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Corpus callosum defects are frequent congenital cerebral disorders caused by mutations in more than 300 genes. These include genes implicated in corpus callosum development or function, as well as genes essential for mitochondrial physiology. However, in utero corpus callosum anomalies rarely raise a suspicion of mitochondrial disease and are characterized by a very large clinical heterogeneity. Here, we report a detailed pathological and neuro-histopathological investigation of nine foetuses from four unrelated families with prenatal onset of corpus callosum anomalies, sometimes associated with other cerebral or extra-cerebral defects. Next generation sequencing allowed the identification of novel pathogenic variants in three different nuclear genes previously reported in mitochondrial diseases: TIMMDC1, encoding a Complex I assembly factor never involved before in corpus callosum defect; MRPS22, a protein of the small mitoribosomal subunit; and EARS2, the mitochondrial tRNA-glutamyl synthetase. The present report describes the antenatal histopathological findings in mitochondrial diseases and expands the genetic spectrum of antenatal corpus callosum anomalies establishing OXPHOS function as an important factor for corpus callosum biogenesis. We propose that, when observed, antenatal corpus callosum anomalies should raise suspicion of mitochondrial disease and prenatal genetic counselling should be considered., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Transcription of mitochondrial DNA generates long polycistronic precursors whose nucleolytic cleavage yields the individual mtDNA-encoded transcripts. In most cases, this cleavage occurs at the 5'- and 3'-ends of tRNA sequences by the concerted action of RNAseP and RNaseZ/ELAC2 endonucleases, respectively. Variants in the ELAC2 gene have been predominantly linked to severe to mild cardiomyopathy that, in its milder forms, is accompanied by variably severe neurological presentations. Here, we report five patients from three unrelated families. Four of the patients presented mild to moderate cardiomyopathy and one died at 1 year of age, one patient had no evidence of cardiomyopathy. The patients had variable neurological presentations that included intellectual disability, ataxia, refractory epilepsy, neuropathy and deafness. All patients carried previously unreported missense and nonsense variants. Enzymatic analyses showed multiple OXPHOS deficiencies in biopsies from two patients, whereas immunoblot analyses revealed a decreased abundance of ELAC2 in fibroblasts from three patients. Northern blot analysis revealed an accumulation of unprocessed mt-tRNA Val -precursor consistent with the role of ELAC2 in transcript processing. Our study expands the genetic spectrum of ELAC2 -linked disease and suggests that cardiomyopathy is not an invariably present clinical hallmark of this pathology.

Image resolution in mass spectrometry imaging (MSI) is governed by the sampling probe, the motion of the stage relative to the probe, and the noise inherent for the sample and instrumentation employed. A new image formation model accounting for these variables is presented here. The model shows that the size of the probe, stage velocity, and the rate at which the probe consumes material from the surface govern the amount of blur present in the image. However, the main limiting factor for resolution is the signal-to-noise ratio (SNR). To evaluate blurring and noise effects, a new computational method for measuring lateral resolution in MSI is proposed. A spectral decomposition of the observed image signal and noise is used to determine a resolution number. To evaluate this technique, a silver step edge was prepared. This device was imaged at different pixels sizes using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). A modulation transfer function (MTF) and a noise power spectrum (NPS) were computed for each single-ion image, and resolution was defined as the point of intersection between the MTF and the NPS. Finally, the algorithm was also applied to a MALDI MSI tissue data set.

