bims-mitdis Biomed News
on Mitochondrial Disorders
Issue of 2021‒04‒18
fifty-five papers selected by
Catalina Vasilescu
University of Helsinki

  1. Genome Med. 2021 Apr 12. 13(1): 55
      BACKGROUND: ATPase family AAA-domain containing protein 3A (ATAD3A) is a nuclear-encoded mitochondrial membrane-anchored protein involved in diverse processes including mitochondrial dynamics, mitochondrial DNA organization, and cholesterol metabolism. Biallelic deletions (null), recessive missense variants (hypomorph), and heterozygous missense variants or duplications (antimorph) in ATAD3A lead to neurological syndromes in humans.METHODS: To expand the mutational spectrum of ATAD3A variants and to provide functional interpretation of missense alleles in trans to deletion alleles, we performed exome sequencing for identification of single nucleotide variants (SNVs) and copy number variants (CNVs) in ATAD3A in individuals with neurological and mitochondrial phenotypes. A Drosophila Atad3a Gal4 knockin-null allele was generated using CRISPR-Cas9 genome editing technology to aid the interpretation of variants.
    RESULTS: We report 13 individuals from 8 unrelated families with biallelic ATAD3A variants. The variants included four missense variants inherited in trans to loss-of-function alleles (p.(Leu77Val), p.(Phe50Leu), p.(Arg170Trp), p.(Gly236Val)), a homozygous missense variant p.(Arg327Pro), and a heterozygous non-frameshift indel p.(Lys568del). Affected individuals exhibited findings previously associated with ATAD3A pathogenic variation, including developmental delay, hypotonia, congenital cataracts, hypertrophic cardiomyopathy, and cerebellar atrophy. Drosophila studies indicated that Phe50Leu, Gly236Val, Arg327Pro, and Lys568del are severe loss-of-function alleles leading to early developmental lethality. Further, we showed that Phe50Leu, Gly236Val, and Arg327Pro cause neurogenesis defects. On the contrary, Leu77Val and Arg170Trp are partial loss-of-function alleles that cause progressive locomotion defects and whose expression leads to an increase in autophagy and mitophagy in adult muscles.
    CONCLUSION: Our findings expand the allelic spectrum of ATAD3A variants and exemplify the use of a functional assay in Drosophila to aid variant interpretation.
    Keywords:  AAA+ protein; ATAD3A; Autophagy; Autosomal recessive; Disease; Drosophila; Mitochondria; Neurogenesis
  2. J Inherit Metab Dis. 2021 Apr 14.
      Glutamyl-tRNA synthetase 2 (encoded by EARS2) is a mitochondrial aminoacyl-tRNA synthetase required to translate the 13 subunits of the electron transport chain encoded by the mitochondrial DNA. Pathogenic EARS2 variants cause combined oxidative phosphorylation deficiency, subtype 12 (COXPD12), an autosomal recessive disorder involving lactic acidosis, intellectual disability and other features of mitochondrial compromise. Patients with EARS2 deficiency present with variable phenotypes ranging from neonatal lethality to a mitigated disease with clinical improvement in early childhood. Here, we report a neonate homozygous for a rare pathogenic variant in EARS2 (c.949G>T; p.G317C). Metabolomics in primary fibroblasts from this patient revealed expected abnormalities in TCA cycle metabolites, as well as numerous changes in purine, pyrimidine and fatty acid metabolism. To examine genotype-phenotype correlations in COXPD12, we compared the metabolic impact of reconstituting these fibroblasts with wild-type EARS2 vs. four additional EARS2 variants from COXPD12 patients with varying clinical severity. Metabolomics identified a group of signature metabolites, mostly from the TCA cycle and amino acid metabolism, that discriminate between EARS2 variants causing relatively mild and severe COXPD12. Taken together, these findings indicate that metabolomics in patient-derived fibroblasts may help establish genotype-phenotype correlations in EARS2 deficiency and likely other mitochondrial disorders. This article is protected by copyright. All rights reserved.
