of AH>GHin warm-blooded (mammals and birds) compared to cold-blooded (Actinopterygii, amphibia, reptilia) vertebrate classes. Overall, our findings suggest that temperature, through its influence on metabolism and oxidative damage, shapes the mutational properties and nucleotide content of the mtDNA in all vertebrates.

Introduction: We aimed to investigate whether sonographic peripheral cross-sectional nerve area (CSA) and progranulin (PGRN), a neuritic growth factor, are related to each other and whether they interact to predict clinical and paraclinical measures in amyotrophic lateral sclerosis (ALS). Methods: We included 55 ALS patients who had forearm median and ulnar nerve CSA, cerebrospinal fluid (CSF) PGRN and serum PGRN measures available. CSF PGRN was normalized against the CSF/serum albumin ratio (Qalb). Using age, sex, height and weight adjusted general linear models we examined CSA x CSF PGRN interaction effects on various measures. Results: There was a medium-effect size inverse relationship between CSA and CSF PGRN, but not between CSA and serum PGRN. Lower CSA values together with higher CSF PGRN levels were linked to smaller motor amplitudes. Discussion: In ALS, the constellation of peripheral nerve atrophy together with higher CSF PGRN levels indicates pronounced axonal damage. This article is protected by copyright. All rights reserved.

Increased oxidative stress has been widely implicated in the pathogenesis in various forms of human epilepsy. Here, we report a homozygous mutation in TXNRD1 (thioredoxin reductase 1) in a family with genetic generalized epilepsy. TXNRD1 is an essential selenium-containing enzyme involved in detoxification of reactive oxygen species (ROS) and redox signaling. The TXNRD1 mutation p.Pro190Leu affecting a highly conserved amino acid residue was identified by whole-exome sequencing of blood DNA from the index patient. The detected mutation and its segregation within the family- all siblings of the index patient were homozygous and the parents heterozygous-were confirmed by Sanger sequencing. TXNRD1 activity was determined in subcellular fractions from a skeletal muscle biopsy and skin fibroblasts of the index patient and the expression levels of the mutated protein were assessed by Se-75 labeling and Western blot analysis. As result of the mutation, the activity of TXNRD1 was reduced in the patient's fibroblasts and skeletal muscle (to 34 +/- 3% and 16 +/- 8% of controls, respectively). In fibroblasts, we detected reduced Se-75-labeling of the enzyme (41 +/- 3% of controls). An in-depth in vitro kinetic analysis of the recombinant mutated TXNRD1 indicated 30-40% lowered k(cat)/Se values. Therefore, a reduced activity of the enzyme in the patient's tissue samples is explained by (i) lower enzyme turnover and (ii) reduced abundance of the mutated enzyme as confirmed by Western blotting and Se-75 labeling. The mutant fibroblasts were also found to be less resistant to a hydrogen peroxide challenge. Our data agree with a potential role of insufficient ROS detoxification for disease manifestation in genetic generalized epilepsy.

A recent report by Luo et al (2018) in PNAS (DOI:10.1073/pnas.1810946115) presented evidence of biparental inheritance of mitochondrial DNA. The pattern of inheritance, however, resembled that of a nuclear gene. The authors explained this peculiarity with Mendelian segregation of a faulty gatekeeper gene that permits survival of paternal mtDNA in the oocyte. Three other groups (Vissing, 2019; Lutz-Bonengel and Parson, 2019; Salas et al, 2019), however, posited the observation was an artifact of inheritance of mtDNA nuclear pseudogenes (NUMTs), present in the father’s nuclear genome. We present justification that both interpretations are incorrect, but that the original authors did, in fact, observe biparental inheritance of mtDNA. Our alternative model assumes that because of initially low paternal mtDNA copy number these copies are randomly partitioned into nascent cell lineages. The paternal mtDNA haplotype must have a selective advantage, so ‘seeded’ cells will tend to proceed to fixation of the paternal haplotype in the course of development. We use modeling to emulate the dynamics of paternal genomes and predict their mode of inheritance and distribution in somatic tissue. The resulting offspring is a mosaic of cells that are purely maternal or purely paternal – including in the germline. This mosaicism explains the quasi-Mendelian segregation of the paternal mDNA. Our model is based on known aspects of mtDNA biology and explains all of the experimental observations outlined in Luo et. al., including maternal inheritance of the grand-paternal mtDNA.