Most mitochondrial proteins are synthesized in the cytosol and targeted to mitochondria via N-terminal mitochondrial targeting signals (MTS) that are proteolytically removed upon import. Sometimes, MTS removal is followed by a cleavage of an octapeptide by the mitochondrial intermediate peptidase (MIP), encoded by the MIPEP gene. Previously, MIPEP variants were linked to four cases of multisystemic disorder presenting with cardiomyopathy, developmental delay, hypotonia and infantile lethality. We report here a patient carrying compound heterozygous MIPEP variants-one was not previously linked to mitochondrial disease-who did not have cardiomyopathy and who is alive at the age of 20 years. This patient had developmental delay, global hypotonia, mild optic neuropathy and mild ataxia. Functional characterization of patient fibroblasts and HEK293FT cells carrying MIPEP hypomorphic alleles demonstrated that deficient MIP activity was linked to impaired post-import processing of subunits from four of the five OXPHOS complexes and decreased abundance and activity of some of these complexes in human cells possibly underlying the development of mitochondrial disease. Thus, our work expands the genetic and clinical spectrum of MIPEP-linked disease and establishes MIP as an important regulator of OXPHOS biogenesis and function in human cells., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest to disclose., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Mitochondrial translation is essential for the biogenesis of the mitochondrial oxidative phosphorylation system (OXPHOS) that synthesizes the bulk of ATP for the cell. Hypomorphic and loss-of-function variants in either mitochondrial DNA or in nuclear genes that encode mitochondrial translation factors can result in impaired OXPHOS biogenesis and mitochondrial diseases with variable clinical presentations. Compound heterozygous or homozygous missense and frameshift variants in the FARS2 gene, that encodes the mitochondrial phenylalanyl-tRNA synthetase, are commonly linked to either early-onset epileptic mitochondrial encephalopathy or spastic paraplegia. Here, we expand the genetic spectrum of FARS2-linked disease with three patients carrying novel compound heterozygous variants in the FARS2 gene and presenting with spastic tetraparesis, axial hypotonia and myoclonic epilepsy in two cases.

Like bacterial and cytoplasmic ribosomes, mitoribosomes are large ribonucleoprotein complexes with molecular weights in the range of several million Daltons. Traditionally, studying the assembly of such high molecular weight complexes is done using ultracentrifugation through linear density gradients, which remains the method of choice due to its versatility and superior resolving power in the high molecular weight range. Here, we present a protocol for the analysis of mitoribosomal assembly in heart mitochondrial extracts using linear density sucrose gradients that we have previously employed to characterize the essential role of different mitochondrial proteins in mitoribosomal biogenesis. This protocol details in a stepwise manner a typical mitoribosomal assembly analysis starting with isolation of mitochondria, preparation and ultracentrifugation of the gradients, fractionation and ending with SDS-PAGE, and immunoblotting of the gradient fractions. Even though we provide an example with heart mitochondria, this protocol can be directly applied to virtually all mouse tissues, as well as cultured cells, with little to no modifications.

Pathogenic GFM1 variants have been linked to neurological phenotypes with or without liver involvement, but only a few cases have been reported in the literature. Here, we report clinical, biochemical, and neuroimaging findings from nine unrelated children carrying GFM1 variants, 10 of which were not previously reported. All patients presented with neurological involvement-mainly axial hypotonia and dystonia during the neonatal period-with five diagnosed with West syndrome; two children had liver involvement with cytolysis episodes or hepatic failure. While two patients died in infancy, six exhibited a stable clinical course. Brain magnetic resonance imaging showed the involvement of basal ganglia, brainstem, and periventricular white matter. Mutant EFG1 and OXPHOS proteins were decreased in patient's fibroblasts consistent with impaired mitochondrial translation. Thus, we expand the genetic spectrum of GFM1-linked disease and provide detailed clinical profiles of the patients that will improve the diagnostic success for other patients carrying GFM1 mutations., (© 2019 Wiley Periodicals, Inc.)

