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Mutations in mitochondrial (mt-)tRNAs frequently cause mitochondrial dysfunction. Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), and myoclonus epilepsy associated with ragged red fibers (MERRF) are major clinical subgroups of mitochondrial diseases caused by pathogenic point mutations in tRNA genes encoded in mtDNA. We previously reported a severe reduction in the frequency of 5-taurinomethyluridine (τm5U) and its 2-thiouridine derivative (τm5s2U) in the anticodons of mutant mt-tRNAs isolated from the cells of patients with MELAS and MERRF, respectively. The hypomodified tRNAs fail to decode cognate codons efficiently, resulting in defective translation of respiratory chain proteins in mitochondria. To restore the mitochondrial activity of MELAS patient cells, we overexpressed MTO1, a τm5U-modifying enzyme, in patient-derived myoblasts. We used a newly developed primer extension method and showed that MTO1 overexpression almost completely restored the τm5U modification of the MELAS mutant mt-tRNALeu(UUR). An increase in mitochondrial protein synthesis and oxygen consumption rate suggested that the mitochondrial function of MELAS patient cells can be activated by restoring the τm5U of the mutant tRNA. In addition, we confirmed that MTO1 expression restored the τm5s2U of the mutant mt-tRNALys in MERRF patient cells. These findings pave the way for epitranscriptomic therapies for mitochondrial diseases.

Funding Information: This work was supported by the Academy of Finland (307431 and 314706 to B.J.B.), the Sigrid Juselius Foundation Senior Investigator Award to B.J.B., and United Mitochondrial Disease Foundation (PI-16-0598 to B.J.B.) and donations from the Hereditary Neuropathy Foundation, Lindsey Flynt, and Medtronic to B.J.B.; the Orion Research Foundation and the Finnish Cultural Foundation to K.Y.N.; the Academy of Finland (321961 to U.R.); the Sigrid Juselius Foundation, the Academy of Finland (331556), and the Jane and Aatos Erkko Foundation to C.D.D.; Action Medical Research (GN2494 to W.G.N.) and the Manchester NIHR Biomedical Research Centre (IS-BRC-1215-20007 to W.G.N.); the Wellcome Centre for Mitochondrial Research (203105/Z/16/Z to R.W.T.), the Mitochondrial Disease Patient Cohort (UK) (G0800674 to R.W.T.), the Medical Research Council International Centre for Genomic Medicine in Neuromuscular Disease (MR/S005021/1 to R.W.T.), the Lily Foundation, the UK NIHR Biomedical Research Centre for Ageing and Age-related disease award to the Newcastle upon Tyne Foundation Hospitals NHS Trust, the Pathological Society, and the UK NHS Highly Specialised Service for Rare Mitochondrial Disorders of Adults and Children to R.W.T.; Medical Research Council (MR/W019027/1 to W.G.N. and R.W.T.); the Academy of Finland (338836 and 314672 to V.O.P.); and the Sigrid Juselius Foundation and the Jane and Aatos Erkko Foundation. Publisher Copyright: Copyright © 2022 The Authors, some rights reserved; A stop codon within the mRNA facilitates coordinated termination of protein synthesis, releasing the nascent polypeptide from the ribosome. This essential step in gene expression is impeded with transcripts lacking a stop codon, generating nonstop ribosome complexes. Here, we use deep sequencing to investigate sources of nonstop mRNAs generated from the human mitochondrial genome. We identify diverse types of nonstop mRNAs on mitochondrial ribosomes that are resistant to translation termination by canonical release factors. Failure to resolve these aberrations by the mitochondrial release factor in rescue (MTRFR) imparts a negative regulatory effect on protein synthesis that is associated with human disease. Our findings reveal a source of underlying noise in mitochondrial gene expression and the importance of responsive ribosome quality control mechanisms for cell fitness and human health.

SummaryMitochondrial ribosomes (mitoribosomes) have undergone substantial structural remodelling throughout evolution. Compared to their prokaryotic counterparts, mitoribosomes show a substantial loss of ribosomal RNA, whilst acquiring unique protein subunits located on the periphery of the ribosomal subunit structures. We set out to investigate the functional properties of all 14 unique (mitochondrial-specific or supernumerary) human mitoribosomal proteins in the small subunit. Using genome editing with CRISPR-Cas9, we made knockouts for each subunit in HEK293 cells to study the effect on mitoribosome assembly and function in protein synthesis. Unexpectedly, we show that each supernumerary knockout leads to a unique mitoribosome assembly defect with variable impact on mitochondrial protein synthesis. Our data demonstrates that all supernumerary subunits are essential structural components except mS37. Surprisingly, we found the stability of mS37 was reduced in all our supernumerary knockouts of the small and large ribosomal subunits as well as patient-derived lines with mitoribosome assembly defects. We identified that a redox regulated CX9C motif in mS37 was essential for protein stability, suggesting a potential mechanism to regulate mitochondrial protein synthesis. Together, our findings support a modular assembly of the human mitochondrial small ribosomal subunit mediated by essential supernumerary subunits and identify a redox regulatory role involving mS37 in mitochondrial protein synthesis in health and disease.

Inonotus obliquus, Chaga mushroom, is a fungal species from Hymenochaetaceae family (Basidiomycota) which has been widely used for traditional medicine in Europe and Asia. Here, chaga genome was sequenced using Pacbio sequencing into a 50.7Mbp assembly consisting of 301 primary contigs with an N50 value of 375 kbp. Genome evolution analyses revealed a lineage-specific whole genome duplication event and an expansion of Cytochrome P450 superfamily. Fungal biosynthetic clusters were enriched for tandemly duplicated genes, suggesting that biosynthetic pathway evolution has proceeded through small-scale duplications. Metabolomic fingerprinting confirmed a highly complex terpene biosynthesis chemistry when compared against related fungal species lacking the genome duplication event.