Aging in postmitotic tissues is associated with clonal expansion of somatic mitochondrial deletions, the origin of which is not well understood. Deletions in mitochondrial DNA (mtDNA) are often flanked by direct nucleotide repeats, but this alone does not fully explain their distribution. Here, we hypothesized that the close proximity of direct repeats on single-stranded DNA might play a role in the formation of deletions. By analyzing human mtDNA deletions in the major arc of mtDNA, which is single-stranded during replication and is characterized by a high number of deletions, we found a non-uniform distribution with a "hot spot" where one deletion breakpoint occurred within the region of 6-9kb and another within 13-16kb of the mtDNA. This distribution was not explained by the presence of direct repeats, suggesting that other factors, such as the spatial proximity of these two regions can be the cause. In silico analyses revealed that the single-stranded major arc may be organized as a large-scale hairpin-like loop with a center close to 11kb and contacting regions between 6-9 kb and 13-16 kb, which would explain the high deletion activity in this contact zone. The direct repeats located within the contact zone, such as the well-known common repeat with a first arm at 8470-8482 bp and a second arm at 13447-13459 bp, are three times more likely to cause deletions compared to direct repeats located outside of the contact zone. An analysis of age- and disease-associated deletions demonstrated that the contact zone plays a crucial role in explaining the age-associated deletions, emphasizing its importance in the rate of healthy aging. Overall, we provide topological insights into the mechanism of age-associated deletion formation in human mtDNA, which could be used to predict somatic deletion burden and maximum lifespan in different human haplogroups and mammalian species.

Mutational spectrum of the mitochondrial genome (mtDNA) does not resemble signatures of any known mutagens and variation in mtDNA mutational spectra between different tissues and organisms is still incomprehensible. Since mitochondria is tightly involved in aerobic energy production, it is expected that mtDNA mutational spectra may be affected by the oxidative damage which is increasing with cellular and organismal aging. However, the well-documented mutational signature of the oxidative damage, G>T substitutions, is typical only for the nuclear genome while it is extremely rare and age-independent in mtDNA. Thus it is still unclear if there is a mitochondria - specific mutational signature of the oxidative damage. Here, reconstructing mtDNA mutational spectra for human cancers originated from 21 tissues with various cell turnover rate, human oocytes fertilized at different ages, and 424 mammalian species with variable generation length which is a proxy for oocyte age, we observed that the frequency of AH>GHsubstitutions (H- heavy chain notation) is positively correlated with cellular and organismal longevity. Moreover, this mutational bias from AHto GHaffects nucleotide content at the fourfold degenerative synonymous positions leading to a deficit of AHand excess of GH, which is especially pronounced in long-lived mammals. Taking into account additionally, that AH>GHis sensitive to time being single stranded during mtDNA asynchronous replication and A>G is associated with oxidative damage of single-stranded DNA in recent bacterial experiments we propose that AH>GHis a mutational signature of oxidative damage in mtDNA.

Juvenile myoclonic epilepsy (JME) is a common syndrome of genetic generalized epilepsies (GGEs). Linkage and association studies suggest that the gene encoding the bromodomain-containing protein 2 (BRD2) may increase risk of JME. The present methylation and association study followed up a recent report highlighting that the BRD2 promoter CpG island (CpG76) is differentially hypermethylated in lymphoblastoid cells from Caucasian patients with JME compared to patients with other GGE subtypes and unaffected relatives. In contrast, we found a uniform low average percentage of methylation (

Juvenile myoclonic epilepsy (JME) is a common syndrome of genetic generalized epilepsies (GGEs). Linkage and association studies suggest that the gene encoding the bromodomain-containing protein 2 (BRD2) may increase risk of JME. The present methylation and association study followed up a recent report highlighting that the BRD2 promoter CpG island (CpG76) is differentially hypermethylated in lymphoblastoid cells from Caucasian patients with JME compared to patients with other GGE subtypes and unaffected relatives. In contrast, we found a uniform low average percentage of methylation (