Mitochondria contain a dedicated translation system, which is responsible for the intramitochondrial synthesis of 13 mitochondrial DNA (mtDNA)-encoded polypeptides essential for the biogenesis of oxidative phosphorylation (OXPHOS) complexes I and III-V. Mutations in nuclear genes encoding factors involved in mitochondrial translation result in isolated or multiple OXPHOS deficiencies and mitochondrial disease. Here, we report the identification of disease-causing variants in the MRPS28 gene, encoding the small mitoribosomal subunit (mtSSU) protein bS1m in a patient with intrauterine growth retardation, craniofacial dysmorphism and developmental delay. Whole exome sequencing helped identify a seemingly homozygous missense variant NM_014018.2:c.356A>G, p.(Lys119Arg) which affected a highly conserved lysine residue. The variant was present in the mother in a heterozygous state, but not in the father who likely carried a large deletion spanning exon 2 and parts of introns 1 and 2 that could account for the apparent homozygosity of the patient. Polymerase chain reaction (PCR) amplification and Sanger sequencing of MRPS28 cDNA from patient fibroblasts revealed the presence of a truncated MRPS28 transcript, which lacked exon 2. Molecular and biochemical characterization of patient fibroblasts revealed a decrease in the abundance of the bS1m protein, decreased abundance of assembled mtSSU and inhibited mitochondrial translation. Consequently, OXPHOS biogenesis and cellular respiration were compromised in these cells. Expression of wild-type MRPS28 restored mitoribosomal assembly, mitochondrial translation and OXPHOS biogenesis, thereby demonstrating the deleterious nature of the identified MRPS28 variants. Thus, MRPS28 joins the increasing number of nuclear genes encoding mitoribosomal structural proteins linked to mitochondrial disease., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Aminoacyl-tRNA synthetases are ubiquitous enzymes, which universally charge tRNAs with their cognate amino acids for use in cytosolic or organellar translation. In humans, mutations in mitochondrial tRNA synthetases have been linked to different tissue-specific pathologies. Mutations in the KARS gene, which encodes both the cytosolic and mitochondrial isoform of lysyl-tRNA synthetase, cause predominantly neurological diseases that often involve deafness, but have also been linked to cardiomyopathy, developmental delay, and lactic acidosis. Using whole exome sequencing, we identified two compound heterozygous mutations, NM_001130089.1:c.683C>T p.(Pro228Leu) and NM_001130089.1:c.1438del p.(Leu480TrpfsX3), in a patient presenting with sensorineural deafness, developmental delay, hypotonia, and lactic acidosis. Nonsense-mediated mRNA decay eliminated the truncated mRNA transcript, rendering the patient hemizygous for the missense mutation. The c.683C>T mutation was previously described, but its pathogenicity remained unexamined. Molecular characterization of patient fibroblasts revealed a multiple oxidative phosphorylation deficiency due to impaired mitochondrial translation, but no evidence of inhibition of cytosolic translation. Reintroduction of wild-type mitochondrial KARS, but not the cytosolic isoform, rescued this phenotype confirming the disease-causing nature of p.(Pro228Leu) exchange and demonstrating the mitochondrial etiology of the disease. We propose that mitochondrial translation deficiency is the probable disease culprit in this and possibly other patients with mutations in KARS., (© 2018 Wiley Periodicals, Inc.)

Background: Because the mitochondrial respiratory chain (RC) is ubiquitous, its deficiency can theoretically give rise to any symptom in any organ or tissue at any age with any mode of inheritance, owing to the twofold genetic origin of respiratory enzyme machinery, that is, nuclear and mitochondrial. Not all respiratory enzyme deficiencies are primary and secondary or artefactual deficiency is frequently observed, leading to a number of misleading conclusions and inappropriate investigations in clinical practice. This study is aimed at investigating the potential role of brain MRI in distinguishing primary RC deficiency from phenocopies and other aetiologies., Methods: Starting from a large series of 189 patients (median age: 3.5 years (8 days-56 years), 58% males) showing signs of RC enzyme deficiency, for whom both brain MRIs and disease-causing mutations were available, we retrospectively studied the positive predictive value (PPV) and the positive likelihood ratio (LR+) of brain MRI imaging and its ability to discriminate between two groups: primary deficiency of the mitochondrial RC machinery and phenocopies., Results: Detection of (1) brainstem hyperintensity with basal ganglia involvement (P≤0.001) and (2) lactate peak with either brainstem or basal ganglia hyperintensity was highly suggestive of primary RC deficiency (P≤0.01). Fourteen items had a PPV>95% and LR+ was greater than 9 for seven signs. Biallelic SLC19A3 mutations represented the main differential diagnosis. Non-significant differences between the two groups were found for cortical/subcortical atrophy, leucoencephalopathy and involvement of caudate nuclei, spinothalamic tract and corpus callosum., Conclusion: Based on these results and owing to invasiveness of skeletal muscle biopsies and cost of high-throughput DNA sequencing, we suggest giving consideration to brain MRI imaging as a diagnostic marker and an informative investigation to be performed in patients showing signs of RC enzyme deficiency., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Biogenesis of the mitochondrial oxidative phosphorylation system, which produces the bulk of ATP for almost all eukaryotic cells, depends on the translation of 13 mtDNA-encoded polypeptides by mitochondria-specific ribosomes in the mitochondrial matrix. These mitoribosomes are dual-origin ribonucleoprotein complexes, which contain mtDNA-encoded rRNAs and tRNAs and ∼80 nucleus-encoded proteins. An increasing number of gene mutations that impair mitoribosomal function and result in multiple OXPHOS deficiencies are being linked to human mitochondrial diseases. Using exome sequencing in two unrelated subjects presenting with sensorineural hearing impairment, mild developmental delay, hypoglycemia, and a combined OXPHOS deficiency, we identified mutations in the gene encoding the mitochondrial ribosomal protein S2, which has not previously been implicated in disease. Characterization of subjects' fibroblasts revealed a decrease in the steady-state amounts of mutant MRPS2, and this decrease was shown by complexome profiling to prevent the assembly of the small mitoribosomal subunit. In turn, mitochondrial translation was inhibited, resulting in a combined OXPHOS deficiency detectable in subjects' muscle and liver biopsies as well as in cultured skin fibroblasts. Reintroduction of wild-type MRPS2 restored mitochondrial translation and OXPHOS assembly. The combination of lactic acidemia, hypoglycemia, and sensorineural hearing loss, especially in the presence of a combined OXPHOS deficiency, should raise suspicion for a ribosomal-subunit-related mitochondrial defect, and clinical recognition could allow for a targeted diagnostic approach. The identification of MRPS2 as an additional gene related to mitochondrial disease further expands the genetic and phenotypic spectra of OXPHOS deficiencies caused by impaired mitochondrial translation., (Copyright © 2018 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Neurodegeneration with brain iron accumulation (NBIA) is a genetically heterogeneous condition characterized by progressive dystonia with iron accumulation in the basal ganglia. How NBIA-associated mutations trigger iron overload remains poorly understood. After studying fibroblast cell lines from subjects carrying both known and unreported biallelic mutations in CRAT and REPS1, we ascribe iron overload to the abnormal recycling of transferrin receptor (TfR1) and the reduction of TfR1 palmitoylation in NBIA. Moreover, we describe palmitoylation as a hitherto unreported level of post-translational TfR1 regulation. A widely used antimalarial agent, artesunate, rescued abnormal TfR1 palmitoylation in cultured fibroblasts of NBIA subjects. These observations suggest therapeutic strategies aimed at targeting impaired TfR1 recycling and palmitoylation in NBIA., (Copyright © 2018 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