Nucleotide repeat sequences are prevalent in the genome and expansion of these sequences is associated with more than 40 neuromuscular disorders. To understand the pathogenic mechanisms underlying RNA-repeat toxicity, we performed a genetic screen in a Caenorhabditis elegans model expressing an expanded CUG repeat specifically in the muscle. Here, we show that expression of this RNA repeat impairs motility by mitochondrial dysfunction, disrupting mitochondrial morphology and respiration. The phenotype is dependent on the RNA-binding factor MBL-1 and requires factors from the ribosome-associated protein quality control complex. Furthermore, Coenzyme Q supplementation rescued the motility impairment and all of the mitochondrial phenotypes. Together, our data reveal the importance of mitochondrial dysfunction in the molecular pathogenesis of RNA repeat expansion disorders.

Pathogenic variants that disrupt human mitochondrial protein synthesis are associated with a clinically heterogenous group of diseases. Despite an impairment in oxidative phosphorylation being a common phenotype, the underlying molecular pathogenesis is more complex than simply a bioenergetic deficiency. Currently, we have limited mechanistic understanding on the scope by which a primary defect in mitochondrial protein synthesis contributes to organelle dysfunction. Since the proteins encoded in the mitochondrial genome are hydrophobic and need co-translational insertion into a lipid bilayer, responsive quality control mechanisms are required to resolve aberrations that arise with the synthesis of truncated and misfolded proteins. Here, we show that defects in the OXA1L-mediated insertion of MT-ATP6 nascent chains into the mitochondrial inner membrane are rapidly resolved by the AFG3L2 protease complex. Using pathogenic MT-ATP6 variants, we then reveal discrete steps in this quality control mechanism and the differential functional consequences to mitochondrial gene expression. The inherent ability of a given cell type to recognize and resolve impairments in mitochondrial protein synthesis may in part contribute at the molecular level to the wide clinical spectrum of these disorders.

Signaling circuits crucial to systemic physiology are widespread, yet uncovering their molecular underpinnings remains a barrier to understanding the etiology of many metabolic disorders. Here, we identify a copper-linked signaling circuit activated by disruption of mitochondrial function in the murine liver or heart that results in atrophy of the spleen and thymus and causes a peripheral white blood cell deficiency. We demonstrate that the leukopenia is caused by α-fetoprotein, which requires copper and the cell surface receptor CCR5 to promote white blood cell death. We further show that α-fetoprotein expression is upregulated in several cell types upon inhibition of oxidative phosphorylation, including a muscle cell model of Barth syndrome. Collectively, our data argue that α-fetoprotein secreted by bioenergetically stressed tissue suppresses the immune system, an effect which may explain the recurrent infections that are observed in a subset of mitochondrial diseases or in other disorders with mitochondrial involvement.

Proteotoxicity has long been considered a key factor in mitochondrial dysfunction and human disease. The origin of the endogenous offending toxic substrates and the regulatory pathways to deal with these insults, however, have remained unclear. Mitochondria maintain a compartmentalized gene expression system that in animals is only responsible for synthesis of 1% of the organelle proteome. Because of the relatively small contribution of the mitochondrial genome to the overall proteome, the synthesis and quality control of these nascent chains to maintain organelle proteostasis has long been overlooked. However, recent research has uncovered mechanisms by which defects to the quality control of mitochondrial gene expression are linked to a novel cellular stress response that impinges upon organelle form and function and cell fitness. In this review, we discuss the mechanisms for a key event in the response: activation of the metalloprotease OMA1. This severs the membrane tether of the dynamin-related GTPase OPA1, which is a critical determinant for mitochondrial morphology and function. We also highlight the evolutionary conservation from bacteria of these quality-control mechanisms to maintain membrane integrity, gene expression, and cell fitness.

International audience; Dysfunction of mitochondrial translation is an increasingly important molecular cause of human disease, but structural defects of mitochondrial ribosomal subunits are rare. We used next-generation sequencing to identify a homozygous variant in the mitochondrial small ribosomal protein 14 (MRPS14, uS14m) in a patient manifesting with perinatal hypertrophic cardiomyopathy, growth retardation, muscle hypotonia, elevated lactate, dysmorphy and mental retardation. In skeletal muscle and fibroblasts from the patient, there was biochemical deficiency in complex IV of the respiratory chain. In fibroblasts, mitochondrial translation was impaired, and ectopic expression of a wild-type MRPS14 cDNA functionally complemented this defect. Surprisingly, the mutant uS14m was stable and did not affect assembly of the small ribosomal subunit. Instead, structural modeling of the uS14m mutation predicted a disruption to the ribosomal mRNA channel.Collectively, our data demonstrate pathogenic mutations in MRPS14 can manifest as a perinatal-onset mitochondrial hypertrophic cardiomyopathy with a novel molecular pathogenic mechanism that impairs the function of mitochondrial ribosomes during translation elongation or mitochondrial mRNA recruitment rather than assembly.

Human mitochondrial RNase P (mt-RNase P) is responsible for 5' end processing of mitochondrial precursor tRNAs, a vital step in mitochondrial RNA maturation, and is comprised of three protein subunits: TRMT10C, SDR5C1 (HSD10), and PRORP. Pathogenic variants in TRMT10C and SDR5C1 are associated with distinct recessive or x-linked infantile onset disorders, resulting from defects in mitochondrial RNA processing. We report four unrelated families with multisystem disease associated with bi-allelic variants in PRORP, the metallonuclease subunit of mt-RNase P. Affected individuals presented with variable phenotypes comprising sensorineural hearing loss, primary ovarian insufficiency, developmental delay, and brain white matter changes. Fibroblasts from affected individuals in two families demonstrated decreased steady state levels of PRORP, an accumulation of unprocessed mitochondrial transcripts, and decreased steady state levels of mitochondrial-encoded proteins, which were rescued by introduction of the wild-type PRORP cDNA. In mt-tRNA processing assays performed with recombinant mt-RNase P proteins, the disease-associated variants resulted in diminished mitochondrial tRNA processing. Identification of disease-causing variants in PRORP indicates that pathogenic variants in all three subunits of mt-RNase P can cause mitochondrial dysfunction, each with distinct pleiotropic clinical presentations.