Secondary mitochondrial dysfunction is a feature in a wide variety of human protein aggregate diseases caused by mutations in different proteins, both in the central nervous system and in striated muscle. The functional relationship between the expression of a mutated protein and mitochondrial dysfunction is largely unknown. In particular, the mechanism how this dysfunction drives the disease process is still elusive. To address this issue for protein aggregate myopathies, we performed a comprehensive, multi-level analysis of mitochondrial pathology in skeletal muscles of human patients with mutations in the intermediate filament protein desmin and in muscles of hetero- and homozygous knock-in mice carrying the R349P desmin mutation. We demonstrate that the expression of mutant desmin causes disruption of the extrasarcomeric desmin cytoskeleton and extensive mitochondrial abnormalities regarding subcellular distribution, number and shape. At the molecular level, we uncovered changes in the abundancy and assembly of the respiratory chain complexes and supercomplexes. In addition, we revealed a marked reduction of mtDNA- and nuclear DNA-encoded mitochondrial proteins in parallel with large-scale deletions in mtDNA and reduced mtDNA copy numbers. Hence, our data demonstrate that the expression of mutant desmin causes multi-level damage of mitochondria already in early stages of desminopathies. Electronic supplementary material The online version of this article (doi:10.1007/s00401-016-1592-7) contains supplementary material, which is available to authorized users.

In this review, we describe key signaling pathways regulating potassium channels present in the inner mitochondrial membrane. The signaling cascades covered here include phosphorylation, redox reactions, modulation by calcium ions and nucleotides. The following types of potassium channels have been identified in the inner mitochondrial membrane of various tissues: ATP-sensitive, Ca2+-activated, voltage-gated and two-pore domain potassium channels. The direct roles of these channels involve regulation of mitochondrial respiration, membrane potential and synthesis of reactive oxygen species (ROS). Changes in channel activity lead to diverse pro-life and pro-death responses in different cell types. Hence, characterizing the signaling pathways regulating mitochondrial potassium channels will facilitate understanding the physiological role of these proteins. Additionally, we describe in this paper certain regulatory mechanisms, which are unique to mitochondrial potassium channels.

Carbon monoxide (CO) is an endogenously synthesized gaseous mediator and is involved in the regulation of numerous physiological processes. Mitochondria, in which hemoproteins are abundant, are among the targets for CO action. Large-conductance calcium-activated (mitoBKCa) channels in the inner mitochondrial membrane share multiple biophysical similarities with the BKCa channels of the plasma membrane and could be a potential target for CO. To test this hypothesis, the activity of the mitoBKCa channels in human astrocytoma U-87 MG cell mitochondria was assessed with the patch-clamp technique. The effects of CO-releasing molecules (CORMs), such as CORM-2, CORM-401, and CORM-A1, were compared to the application of a CO-saturated solution to the mitoBKCa channels in membrane patches. The applied CORMs showed pleiotropic effects including channel inhibition, while the CO-containing solution did not significantly modulate channel activity. Interestingly, CO applied to the mitoBKCa channels, which were inhibited by exogenously added heme, stimulated the channel. To summarize, our findings indicate a requirement of heme binding to the mitoBKCa channel for channel modulation by CO and suggest that CORMs might have complex unspecific effects on mitoBKCa channels.