The synthesis of all 13 mitochondrial DNA (mtDNA)-encoded protein subunits of the human oxidative phosphorylation (OXPHOS) system is carried out by mitochondrial ribosomes (mitoribosomes). Defects in the stability of mitoribosomal proteins or mitoribosome assembly impair mitochondrial protein translation, causing combined OXPHOS enzyme deficiency and clinical disease. Here we report four autosomal-recessive pathogenic mutations in the gene encoding the small mitoribosomal subunit protein, MRPS34, in six subjects from four unrelated families with Leigh syndrome and combined OXPHOS defects. Whole-exome sequencing was used to independently identify all variants. Two splice-site mutations were identified, including homozygous c.321+1G>T in a subject of Italian ancestry and homozygous c.322-10G>A in affected sibling pairs from two unrelated families of Puerto Rican descent. In addition, compound heterozygous MRPS34 mutations were identified in a proband of French ancestry; a missense (c.37G>A [p.Glu13Lys]) and a nonsense (c.94C>T [p.Gln32 ∗ ]) variant. We demonstrated that these mutations reduce MRPS34 protein levels and the synthesis of OXPHOS subunits encoded by mtDNA. Examination of the mitoribosome profile and quantitative proteomics showed that the mitochondrial translation defect was caused by destabilization of the small mitoribosomal subunit and impaired monosome assembly. Lentiviral-mediated expression of wild-type MRPS34 rescued the defect in mitochondrial translation observed in skin fibroblasts from affected subjects, confirming the pathogenicity of MRPS34 mutations. Our data establish that MRPS34 is required for normal function of the mitoribosome in humans and furthermore demonstrate the power of quantitative proteomic analysis to identify signatures of defects in specific cellular pathways in fibroblasts from subjects with inherited disease., (Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Mitochondrial diseases are heterogeneous and incurable conditions typically resulting from deficient ATP production in the cells. Mice, owing to their genetic and physiological similarity to humans as well as their relatively easy maintenance and propagation, are extremely valuable for studying mitochondrial diseases and are also indispensable for the preclinical evaluation of novel therapies for these devastating conditions. Here, we review the recent exciting developments in the field focusing on mouse models for mitochondrial disease genes although models for genes not involved in the pathogenesis of mitochondrial disease and therapeutic proof-of-concept studies using mouse models are also discussed., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Acyl-CoA dehydrogenase family, member 9 (ACAD9) mutation is a frequent, usually fatal cause of early-onset cardiac hypertrophy and mitochondrial respiratory chain complex I deficiency in early childhood. We retrospectively studied a series of 20 unrelated children with cardiac hypertrophy and isolated complex I deficiency and identified compound heterozygosity for missense, splice site or frame shift ACAD9 variants in 8/20 patients (40%). Age at onset ranged from neonatal period to 9 years and 5/8 died in infancy. Heart transplantation was possible in 3/8. Two of them survived and one additional patient improved spontaneously. Importantly, the surviving patients later developed delayed-onset neurologic or muscular symptoms, namely cognitive impairment, seizures, muscle weakness and exercise intolerance. Other organ involvement included proximal tubulopathy, renal failure, secondary ovarian failure and optic atrophy. We conclude that ACAD9 mutation is the most frequent cause of cardiac hypertrophy and isolated complex I deficiency. Heart transplantation in children surviving neonatal period should be considered with caution, as delayed-onset muscle and brain involvement of various severity may occur, even if absent prior to transplantation.