Mitochondrial ribosomes (mitoribosomes) are tethered to the mitochondrial inner membrane to facilitate the cotranslational membrane insertion of the synthesized proteins. We report cryo-electron microscopy structures of human mitoribosomes with nascent polypeptide, bound to the insertase oxidase assembly 1-like (OXA1L) through three distinct contact sites. OXA1L binding is correlated with a series of conformational changes in the mitoribosomal large subunit that catalyze the delivery of newly synthesized polypeptides. The mechanism relies on the folding of mL45 inside the exit tunnel, forming two specific constriction sites that would limit helix formation of the nascent chain. A gap is formed between the exit and the membrane, making the newly synthesized proteins accessible. Our data elucidate the basis by which mitoribosomes interact with the OXA1L insertase to couple protein synthesis and membrane delivery.

Post-transcriptional RNA modifications play a critical role in the pathogenesis of human mitochondrial disorders, but the mechanisms by which specific modifications affect mitochondrial protein synthesis remain poorly understood. Here we used a quantitative RNA sequencing approach to investigate, at nucleotide resolution, the stoichiometry and methyl modifications of the entire mitochondrial tRNA pool, and establish the relevance to human disease. We discovered that a N1-methyladenosine (m1A) modification is missing at position 58 in the mitochondrial tRNALys of patients with the mitochondrial DNA mutation m.8344 A > G associated with MERRF (myoclonus epilepsy, ragged-red fibers). By restoring the modification on the mitochondrial tRNALys, we demonstrated the importance of the m1A58 to translation elongation and the stability of selected nascent chains. Our data indicates regulation of post-transcriptional modifications on mitochondrial tRNAs is finely tuned for the control of mitochondrial gene expression. Collectively, our findings provide novel insight into the regulation of mitochondrial tRNAs and reveal greater complexity to the molecular pathogenesis of MERRF., Mutations in mitochondrially-encoded tRNA genes can lead to mitochondrial disorders. Here the authors use next generation RNA sequencing to reveal the role of a N1 -methyladenosine modification in tRNALys MERR patients for translation elongation and the stability of selected nascent chains.

Mitochondrial dysfunction elicits stress responses that safeguard cellular homeostasis against metabolic insults. Mitochondrial integrated stress response (ISRmt) is a major response to mitochondrial (mt)DNA expression stress (mtDNA maintenance, translation defects), but the knowledge of dynamics or interdependence of components is lacking. We report that in mitochondrial myopathy, ISRmt progresses in temporal stages and development from early to chronic and is regulated by autocrine and endocrine effects of FGF21, a metabolic hormone with pleiotropic effects. Initial disease signs induce transcriptional ISRmt (ATF5, mitochondrial one-carbon cycle, FGF21, and GDF15). The local progression to 2nd metabolic ISRmt stage (ATF3, ATF4, glucose uptake, serine biosynthesis, and transsulfuration) is FGF21 dependent. Mitochondrial unfolded protein response marks the 3rd ISRmt stage of failing tissue. Systemically, FGF21 drives weight loss and glucose preference, and modifies metabolism and respiratory chain deficiency in a specific hippocampal brain region. Our evidence indicates that FGF21 is a local and systemic messenger of mtDNA stress in mice and humans with mitochondrial disease.

Quality control defects of mitochondrial nascent chain synthesis trigger a sequential stress response characterized by OMA1 activation and ribosome decay, determining mitochondrial form and function., Mitochondria have a compartmentalized gene expression system dedicated to the synthesis of membrane proteins essential for oxidative phosphorylation. Responsive quality control mechanisms are needed to ensure that aberrant protein synthesis does not disrupt mitochondrial function. Pathogenic mutations that impede the function of the mitochondrial matrix quality control protease complex composed of AFG3L2 and paraplegin cause a multifaceted clinical syndrome. At the cell and molecular level, defects to this quality control complex are defined by impairment to mitochondrial form and function. Here, we establish the etiology of these phenotypes. We show how disruptions to the quality control of mitochondrial protein synthesis trigger a sequential stress response characterized first by OMA1 activation followed by loss of mitochondrial ribosomes and by remodelling of mitochondrial inner membrane ultrastructure. Inhibiting mitochondrial protein synthesis with chloramphenicol completely blocks this stress response. Together, our data establish a mechanism linking major cell biological phenotypes of AFG3L2 pathogenesis and show how modulation of mitochondrial protein synthesis can exert a beneficial effect on organelle homeostasis.