Each model used in the work referred to by Rutter et al. (1) addressed certain aspects of mitochondrial biology, and together, they fully support the conclusions made. Please note that we describe Ca2+-mediated regulation of oxidative phosphorylation (OXPHOS) fluxes (2, 3) and do not question Ca2+-responsiveness of pyruvate dehydrogenase en-zyme activity (4). To address concerns such as those raised by Rutter et al. (1), we studied glutamate/malate-dependent OXPHOS in the absence of exogenous pyruvate in mitochondria, omitted pyruvate from cell experiments, and implemented the working rat heart model perfused by Krebs–Henseleit (glucose) buffer. This unequivocally demonstrates in a broad range of models that MAS (malate-aspartate shuttle) inhibition induces a state of mitochondrial pyruvate starvation (3).An unresolved observation is that mitochondria of MCU knockout mice show negligible activity of Ca2+-uptake (5), which we confirm (3). We attributed this activity to residual expression of wild-type Mcu transcripts (3) as the result of a rare event of gene-trap excision during mRNA splicing, since this activity was sensitive to ruthenium red, an inhibitor of the MCU. Besides, please also note the low MCU Ca2+ affinity (6). In vivo, the endoplasmic reticulum is thought to facilitate the generation of microcompartments of high Ca2+ concentration to allow Ca2+ uptake via MCU (6). This mechanism is compromised in MCU knockout mice and can be ruled out in isolated mitochondria. Thus, our data support the notion that, depending on tissue, model system and pathophysiological status, a combination of mechanisms (e.g., mitochondrial gas pedal and MCU) control OXPHOS substrate supply.Notably, the viability of MCU knockout mice (3, 5), albeit living in a laboratory cage, indicates that MCU-dependent pathways are dispensable for a sedentary life. It remains interestingto elucidate, however, why MCU-dependent activation of matrix dehydrogenases is indispensable for high activity states (7) and how this may allow stressful life in the wild.

Chronic progressive external ophthalmoplegia (CPEO) is a frequent clinical manifestation of disorders caused by pathogenic mitochondrial DNA mutations. However, for diagnostic purposes skeletal muscle tissue is used, since extraocular muscle tissue is usually not available for work-up. In the present study we aimed to identify causative factors that are responsible for extraocular muscle to be primarily affected in CPEO. We performed comparative histochemical and molecular genetic analyses of extraocular muscle and skeletal muscle single fibers in a case of isolated CPEO caused by the heteroplasmic m.5667GA mutation in the mitochondrial tRNA

Isolated cytochrome c oxidase (complex IV) deficiency is one of the most frequent respiratory chain defects in humans and is usually caused by mutations in proteins required for assembly of the complex. Mutations in nuclear-encoded structural subunits are very rare. In a patient with Leigh-like syndrome presenting with leukodystrophy and severe epilepsy, we identified a homozygous splice site mutation in COX8A, which codes for the ubiquitously expressed isoform of subunit VIII, the smallest nuclear-encoded subunit of complex IV. The mutation, affecting the last nucleotide of intron 1, leads to aberrant splicing, a frame-shift in the highly conserved exon 2, and decreased amount of the COX8A transcript. The loss of the wild-type COX8A protein severely impairs the stability of the entire cytochrome c oxidase enzyme complex and manifests in isolated complex IV deficiency in skeletal muscle and fibroblasts, similar to the frequent c.845_846delCT mutation in the assembly factor SURF1 gene. Stability and activity of complex IV could be rescued in the patient’s fibroblasts by lentiviral expression of wild-type COX8A. Our findings demonstrate that COX8A is indispensable for function of human complex IV and its mutation causes human disease.

Seizures are often the key manifestation of neurological diseases caused by pathogenic mutations in 169 of the genes that have so far been identified to affect mitochondrial function. Mitochondria are the main producers of ATP needed for normal electrical activities of neurons and synaptic transmission. Additionally, they have a central role in neurotransmitter synthesis, calcium homoeostasis, redox signalling, production and modulation of reactive oxygen species, and neuronal death. Hypotheses link mitochondrial failure to seizure generation through changes in calcium homoeostasis, oxidation of ion channels and neurotransmitter transporters by reactive oxygen species, a decrease in neuronal plasma membrane potential, and reduced network inhibition due to interneuronal dysfunction. Seizures, irrespective of their origin, represent an excessive acute energy demand in the brain. Accordingly, secondary mitochondrial dysfunction has been described in various epileptic disorders, including disorders that are mainly of non-mitochondrial origin. An understanding of the reciprocal relation between mitochondrial dysfunction and epilepsy is crucial to select appropriate anticonvulsant treatment and has the potential to open up new therapeutic approaches in the subset of epileptic disorders caused by mitochondrial dysfunction.