Mitochondrial complex I deficiency results in a plethora of often severe clinical phenotypes manifesting in early childhood. Here, we report on three complex-I-deficient adult subjects with relatively mild clinical symptoms, including isolated, progressive exercise-induced myalgia and exercise intolerance but with normal later development. Exome sequencing and targeted exome sequencing revealed compound-heterozygous mutations in TMEM126B, encoding a complex I assembly factor. Further biochemical analysis of subject fibroblasts revealed a severe complex I deficiency caused by defective assembly. Lentiviral complementation with the wild-type cDNA restored the complex I deficiency, demonstrating the pathogenic nature of these mutations. Further complexome analysis of one subject indicated that the complex I assembly defect occurred during assembly of its membrane module. Our results show that TMEM126B defects can lead to complex I deficiencies and, interestingly, that symptoms can occur only after exercise., (Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Mitochondrial disorders are clinically and genetically diverse, with mutations in mitochondrial or nuclear genes able to cause defects in mitochondrial gene expression. Recently, mutations in several genes encoding factors involved in mt-tRNA processing have been identified to cause mitochondrial disease. Using whole-exome sequencing, we identified mutations in TRMT10C (encoding the mitochondrial RNase P protein 1 [MRPP1]) in two unrelated individuals who presented at birth with lactic acidosis, hypotonia, feeding difficulties, and deafness. Both individuals died at 5 months after respiratory failure. MRPP1, along with MRPP2 and MRPP3, form the mitochondrial ribonuclease P (mt-RNase P) complex that cleaves the 5' ends of mt-tRNAs from polycistronic precursor transcripts. Additionally, a stable complex of MRPP1 and MRPP2 has m(1)R9 methyltransferase activity, which methylates mt-tRNAs at position 9 and is vital for folding mt-tRNAs into their correct tertiary structures. Analyses of fibroblasts from affected individuals harboring TRMT10C missense variants revealed decreased protein levels of MRPP1 and an increase in mt-RNA precursors indicative of impaired mt-RNA processing and defective mitochondrial protein synthesis. The pathogenicity of the detected variants-compound heterozygous c.542G>T (p.Arg181Leu) and c.814A>G (p.Thr272Ala) changes in subject 1 and a homozygous c.542G>T (p.Arg181Leu) variant in subject 2-was validated by the functional rescue of mt-RNA processing and mitochondrial protein synthesis defects after lentiviral transduction of wild-type TRMT10C. Our study suggests that these variants affect MRPP1 protein stability and mt-tRNA processing without affecting m(1)R9 methyltransferase activity, identifying mutations in TRMT10C as a cause of mitochondrial disease and highlighting the importance of RNA processing for correct mitochondrial function., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Mitochondrial dysfunction is a well-established cause of sensorineural deafness, but the pathophysiological events are poorly understood. Non-syndromic deafness and predisposition to aminoglycoside-induced deafness can be caused by specific mutations in the 12S rRNA gene of mtDNA and are thus maternally inherited traits. The pathophysiology induced by mtDNA mutations has traditionally been attributed to deficient oxidative phosphorylation, which causes energy crisis with functional impairment of multiple cellular processes. In contrast, it was recently reported that signaling induced by 'hypermethylation' of two conserved adenosines of 12S rRNA in the mitoribosome is of key pathophysiological importance in sensorineural deafness. In support for this concept, it was reported that overexpression of the essential mitochondrial methyltransferase TFB1M in the mouse was sufficient to induce mitoribosomal hypermethylation and deafness. At variance with this model, we show here that 12S rRNA is near fully methylated in vivo in the mouse and thus cannot be further methylated to any significant extent. Furthermore, bacterial artificial chromosome transgenic mice overexpressing TFB1M have no increase of 12S rRNA methylation levels and hear normally. We thus conclude that therapies directed against mitoribosomal methylation are unlikely to be beneficial to patients with sensorineural hearing loss or other types of mitochondrial disease., (© The Author 2015. Published by Oxford University Press.)