Spinocerebellar ataxia 28 is an autosomal dominant neurodegenerative disorder caused by missense mutations affecting the proteolytic domain of AFG3L2, a major component of the mitochondrial m-AAA protease. However, little is known of the underlying pathogenetic mechanisms or how to treat patients with SCA28. Currently available Afg3l2 mutant mice harbour deletions that lead to severe, early-onset neurological phenotypes that do not faithfully reproduce the late-onset and slowly progressing SCA28 phenotype. Here we describe production and detailed analysis of a new knock-in murine model harbouring an Afg3l2 allele carrying the p.Met665Arg patient-derived mutation. Heterozygous mutant mice developed normally but signs of ataxia were detectable by beam test at 18 months. Cerebellar pathology was negative; electrophysiological analysis showed increased spontaneous firing in Purkinje cells from heterozygous mutants with respect to wild-type controls, although not statistically significant. As homozygous mutants died perinatally with evidence of cardiac atrophy, for each genotype we generated mouse embryonic fibroblasts (MEFs) to investigate mitochondrial function. MEFs from mutant mice showed altered mitochondrial bioenergetics, with decreased basal oxygen consumption rate, ATP synthesis and mitochondrial membrane potential. Mitochondrial network formation and morphology was also altered, in line with greatly reduced expression of Opa1 fusogenic protein L-isoforms. The mitochondrial alterations observed in MEFs were also detected in cerebella of 18-month-old heterozygous mutants, suggesting they may be a hallmark of disease. Pharmacological inhibition of de novo mitochondrial protein translation with chloramphenicol caused reversal of mitochondrial morphology in homozygous mutant MEFs, supporting the relevance of mitochondrial proteotoxicity for SCA28 pathogenesis and therapy development.

Impaired turnover of newly synthesized mitochondrial proteins of the oxidative phosphorylation complexes leads to protein over-accumulation in the inner mitochondrial membrane, thereby generating a stress that dissipates the mitochondrial membrane potential and therefore compromises organelle and cellular fitness., Mitochondrial ribosomes synthesize a subset of hydrophobic proteins required for assembly of the oxidative phosphorylation complexes. This process requires temporal and spatial coordination and regulation, so quality control of mitochondrial protein synthesis is paramount to maintain proteostasis. We show how impaired turnover of de novo mitochondrial proteins leads to aberrant protein accumulation in the mitochondrial inner membrane. This creates a stress in the inner membrane that progressively dissipates the mitochondrial membrane potential, which in turn stalls mitochondrial protein synthesis and fragments the mitochondrial network. The mitochondrial m-AAA protease subunit AFG3L2 is critical to this surveillance mechanism that we propose acts as a sensor to couple the synthesis of mitochondrial proteins with organelle fitness, thus ensuring coordinated assembly of the oxidative phosphorylation complexes from two sets of ribosomes.

Mammalian mitochondrial DNA (mtDNA) is a high-copy maternally inherited genome essential for aerobic energy metabolism. Mutations in mtDNA can lead to heteroplasmy, the co-occurence of two different mtDNA variants in the same cell, which can segregate in a tissue-specific manner affecting the onset and severity of mitochondrial dysfunction. To investigate mechanisms regulating mtDNA segregation we use a heteroplasmic mouse model with two polymorphic neutral mtDNA haplotypes (NZB and BALB) that displays tissue-specific and age-dependent selection for mtDNA haplotypes. In the hematopoietic compartment there is selection for the BALB mtDNA haplotype, a phenotype that can be modified by allelic variants of Gimap3. Gimap3 is a tail-anchored member of the GTPase of the immunity-associated protein (Gimap) family of protein scaffolds important for leukocyte development and survival. Here we show how the expression of two murine Gimap3 alleles from Mus musculus domesticus and M. m. castaneus differentially affect mtDNA segregation. The castaneus allele has incorporated a uORF (upstream open reading frame) in-frame with the Gimap3 mRNA that impairs translation and imparts a negative effect on the steady-state protein abundance. We found that quantitative changes in the expression of Gimap3 and the paralogue Gimap5, which encodes a lysosomal protein, affect mtDNA segregation in the mouse hematopoietic tissues. We also show that Gimap3 localizes to the endoplasmic reticulum and not mitochondria as previously reported. Collectively these data show that the abundance of protein scaffolds on the endoplasmic reticulum and lysosomes are important to the segregation of the mitochondrial genome in the mouse hematopoietic compartment.

We describe a novel frameshift mutation in the mitochondrial ATP6 gene in a 4-year-old girl associated with ataxia, microcephaly, developmental delay and intellectual disability. A heteroplasmic frameshift mutation in the MT-ATP6 gene was confirmed in the patient's skeletal muscle and blood. The mutation was not detectable in the mother's DNA extracted from blood or buccal cells. Enzymatic and oxymetric analysis of the mitochondrial respiratory system in the patients' skeletal muscle and skin fibroblasts demonstrated an isolated complex V deficiency. Native PAGE with subsequent immunoblotting for complex V revealed impaired complex V assembly and accumulation of ATPase subcomplexes. Whilst northern blotting confirmed equal presence of ATP8/6 mRNA, metabolic S-35-labelling of mitochondrial translation products showed a severe depletion of the ATP6 protein together with aberrant translation product accumulation. In conclusion, this novel isolated complex V defect expands the clinical and genetic spectrum of mitochondrial defects of complex V deficiency. Furthermore, this work confirms the benefit of native PAGE as an additional diagnostic method for the identification of OXPHOS defects, as the presence of complex V subcomplexes is associated with pathogenic mutations of mtDNA. (C) 2017 Elsevier Masson SAS. All rights reserved.

SummaryProliferating cells require coordinated gene expression between the nucleus and mitochondria in order to divide, ensuring sufficient organelle number in daughter cells [1]. However, the machinery and mechanisms whereby proliferating cells monitor mitochondria and coordinate organelle biosynthesis remain poorly understood. Antibiotics inhibiting mitochondrial translation have emerged as therapeutics for human cancers because they block cell proliferation [2, 3]. These proliferative defects were attributable to modest decreases in mitochondrial respiration [3, 4], even though tumors are mainly glycolytic [5] and mitochondrial respiratory chain function appears to play a minor role in cell proliferation in vivo [6]. Here we challenge this interpretation by demonstrating that one class of antiproliferative antibiotic induces stalled mitochondrial ribosomes, which triggers a mitochondrial ribosome and RNA decay pathway. Rescue of the stalled mitochondrial ribosomes initiates a retrograde signaling response to block cell proliferation and occurs prior to any loss of mitochondrial respiration. The loss of respiratory chain function is simply a downstream effect of impaired mitochondrial translation and not the antiproliferative signal. This mitochondrial ribosome quality-control pathway is actively monitored in cells and constitutes an important organelle checkpoint for cell division.