In the recent years, several neurological syndromes related to defects of the glucose transporter type 1 (Glut1) have been descried. They include the glucose transporter deficiency syndrome (Glut1-DS) as the most severe form, the paroxysmal exertion-induced dyskinesia (FED), a form of spastic paraparesis (CSE) as well as the childhood (CAE) and the early-onset absence epilepsy (EOAE). Glut1, encoded by the gene SLC2A1, is the most relevant glucose transporter in the brain. All Glut1 syndromes respond well to a ketogenic diet (KD) and most of the patients show a rapid seizure control. Ketogenic Diet developed to an established treatment for other forms of pharmaco-resistant epilepsies. Since we were interested in the question if those patients might have an underlying Glut1 defect, we sequenced SLC2A1 in a cohort of 28 patients with different forms of pharmaco-resistant epilepsies responding well to a KD. Unfortunately, we could not detect any mutations in SLC2A1. The exact action mechanisms of MD in pharmaco-resistant epilepsy are not well understood, but bypassing the Glut1 transporter seems not to play an important role. (C) 2015 Elsevier B.V. All rights reserved.

MGME1, also known as Ddk1 or C20orf72, is a mitochondrial exonuclease found to be involved in the processing of mitochondrial DNA (mtDNA) during replication. Here, we present detailed insights on the role of MGME1 in mtDNA maintenance. Upon loss of MGME1, elongated 7S DNA species accumulate owing to incomplete processing of 5' ends. Moreover, an 11-kb linear mtDNA fragment spanning the entire major arc of the mitochondrial genome is generated. In contrast to control cells, where linear mtDNA molecules are detectable only after nuclease S1 treatment, the 11-kb fragment persists in MGME1-deficient cells. In parallel, we observed characteristic mtDNA duplications in the absence of MGME1. The fact that the breakpoints of these mtDNA rearrangements do not correspond to either classical deletions or the ends of the linear 11-kb fragment points to a role of MGME1 in processing mtDNA ends, possibly enabling their repair by homologous recombination. In agreement with its functional involvement in mtDNA maintenance, we show that MGME1 interacts with the mitochondrial replicase PolgA, suggesting that it is a constituent of the mitochondrial replisome, to which it provides an additional exonuclease activity. Thus, our results support the viewpoint that MGME1-mediated mtDNA processing is essential for faithful mitochondrial genome replication and might be required for intramolecular recombination of mtDNA.

Epilepsy is a very frequent, severe, and disabling neurological disorder with has a considerable disease burden worldwide [...].

Genetic generalised epilepsy is the most common type of inherited epilepsy. Despite a high concordance rate of 80% in monozygotic twins, the genetic background is still poorly understood. We aimed to investigate the burden of rare genetic variants in genetic generalised epilepsy.For this exome-based case-control study, we used three different genetic generalised epilepsy case cohorts and three independent control cohorts, all of European descent. Cases included in the study were clinically evaluated for genetic generalised epilepsy. Whole-exome sequencing was done for the discovery case cohort, a validation case cohort, and two independent control cohorts. The replication case cohort underwent targeted next-generation sequencing of the 19 known genes encoding subunits of GABAStatistical comparison of 152 familial index cases with genetic generalised epilepsy in the discovery cohort to 549 ethnically matched controls suggested an enrichment of rare missense (Nonsyn) variants in the ensemble of 19 genes encoding GABAFunctionally relevant variants in genes encoding GABAEuroEPINOMICS (European Science Foundation through national funding organisations), Epicure and EpiPGX (Sixth Framework Programme and Seventh Framework Programme of the European Commission), Research Unit FOR2715 (German Research Foundation and Luxembourg National Research Fund).

Oxyphil cell transformation of epithelial cells due to the accumulation of mitochondria occurs often during cellular aging. To understand the pathogenic mechanisms, we studied mitochondrial DNA (mtDNA) alterations in the three cell types of the parathyroids using multiplex real-time PCR and next-generation sequencing. mtDNA was analyzed from cytochrome c oxidase (COX)-positive and COX-negative areas of 19 parathyroids. Mitochondria-rich pre-oxyphil/oxyphil cells were more prone to develop COX defects than the mitochondria-poor clear chief cells (P < 0.001). mtDNA increased approximately 2.5-fold from clear chief to oxyphil cells. In COX deficiency, the increase was even more pronounced, and COX-negative oxyphil cells had approximately two times more mtDNA than COX-positive oxyphil cells (P < 0.001), illustrating the influence of COX deficiency on mtDNA biosynthesis, probably as a consequence of insufficient ATP synthesis. Next-generation sequencing revealed a broad spectrum of putative pathogenic mtDNA point mutations affecting NADH dehydrogenase and COX genes as well as regulatory elements of mtDNA. NADH dehydrogenase gene mutations preferentially accumulated in COX-positive pre-oxyphil/oxyphil cells and, therefore, could be essential for inducing oxyphil cell transformation by increasing mtDNA/mitochondrial biogenesis. In contrast, COX-negative cells predominantly harbored mutations in the MT-CO1 and MT-CO3 genes and in regulatory mtDNA elements, but only rarely NADH dehydrogenase mutations. Thus, multiple hits in NADH dehydrogenase and COX activity-impairing genes represent the molecular basis of oxyphil cell transformation in the parathyroids.