A homozygote missense mutation of the pseudouridylate synthase gene was found in an adult patient with chronic sideroblastic anemia, diarrhea, microcephaly and failure to thrive. Moderate muscle weakness occurred in adulthood (6-min walk distance at 26 years: 240 m, control range 380-782 m) but a profound deficiency of mitochondrial respiratory chain complexes I and IV were found in her skeletal muscle. This, to our knowledge, is the first example of long survival of this usually fatal mitochondrial deficiency into adulthood. We suggest giving consideration to mitochondrial translation deficiency in unexplained syndromic sideroblastic anemia in adulthood.

Respiratory chain deficiencies exhibit a wide variety of clinical phenotypes resulting from defective mitochondrial energy production through oxidative phosphorylation. These defects can be caused by either mutations in the mtDNA or mutations in nuclear genes coding for mitochondrial proteins. The underlying pathomechanisms can affect numerous pathways involved in mitochondrial physiology. By whole-exome and candidate gene sequencing, we identified 11 individuals from 9 families carrying compound heterozygous or homozygous mutations in GTPBP3, encoding the mitochondrial GTP-binding protein 3. Affected individuals from eight out of nine families presented with combined respiratory chain complex deficiencies in skeletal muscle. Mutations in GTPBP3 are associated with a severe mitochondrial translation defect, consistent with the predicted function of the protein in catalyzing the formation of 5-taurinomethyluridine (τm(5)U) in the anticodon wobble position of five mitochondrial tRNAs. All case subjects presented with lactic acidosis and nine developed hypertrophic cardiomyopathy. In contrast to individuals with mutations in MTO1, the protein product of which is predicted to participate in the generation of the same modification, most individuals with GTPBP3 mutations developed neurological symptoms and MRI involvement of thalamus, putamen, and brainstem resembling Leigh syndrome. Our study of a mitochondrial translation disorder points toward the importance of posttranscriptional modification of mitochondrial tRNAs for proper mitochondrial function., (Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Background: Inherited optic neuropathy has been ascribed to mutations in mitochondrial fusion/fission dynamics genes, nuclear and mitochondrial DNA-encoded respiratory enzyme genes or nuclear genes of poorly known mitochondrial function. However, the disease causing gene remains unknown in many families., Methods: We used exome sequencing in order to identify the gene responsible for isolated or syndromic optic atrophy in five patients from three independent families., Results: We found homozygous or compound heterozygous missense and frameshift mutations in the gene encoding mitochondrial aconitase (ACO2), a tricarboxylic acid cycle enzyme, catalysing interconversion of citrate into isocitrate. Unlike wild type ACO2, all mutant ACO2 proteins failed to complement the respiratory growth of a yeast aco1-deletion strain. Retrospective studies using patient-derived cultured skin fibroblasts revealed various degrees of deficiency in ACO2 activity, but also in ACO1 cytosolic activity., Conclusions: Our study shows that autosomal recessive ACO2 mutations can cause either isolated or syndromic optic neuropathy. This observation identifies ACO2 as the second gene responsible for non-syndromic autosomal recessive optic neuropathies and provides evidence for a genetic overlap between isolated and syndromic forms, giving further support to the view that optic atrophy is a hallmark of defective mitochondrial energy supply., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