Background The genetic complexity of infantile cardiomyopathies is remarkable, and the importance of mitochondrial translation defects as a causative factor is only starting to be recognised. We investigated the genetic basis for infantile onset recessive hypertrophic cardiomyopathy in two siblings. Methods and results Analysis of respiratory chain enzymes revealed a combined deficiency of complexes I and IV in the heart and skeletal muscle. Exome sequencing uncovered a homozygous mutation (L156R) in MRPL44 of both siblings. MRPL44 encodes a protein in the large subunit of the mitochondrial ribosome and is suggested to locate in close proximity to the tunnel exit of the yeast mitochondrial ribosome. We found severely reduced MRPL44 levels in the patient9s heart, skeletal muscle and fibroblasts suggesting that the missense mutation affected the protein stability. In patient fibroblasts, decreased MRPL44 affected assembly of the large ribosomal subunit and stability of 16S rRNA leading to complex IV deficiency. Despite this assembly defect, de novo mitochondrial translation was only mildly affected in fibroblasts suggesting that MRPL44 may have a function in the assembly/stability of nascent mitochondrial polypeptides exiting the ribosome. Retroviral expression of wild-type MRPL44 in patient fibroblasts rescued the large ribosome assembly defect and COX deficiency. Conclusions These findings indicate that mitochondrial ribosomal subunit defects can generate tissue-specific manifestations, such as cardiomyopathy.

Mitochondria are dynamic organelles that divide and fuse by remodeling an outer and inner membrane in response to developmental, physiological and stress stimuli. These events are coordinated by conserved dynamin-related GTPases. The dynamics of mitochondrial morphology require coordination with mitochondrial DNA (mtDNA) to ensure faithful genome transmission, however, this process remains poorly understood. Mitochondrial division is linked to the segregation of mtDNA but how it affects cases of mtDNA heteroplasmy, where two or more mtDNA variants/mutations co-exist in a cell, is unknown. Segregation of heteroplasmic human pathogenic mtDNA mutations is a critical factor in the onset and severity of human mitochondrial diseases. Here, we investigated the coupling of mitochondrial morphology to the transmission and segregation of mtDNA in mammals by taking advantage of two genetically modified mouse models: one with a dominant-negative mutation in the dynamin-related protein 1 (Drp1 or Dnm1l) that impairs mitochondrial fission and the other, heteroplasmic mice segregating two neutral mtDNA haplotypes (BALB and NZB). We show a tissue-specific response to mtDNA segregation from a defect in mitochondrial fission. Only mtDNA segregation in the hematopoietic compartment is modulated from impaired Dnm1l function. In contrast, no effect was observed in other tissues arising from the three germ layers during development and in mtDNA transmission through the female germline. Our data suggest a robust organization of a heteroplasmic mtDNA segregating unit across mammalian cell types that can overcome impaired mitochondrial division to ensure faithful transmission of the mitochondrial genome.

Mitochondrial DNA (mtDNA) is a multi-copy genome encoding for proteins essential for aerobic energy metabolism. Mutations in mtDNA can lead to a variety of human diseases, from mild metabolic syndromes to severe fatal encephalomyopathies. Most mtDNA mutations co-exist with wild type genomes in a state known as heteroplasmy. The segregation of these pathogenic mutants is tissue and mutation specific, and a key determinant in the onset and severity of human mitochondrial disorders. We used a forward genetic approach in mice to identify and demonstrate that Gimap3 (GTP ase of immunity associated protein) is a key regulator of mtDNA segregation in leukocytes. The Gimap gene cluster is found only in vertebrates and appear to be a class of nucleotide-dependent dimerization GTP ases. Gimap3 is a membrane-anchored GTP ase with a critical role in T cell development. Here, we summarize our genetic findings and postulate how Gimap3 might regulate mtDNA genetics.

Summary Mitochondrial dysfunction affects cellular energy metabolism, but less is known about the consequences for cytoplasmic biosynthetic reactions. We report that mtDNA replication disorders caused by TWINKLE mutations—mitochondrial myopathy (MM) and infantile onset spinocerebellar ataxia (IOSCA)—remodel cellular dNTP pools in mice. MM muscle shows tissue-specific induction of the mitochondrial folate cycle, purine metabolism, and imbalanced and increased dNTP pools, consistent with progressive mtDNA mutagenesis. IOSCA-TWINKLE is predicted to hydrolyze dNTPs, consistent with low dNTP pools and mtDNA depletion in the disease. MM muscle also modifies the cytoplasmic one-carbon cycle, transsulfuration, and methylation, as well as increases glucose uptake and its utilization for de novo serine and glutathione biosynthesis. Our evidence indicates that the mitochondrial replication machinery communicates with cytoplasmic dNTP pools and that upregulation of glutathione synthesis through glucose-driven de novo serine biosynthesis contributes to the metabolic stress response. These results are important for disorders with primary or secondary mtDNA instability and offer targets for metabolic therapy.