Intracerebral hemorrhage (ICH) is a pathological condition that accompanies certain neurological diseases like hemorrhagic stroke or brain trauma. Its effects are severely destructive to the brain and can be fatal. There is an entire spectrum of harmful factors which are associated with the pathogenesis of ICH. One of them is a massive release of hemin from the decomposed erythrocytes. It has been previously shown, that hemin can inhibit the large-conductance Ca(2+)-regulated potassium channel in the plasma membrane. However, it remained unclear whether this phenomenon applies also to the mitochondrial large-conductance Ca(2+)-regulated potassium channel. The aim of the present study was to determine the impact of hemin on the activity of the large conductance Ca(2+)-regulated potassium channel in the brain mitochondria (mitoBKCa). In order to do so, we have used a patch-clamp technique and shown that hemin inhibits mitoBKCa in human astrocytoma U-87 MG cell line mitochondria. Since opening of the mitochondrial potassium channels is known to be cytoprotective, we have elucidated whether hemin can attenuate some of the beneficiary effects of potassium channel opening. We have studied the effect of hemin on reactive oxygen species synthesis, and mild mitochondrial uncoupling in isolated rat brain mitochondria. Taken together, our data show that hemin inhibits mitoBKCa and partially abolishes some of the cytoprotective properties of potassium channel opening. Considering the role of the mitoBKCa in cytoprotection, it can be presumed that its inhibition by hemin may be a novel mechanism contributing to the severity of the ICH symptoms. However, the validity of the presented results shall be further verified in an experimental model of ICH.

Gephyrin is a postsynaptic scaffolding protein, essential for the clustering of glycine and γ-aminobutyric acid type-A receptors (GABAARs) at inhibitory synapses. An impairment of GABAergic synaptic inhibition represents a key pathway of epileptogenesis. Recently, exonic microdeletions in the gephyrin (GPHN) gene have been associated with neurodevelopmental disorders including autism spectrum disorder, schizophrenia and epileptic seizures. Here we report the identification of novel exonic GPHN microdeletions in two patients with idiopathic generalized epilepsy (IGE), representing the most common group of genetically determined epilepsies. The identified GPHN microdeletions involve exons 5–9 (Δ5–9) and 2–3 (Δ2–3), both affecting the gephyrin G-domain. Molecular characterization of the GPHN Δ5–9 variant demonstrated that it perturbs the clustering of regular gephyrin at inhibitory synapses in cultured mouse hippocampal neurons in a dominant-negative manner, resulting in a significant loss of γ2-subunit containing GABAARs. GPHN Δ2–3 causes a frameshift resulting in a premature stop codon (p.V22Gfs*7) leading to haplo-insufficiency of the gene. Our results demonstrate that structural exonic microdeletions affecting the GPHN gene constitute a rare genetic risk factor for IGE and other neuropsychiatric disorders by an impairment of the GABAergic inhibitory synaptic transmission.