We have previously identified transcription factor B1 mitochondrial (TFB1M) as a type 2 diabetes (T2D) risk gene, using human and mouse genetics. To further understand the function of TFB1M and how it is associated with T2D, we created a β-cell-specific knockout of Tfb1m, which gradually developed diabetes. Prior to the onset of diabetes, β-Tfb1m(-/-) mice exhibited retarded glucose clearance owing to impaired insulin secretion. β-Tfb1m(-/-) islets released less insulin in response to fuels, contained less insulin and secretory granules and displayed reduced β-cell mass. Moreover, mitochondria in Tfb1m-deficient β-cells were more abundant with disrupted architecture. TFB1M is known to control mitochondrial protein translation by adenine dimethylation of 12S ribosomal RNA (rRNA). Here, we found that the levels of TFB1M and mitochondrial-encoded proteins, mitochondrial 12S rRNA methylation, ATP production and oxygen consumption were reduced in β-Tfb1m(-/-) islets. Furthermore, the levels of reactive oxygen species (ROS) in response to cellular stress were increased whereas induction of defense mechanisms was attenuated. We also show increased apoptosis and necrosis as well as infiltration of macrophages and CD4(+) cells in the islets. Taken together, our findings demonstrate that Tfb1m-deficiency in β-cells caused mitochondrial dysfunction and subsequently diabetes owing to combined loss of β-cell function and mass. These observations reflect pathogenetic processes in human islets: using RNA sequencing, we found that the TFB1M risk variant exhibited a negative gene-dosage effect on islet TFB1M mRNA levels, as well as insulin secretion. Our findings highlight the role of mitochondrial dysfunction in impairments of β-cell function and mass, the hallmarks of T2D., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Biogenesis of mammalian mitochondrial ribosomes requires a concerted maturation of both the small (SSU) and large subunit (LSU). We demonstrate here that the m(5)C methyltransferase NSUN4, which forms a complex with MTERF4, is essential in mitochondrial ribosomal biogenesis as mitochondrial translation is abolished in conditional Nsun4 mouse knockouts. Deep sequencing of bisulfite-treated RNA shows that NSUN4 methylates cytosine 911 in 12S rRNA (m5C911) of the SSU. Surprisingly, NSUN4 does not need MTERF4 to generate this modification. Instead, the NSUN4/MTERF4 complex is required to assemble the SSU and LSU to form a monosome. NSUN4 is thus a dual function protein, which on the one hand is needed for 12S rRNA methylation and, on the other hand interacts with MTERF4 to facilitate monosome assembly. The presented data suggest that NSUN4 has a key role in controlling a final step in ribosome biogenesis to ensure that only the mature SSU and LSU are assembled.

Regulation of mitochondrial DNA (mtDNA) expression is critical for the control of oxidative phosphorylation in response to physiological demand, and this regulation is often impaired in disease and aging. We have previously shown that mitochondrial transcription termination factor 3 (MTERF3) is a key regulator that represses mtDNA transcription in the mouse, but its molecular mode of action has remained elusive. Based on the hypothesis that key regulatory mechanisms for mtDNA expression are conserved in metazoans, we analyzed Mterf3 knockout and knockdown flies. We demonstrate here that decreased expression of MTERF3 not only leads to activation of mtDNA transcription, but also impairs assembly of the large mitochondrial ribosomal subunit. This novel function of MTERF3 in mitochondrial ribosomal biogenesis is conserved in the mouse, thus we identify a novel and unexpected role for MTERF3 in coordinating the crosstalk between transcription and translation for the regulation of mammalian mtDNA gene expression., Competing Interests: The authors have declared that no competing interests exist.

Regulation of mtDNA expression is critical for maintaining cellular energy homeostasis and may, in principle, occur at many different levels. The leucine-rich pentatricopeptide repeat containing (LRPPRC) protein regulates mitochondrial mRNA stability and an amino-acid substitution of this protein causes the French-Canadian type of Leigh syndrome (LSFC), a neurodegenerative disorder characterized by complex IV deficiency. We have generated conditional Lrpprc knockout mice and show here that the gene is essential for embryonic development. Tissue-specific disruption of Lrpprc in heart causes mitochondrial cardiomyopathy with drastic reduction in steady-state levels of most mitochondrial mRNAs. LRPPRC forms an RNA-dependent protein complex that is necessary for maintaining a pool of non-translated mRNAs in mammalian mitochondria. Loss of LRPPRC does not only decrease mRNA stability, but also leads to loss of mRNA polyadenylation and the appearance of aberrant mitochondrial translation. The translation pattern without the presence of LRPPRC is misregulated with excessive translation of some transcripts and no translation of others. Our findings point to the existence of an elaborate machinery that regulates mammalian mtDNA expression at the post-transcriptional level.