Frequently, mtDNA with pathogenic mutations coexist with wild-type genomes (mtDNA heteroplasmy). Mitochondrial dysfunction and disease ensue only when the proportion of mutated mtDNAs is high, thus a reduction in this proportion should provide an effective therapy for these disorders. We developed a system to decrease specific mtDNA haplotypes by expressing a mitochondrially targeted restriction endonuclease, ApaLI, in cells of heteroplasmic mice. These mice have two mtDNA haplotypes, of which only one contains an ApaLI site. After transfection of cultured hepatocytes with mitochondrially targeted ApaLI, we found a rapid, directional, and complete shift in mtDNA heteroplasmy (2-6 h). We tested the efficacy of this approach in vivo , by using recombinant viral vectors expressing the mitochondrially targeted ApaLI. We observed a significant shift in mtDNA heteroplasmy in muscle and brain transduced with recombinant viruses. This strategy could prevent disease onset or reverse clinical symptoms in patients harboring certain heteroplasmic pathogenic mutations in mtDNA.

Mutations in mitochondrial DNA (mtDNA) are associated with a broad spectrum of clinical disorders. The segregation pattern of pathogenic mtDNAs is an important determinant of both the onset and the severity of the disease phenotype, but the mechanisms controlling mtDNA segregation remain poorly understood. To investigate this, we previously generated heteroplasmic mice containing two different mtDNA haplotypes and showed that BALB/c mtDNA was invariably selected over NZB mtDNA in blood and spleen. Here, we have characterized this process in hematopoietic tissues and tested whether it involves the presentation of mtDNA-encoded peptides by MHC class Ib molecules. Selection against NZB mtDNA was widespread across different hematopoietic cell lineages and proportional to heteroplasmy levels. Backcrossing heteroplasmic mice with CAST/Ei, a strain in which the MHC class Ib molecule H2-M3 is silent, completely abolished selection against NZB mtDNA in the spleen. To test whether this effect depended on an intact immune system, we generated heteroplasmic mice missing functional copies of Tap1, beta2m or Rag1 to impair presentation or recognition of mtDNA-encoded peptides. The kinetics of selection against NZB mtDNA were unaltered in these mice compared with their wild-type littermates. We conclude that mtDNA selection in hematopoietic tissues is not based on an immune mechanism, but likely involves metabolic signaling.

Variants of mitochondrial DNA (mtDNA) are commonly used as markers to track human evolution because of the high sequence divergence and exclusive maternal inheritance. It is assumed that the inheritance is clonal, i.e. that mtDNA is transmitted between generations without germline recombination. In contrast to this assumption, a number of studies have reported the presence of recombinant mtDNA molecules in cell lines and animal tissues, including humans. If germline recombination of mtDNA is frequent, it would strongly impact phylogenetic and population studies by altering estimates of coalescent time and branch lengths in phylogenetic trees. Unfortunately, this whole area is controversial and the experimental approaches have been widely criticized as they often depend on polymerase chain reaction (PCR) amplification of mtDNA and/or involve studies of transformed cell lines. In this study, we used an in vivo mouse model that has had germline heteroplasmy for a defined set of mtDNA mutations for more than 50 generations. To assess recombination, we adapted and validated a method based on cloning of single mtDNA molecules in the lambda phage, without prior PCR amplification, followed by subsequent mutation analysis. We screened 2922 mtDNA molecules and found no germline recombination after transmission of mtDNA under genetically and evolutionary relevant conditions in mammals.

This study examined the seasonal and reproductive influences on individual plasma amino acid concentrations and nitrogen metabolites in a black bear population (Ontario, Canada). During hibernation, 11 of 23 plasma amino acids were significantly higher (13%–108%) in lactating than in nonlactating females, without an alteration in plasma total protein or total essential or nonessential amino acid levels. The greatest changes were observed in glutamine, arginine, and glycine levels. Plasma urea, urea/creatinine, and ammonia levels were significantly lower in hibernating compared with active female bears, but lactation had no effect on these parameters. Taken together these results show that lactation during hibernation is an additional metabolic challenge that results in increased mobilization of individual plasma amino acids and no accumulation of nitrogen end products, underlining the remarkable efficiency of amino acid and urea recycling in denning female black bears.

We examined whether the relationships between mitochondrial enzyme activity, mitochondrial DNA (mtDNA) and mitochondrial RNA (mtRNA) were conserved in rainbow trout (Oncorhynchus mykiss) tissues that differ widely in their metabolic and molecular organization. The activity of citrate synthase (CS), expressed either per gram of tissue or per milligram of total DNA, indicated that these tissues (blood, brain, kidney, liver, cardiac, red and white muscles) varied more than 100-fold in mitochondrial content. Several-fold differences in the levels of CS mRNA per milligram of DNA and CS activity per CS mRNA were also observed, suggesting that fundamental differences exist in the regulation of CS levels across tissues. Although tissues varied 14-fold in RNA g−1, poly(A+) RNA (mRNA) was approximately 2 % of total RNA in all tissues. DNA g−1 also varied 14-fold across tissues, but RNA:DNA ratios varied only 2.5-fold. The relationship between two mitochondrial mRNA species (COX I, ATPase VI) and one mitochondrial rRNA (16S) species was constant across tissues. The ratio of mtRNA to mtDNA was also preserved across most tissues; red and white muscle had 10-to 20-fold lower levels of mtDNA g−1 but 7-to 10-fold higher mtRNA:mtDNA ratios, respectively. Collectively, these data suggest that the relationship between mitochondrial parameters is highly conserved across most tissues, but that skeletal muscles differ in a number of important aspects of respiratory gene expression (‘respiratory genes’ include genes located on mtDNA and genes located in the nucleus that encode mitochondrial protein) and mtDNA transcriptional regulation.