MGME1, also known as Ddk1 or C20orf72, is a mitochondrial exonuclease found to be involved in the processing of mitochondrial DNA (mtDNA) during replication. Here, we present detailed insights on the role of MGME1 in mtDNA maintenance. Upon loss of MGME1, elongated 7S DNA species accumulate owing to incomplete processing of 5′ ends. Moreover, an 11-kb linear mtDNA fragment spanning the entire major arc of the mitochondrial genome is generated. In contrast to control cells, where linear mtDNA molecules are detectable only after nuclease S1 treatment, the 11-kb fragment persists in MGME1-deficient cells. In parallel, we observed characteristic mtDNA duplications in the absence of MGME1. The fact that the breakpoints of these mtDNA rearrangements do not correspond to either classical deletions or the ends of the linear 11-kb fragment points to a role of MGME1 in processing mtDNA ends, possibly enabling their repair by homologous recombination. In agreement with its functional involvement in mtDNA maintenance, we show that MGME1 interacts with the mitochondrial replicase PolgA, suggesting that it is a constituent of the mitochondrial replisome, to which it provides an additional exonuclease activity. Thus, our results support the viewpoint that MGME1-mediated mtDNA processing is essential for faithful mitochondrial genome replication and might be required for intramolecular recombination of mtDNA.

Anfalle sind haufig die Hauptmanifestation von neurologischen Erkrankungen durch Mutationen in bisher 169 Genen, die die mitochondriale Funktion beeinflussen. Mitochondrien sind die Hauptproduzenten des ATP, das fur die normale neuronale Aktivitat und die synaptische Transmission benotigt wird. Gegenwartige Hypothesen verknupfen mitochondriale Dysfunktion und Anfalle mit einer Anderung der Calciumhomoostase, der ROS (reaktiven Sauerstoffspecies)-abhangigen Oxidation von Ionenkanalen und Rezeptoren, der Reduktion des neuronalen Membranpotenzials sowie einer verringerten Netzwerkhemmung durch Fehlfunktion von Interneuronen.

Sialic acid-binding Ig-like lectins (Siglecs) are members of the Ig superfamily that recognize sialic acid residues of glycoproteins. Siglec-E is a mouse CD33-related Siglec that preferentially binds to sialic acid residues of the cellular glycocalyx. Here, we demonstrate gene transcription and protein expression of Siglec-E by cultured mouse microglia. Siglec-E on microglia inhibited phagocytosis of neural debris and prevented the production of superoxide radicals induced by challenge with neural debris. Soluble extracellular Siglec-E receptor protein bound to the neural glycocalyx. Coculture of mouse microglia and neurons demonstrated a neuroprotective effect of microglial Siglec-E that was dependent on neuronal sialic acid residues. Increased neurotoxicity of microglia after knockdown ofSiglecemRNA was neutralized by the reactive oxygen species scavenger Trolox. Data suggest that Siglec-E recognizes the intact neuronal glycocalyx and has neuroprotective function by preventing phagocytosis and the associated oxidative burst.

A B S T R A C T Purpose: The group of the rare progressive myoclonic epilepsies (PME) include a wide spectrum of mitochondrial and metabolic diseases. In juvenile and adult ages, MERRF (myoclonic epilepsy with ragged red fibres) is the most common form. The underlying genetic defect in most patients with the syndrome of MERRF is a mutation in the tRNALys gene, but mutations were also detected in the tRNAPhe gene. Method: Here, we describe a 40 year old patient with prominent myoclonic seizures since 39 years of age without a mutation in the known genes who underwent intensive clinical, genetic and functional workup. Results: The patient had a slight mental retardation and a severe progressive hearing loss based on a defect of the inner ear on both sides. Ictal electroencephalography (EEG) showed bilateral occipital and generalized spikes and polyspikes induced and aggravated by photostimulation. A cranial magnetic resonance imaging (cMRI) detected a global cortical atrophy of the brain and mild periventricular white matter lesions. The electromyography (EMG) was normal but the muscle biopsy showed abundant ragged red fibres. Sequencing of the mitochondrial DNA from the skeletal muscle biopsy revealed a novel heteroplasmic mutation (m.4279A>G) in the tRNAIle gene which was functionally relevant as tested in single skeletal muscle fibre investigations. Conclusion: Mutations in tRNAIle were described in patients with chronic progressive external ophthalmoplegia (CPEO), prominent deafness or cardiomyopathy but, up to now, not in patients with myoclonic epilepsy. The degree of heteroplasmy of this novel mitochondrial DNA mutation was 70% in skeletal muscle but only 15% in blood, pointing to the diagnostic importance of a skeletal muscle biopsy also in patients with myoclonic epilepsy.