Precise control of mitochondrial DNA gene expression is critical for regulation of oxidative phosphorylation capacity in mammals. The MTERF protein family plays a key role in this process, and its members have been implicated in regulation of transcription initiation and site-specific transcription termination. We now demonstrate that a member of this family, MTERF4, directly controls mitochondrial ribosomal biogenesis and translation. MTERF4 forms a stoichiometric complex with the ribosomal RNA methyltransferase NSUN4 and is necessary for recruitment of this factor to the large ribosomal subunit. Loss of MTERF4 leads to defective ribosomal assembly and a drastic reduction in translation. Our results thus show that MTERF4 is an important regulator of translation in mammalian mitochondria., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Type 2 diabetes (T2D) evolves when insulin secretion fails. Insulin release from the pancreatic β cell is controlled by mitochondrial metabolism, which translates fluctuations in blood glucose into metabolic coupling signals. We identified a common variant (rs950994) in the human transcription factor B1 mitochondrial (TFB1M) gene associated with reduced insulin secretion, elevated postprandial glucose levels, and future risk of T2D. Because islet TFB1M mRNA levels were lower in carriers of the risk allele and correlated with insulin secretion, we examined mice heterozygous for Tfb1m deficiency. These mice displayed lower expression of TFB1M in islets and impaired mitochondrial function and released less insulin in response to glucose in vivo and in vitro. Reducing TFB1M mRNA and protein in clonal β cells by RNA interference impaired complexes of the mitochondrial oxidative phosphorylation system. Consequently, nutrient-stimulated ATP generation was reduced, leading to perturbed insulin secretion. We conclude that a deficiency in TFB1M and impaired mitochondrial function contribute to the pathogenesis of T2D., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The 3' end of the rRNA of the small ribosomal subunit contains two extremely highly conserved dimethylated adenines. This modification and the responsible methyltransferases are present in all three domains of life, but its function has remained elusive. We have disrupted the mouse Tfb1m gene encoding a mitochondrial protein homologous to bacterial dimethyltransferases and demonstrate here that loss of TFB1M is embryonic lethal. Disruption of Tfb1m in heart leads to complete loss of adenine dimethylation of the rRNA of the small mitochondrial ribosomal subunit, impaired assembly of the mitochondrial ribosome, and abolished mitochondrial translation. In addition, we present biochemical evidence that TFB1M does not activate or repress transcription in the presence of TFB2M. Our results thus show that TFB1M is a nonredundant dimethyltransferase in mammalian mitochondria. In addition, we provide a possible explanation for the universal conservation of adenine dimethylation of rRNA by showing a critical role in ribosome maintenance.

The m-AAA protease, an ATP-dependent proteolytic complex in the mitochondrial inner membrane, controls protein quality and regulates ribosome assembly, thus exerting essential housekeeping functions within mitochondria. Mutations in the m-AAA protease subunit paraplegin cause axonal degeneration in hereditary spastic paraplegia (HSP), but the basis for the unexpected tissue specificity is not understood. Paraplegin assembles with homologous Afg3l2 subunits into hetero-oligomeric complexes which can substitute for yeast m-AAA proteases, demonstrating functional conservation. The function of a third paralogue, Afg3l1 expressed in mouse, is unknown. Here, we analyze the assembly of paraplegin into m-AAA complexes and monitor consequences of paraplegin deficiency in HSP fibroblasts and in a mouse model for HSP. Our findings reveal variability in the assembly of m-AAA proteases in mitochondria in different tissues. Homo-oligomeric Afg3l1 and Afg3l2 complexes and hetero-oligomeric assemblies of both proteins with paraplegin can be formed. Yeast complementation studies demonstrate the proteolytic activity of these assemblies. Paraplegin deficiency in HSP does not result in the loss of m-AAA protease activity in brain mitochondria. Rather, homo-oligomeric Afg3l2 complexes accumulate, and these complexes can substitute for housekeeping functions of paraplegin-containing m-AAA complexes. We therefore propose that the formation of m-AAA proteases with altered substrate specificities leads to axonal degeneration in HSP.