Skeletal muscle fibers typically undergo modifications in their mitochondrial content, concomitant with alterations in oxidative metabolism that occur during the development of muscle fiber and in response to physiological stimuli. We examined how cold acclimation affects the mitochondrial properties of two fish skeletal muscle fiber types and how the regulators of mitochondrial content differed between tissues. After 2 mo of acclimation to either 4 or 18°C, mitochondrial enzyme activities in both red and white muscle were higher in cold-acclimated fish. No significant differences were detected between acclimation temperatures in the abundance of steady-state mitochondrial mRNA (cytochrome- c oxidase 1, subunit 6 of F0F1-ATPase), rRNA (16S), or DNA copy number. Steady-state mRNA for nuclear-encoded respiratory (adenine nucleotide translocase 1) and glycolytic genes showed high interindividual variability, particularly in the cold-acclimated fish. Although mitochondrial enzymes were 10-fold different between the two muscle types, mitochondrial DNA copy number differed only 4-fold. The relative abundance of mitochondrial mRNA and nuclear mRNA in red and white muscle reflected the differences in copy number of their respective genes. These data suggest that the response to physiological stimuli and determination of tissue-specific mitochondrial properties likely result from the regulation of nuclear-encoded genes.

We studied the interaction between energy metabolism and mitochondrial biogenesis during myogenesis in C2C12 myoblasts. Metabolic rate was nearly constant throughout differentiation, although there was a shift in the relative importance of glycolytic and oxidative metabolism, accompanied by increases in pyruvate dehydrogenase activation state and total activity. These changes in mitochondrial bioenergetic parameters observed during differentiation occurred in the absence of a hypermetabolic stress. A chronic (3 day) energetic stress was imposed on differentiated myotubes using sodium azide to inhibit oxidative metabolism. When used at low concentrations, azide inhibited more than 70% of cytochrome oxidase (COX) activity without changes in bioenergetics (either lactate production or creatine phosphorylation) or mRNA for mitochondrial enzymes. Higher azide concentrations resulted in changes in bioenergetic parameters and increases in steady state COX II mRNA levels. Azide did not affect mtDNA copy number or mRNA levels for other mitochondrial transcripts, suggesting azide affects stability, rather than synthesis, of COX II mRNA. These results indicate that changes in bioenergetics can alter mitochondrial genetic regulation, but that mitochondrial biogenesis accompanying differentiation occurs in the absence of hypermetabolic challenge.

Mitochondria are responsible for the generation of ATP to fuel muscle contraction. Hypermetabolic stresses imposed upon muscles can lead to mitochondrial proliferation, but the resulting mitochondria greatly resemble their progenitors. During the mitochondrial biogenesis that accompanies phenotypic adaptation, the stoichiometric relationships between functional elements are preserved through shared sensitivities of respiratory genes to specific transcription factors. Although the properties of muscle mitochondria are generally thought to be highly conserved across species, there are many examples of mitochondrial differences between muscle types, species and developmental states and even within single cells. In this review, we discuss (1) the nature and regulation of gene families that allow coordinated expression of genes for mitochondrial products and (2) the regulatory mechanisms by which mitochondrial differences can arise over physiological and evolutionary time.

Summary Organelle biosynthesis is a key requirement for cell growth and division. The regulation of mitochondrial biosynthesis exhibits additional layers of complexity compared with that of other organelles because they contain their own genome and dedicated ribosomes. Maintaining these components requires gene expression to be coordinated between the nucleo-cytoplasmic compartment and mitochondria in order to monitor organelle homeostasis and to integrate the responses to the physiological and developmental demands of the cell. Surprisingly, the parameters that are used to monitor or count mitochondrial abundance are not known, nor are the signalling pathways. Inhibiting the translation on mito-ribosomes genetically or with antibiotics can impair cell proliferation and has been attributed to defects in aerobic energy metabolism, even though proliferating cells rely primarily on glycolysis to fuel their metabolic demands. However, a recent study indicates that mitochondrial translational stress and the rescue mechanisms that relieve this stress cause the defect in cell proliferation and occur before any impairment of oxidative phosphorylation. Therefore, the process of mitochondrial translation in itself appears to be an important checkpoint for the monitoring of mitochondrial homeostasis and might have a role in establishing mitochondrial abundance within a cell. This hypothesis article will explore the evidence supporting a role for mito-ribosomes and translation in a mitochondria-counting mechanism.

Plasma 3,5,3'-triiodothyronine (T3) concentration decreased significantly (P < 0.05), during 1-5 days of captivity, from levels in the freshly caught dogfish shark Squalus acanthias. The short-term effects of T3 treatment on the intermediary metabolism of S. acanthias were measured in the gill, kidney, liver, and white muscle. Animals were kept for 1-5 days before experimentation. Three hours after an intraperitoneal injection with either a low T3 dose (8.3 pmol T3/kg fish) or a high T3 dose (830 pmol T3/kg fish), selected enzymes of amino acid metabolism, lipid catabolism, ketone body metabolism, glycolysis, and oxidative metabolism were measured. Activity of enzymes of amino acid metabolism and lipid catabolism increased significantly (P < 0.05) in the liver of fish treated with a low T3 dose. The low dose of T3 apparently influences glycolysis as pyruvate kinase activity significantly increase (P < 0.05) in the kidney and white muscle.

Mitochondrial DNA (mtDNA) is essential for aerobic energy production in eukaryotic cells, and mutations in this genome can lead to mitochondrial dysfunction. Human mtDNA mutations are typically heteroplasmic, a mix of mutant and wild-type genomes, which can present as a heterogeneous group of disorders ranging in severity from mild to fatal, and commonly affecting highly aerobic tissues such as heart, skeletal muscle, and neurons. During the 1990s, many research groups started to notice that mtDNA mutations could segregate depending upon the mutation and tissue. This segregation pattern can have a direct effect on the onset and severity of these mutations. However, these segregation patterns could not be easily explained by respiratory chain function, implying that there is regulation of mtDNA independent of its bioenergetic role. A lot of research on this topic has been largely descriptive, but over the last several years advances in mitochondrial biology have provided some mechanistic insight into the regulation of the organelle and its genome. This review addresses these advances with respect to somatic segregation of mtDNA in mammals.

Mitochondrial DNA (mtDNA) sequence variants segregate in mutation and tissue-specific manners, but the mechanisms remain unknown. The segregation pattern of pathogenic mtDNA mutations is a major determinant of the onset and severity of disease. Using a heteroplasmic mouse model, we demonstrate that Gimap3, an outer mitochondrial membrane GTPase, is a critical regulator of this process in leukocytes. Gimap3 is important for T cell development and survival, suggesting that leukocyte survival may be a key factor in the genetic regulation of mtDNA sequence variants and in modulating human mitochondrial diseases., Author Summary Mitochondria are essential cellular organelles, which contain their own small circular genome in mammals. The mitochondrial genome is maternally inherited and encodes proteins critical for aerobic energy production. Mutations in mitochondrial DNA can lead to disruptions in aerobic energy production, which manifest as a wide spectrum of human clinical disorders. These mutations can segregate in mitotic and post-mitotic tissues, so that the proportion of the mutant genome can change with age, thereby affecting the clinical onset and severity of disease. The mechanisms regulating mtDNA segregation in mammals are not understood. We have used a mouse model of mtDNA segregation to elucidate the molecular basis of this process. Here, we demonstrate that the gene, Gimap3, can regulate mtDNA segregation in mouse white blood cells. This is the first gene identified to regulate mtDNA segregation in mammals, which is also an important factor in cell survival. Future work on this protein will provide critical insight into the pathways that regulate mitochondrial DNA in white blood cells, an important factor in the pathogenesis of human mitochondrial DNA mutations.

To perform preimplantation genetic diagnosis for women carrying heteroplasmic mitochondrial DNA (mtDNA) mutations, it is necessary to ensure that the proportion of mutant mtDNA diagnosed in the biopsied cell gives an accurate indication of the mutant load in the remaining embryo. A heteroplasmic mouse model, carrying NZB and BALB mtDNA genotypes, was used to study the relative proportions of each mtDNA genotype in the ooplasm and first polar body of mature oocytes, and between blastomeres of early cleavage stage embryos. The levels of heteroplasmy varied widely in the gametes compared with the maternal genotype. However, the distribution of the two mtDNA genotypes was virtually identical between the ooplasm and polar body of a mature oocyte, and also between the blastomeres of each 2-, 4- and 6-8-cell embryo. Therefore, the level of heteroplasmy diagnosed from the polar body of an unfertilized oocyte or from a single blastomere of an embryo is representative of the level in the embryo as a whole. Reliable results were obtained from both polar bodies and blastomeres, but the efficiency of diagnosis was greater with blastomeres. We conclude that preimplantation genetic diagnosis is feasible for mtDNA diseases, although it should be approached with caution, as it is possible that transmission of some pathogenic mutations could behave in a different manner.

Mammalian mitochondrial DNA (mtDNA) is a high copy-number, maternally inherited genome that codes for a small number of essential proteins involved in oxidative phosphorylation. Mutations in mtDNA are responsible for a broad spectrum of clinical disorders1. The segregation pattern of pathogenic mtDNA mutants is an important determinant of the nature and severity of mitochondrial disease, but it varies with the specific mutation, cell type and nuclear background and generally does not correlate well with mitochondrial dysfunction2, 3, 4, 5, 6, 7, 8, 9, 10, 11. To identify nuclear genes that modify the segregation behavior of mtDNA, we used a heteroplasmic mouse model derived from two inbred strains (BALB/c and NZB; ref. 12), in which we had previously demonstrated tissue-specific and age-dependent directional selection for different mtDNA genotypes in the same mouse13. Here we show that this phenotype segregates in F2 mice from a genetic cross (BALB/c CAST/Ei) and that it maps to at least three quantitative-trait loci (QTLs). Genome-wide scans showed linkage of the trait to loci on Chromosomes 2, 5 and 6, accounting for 16–35% of the variance in the trait, depending on the tissue and age of the mouse. This is the first genetic evidence for nuclear control of mammalian mtDNA segregation.

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We have previously constructed lines of heteroplasmic mice from two inbred strains (NZB/BinJ and BALB/c) to investigate the mechanisms of segregation of mtDNA sequence variants. Analysis of the segregation behaviour of mtDNA in several tissues showed that the NZB genotype was invariably selected in liver/kidney and the BALB genotype in blood/spleen. Segregation was not significant in post-mitotic tissues. Here we have investigated this novel pattern of mtDNA segregation in isolated hepatocytes to determine the mechanism of selection. Polarographic measurements of respiratory chain function showed no difference between mitochondria containing either 0 or 91-97% NZB mtDNAs on a BALB nuclear background. Single-cell PCR analysis of mtDNA in isolated hepatocytes demonstrated that most hepatocytes eventually fix the NZB genotype. The rate of selection was constant with time and independent of the initial genotype frequency. Based on a mtDNA replication rate of 9.4 days, NZB mtDNA has an approximately 14% selective advantage over BALB mtDNA; however, in vivo pulse labelling with BrdU demonstrated that this was not based on efficiency of replication. Surprisingly, when hepatocytes were cultured in vitro, the majority of independent colonies selected BALB mtDNA, even if they were nearly fixed for the NZB mtDNA genotype when initially plated. These data suggest that selection for NZB mtDNA in the liver of these mice is not based on respiratory chain function at the cellular or organellar level, or a simple replicative advantage, but on a factor(s) involved with mtDNA maintenance.