    Keywords:  EARS2; Mitochondria; genotype-phenotype correlation; inborn errors of metabolism; lactic acidosis; metabolomics
  3. Nat Commun. 2021 Apr 16. 12(1): 2304
      Mitochondria play a pivotal role in the generation of signals coupling metabolism with neurotransmitter release, but a role for mitochondrial-produced ROS in regulating neurosecretion has not been described. Here we show that endogenously produced hydrogen peroxide originating from axonal mitochondria (mtH2O2) functions as a signaling cue to selectively regulate the secretion of a FMRFamide-related neuropeptide (FLP-1) from a pair of interneurons (AIY) in C. elegans. We show that pharmacological or genetic manipulations that increase mtH2O2 levels lead to increased FLP-1 secretion that is dependent upon ROS dismutation, mitochondrial calcium influx, and cysteine sulfenylation of the calcium-independent PKC family member PKC-1. mtH2O2-induced FLP-1 secretion activates the oxidative stress response transcription factor SKN-1/Nrf2 in distal tissues and protects animals from ROS-mediated toxicity. mtH2O2 levels in AIY neurons, FLP-1 secretion and SKN-1 activity are rapidly and reversibly regulated by exposing animals to different bacterial food sources. These results reveal a previously unreported role for mtH2O2 in linking diet-induced changes in mitochondrial homeostasis with neuropeptide secretion.
  4. Biosystems. 2021 Apr 12. pii: S0303-2647(21)00081-2. [Epub ahead of print] 104426
      Mitochondria's significance in human diseases and in functioning, health and death of eukaryotic cell has been acknowledged widely. Yet our perspective in cell biology and evolution remains nucleocentric. Mitochondrial DNA, by virtue of its omnipresence and species-level conservation, is used as a barcode in animal taxonomy. This article analyses various levels of containment structures that enclose mitochondrial DNA and advocates a fresh perspective wherein evolution of organic structures of the eukarya domain seem to support and facilitate survival and proliferation of mitochondrial DNA by splitting containers as they age and by directing them along two distinct pathways: destruction of containers with more mutant mitochondrial DNA and rejuvenation of containers with less mutant mitochondrial DNA.
    Keywords:  Eukaryotes; Eukaryotic cell; Evolution; Mitochondria; Mitochondrial DNA
  5. Proc Natl Acad Sci U S A. 2021 Apr 20. pii: e2004253118. [Epub ahead of print]118(16):
      Mitochondrial ATP production is a well-known regulator of neuronal excitability. The reciprocal influence of plasma-membrane potential on ATP production, however, remains poorly understood. Here, we describe a mechanism by which depolarized neurons elevate the somatic ATP/ADP ratio in Drosophila glutamatergic neurons. We show that depolarization increased phospholipase-Cβ (PLC-β) activity by promoting the association of the enzyme with its phosphoinositide substrate. Augmented PLC-β activity led to greater release of endoplasmic reticulum Ca2+ via the inositol trisphosphate receptor (IP3R), increased mitochondrial Ca2+ uptake, and promoted ATP synthesis. Perturbations that decoupled membrane potential from this mode of ATP synthesis led to untrammeled PLC-β-IP3R activation and a dramatic shortening of Drosophila lifespan. Upon investigating the underlying mechanisms, we found that increased sequestration of Ca2+ into endolysosomes was an intermediary in the regulation of lifespan by IP3Rs. Manipulations that either lowered PLC-β/IP3R abundance or attenuated endolysosomal Ca2+ overload restored animal longevity. Collectively, our findings demonstrate that depolarization-dependent regulation of PLC-β-IP3R signaling is required for modulation of the ATP/ADP ratio in healthy glutamatergic neurons, whereas hyperactivation of this axis in chronically depolarized glutamatergic neurons shortens animal lifespan by promoting endolysosomal Ca2+ overload.
    Keywords:  ER Ca2+ signaling; aging; longevity; lysosomes; neuronal excitability
  6. Methods Mol Biol. 2021 ;2281 265-272
      The mitochondrial single-stranded DNA-binding protein (mtSSB) regulates the function of the mitochondrial DNA (mtDNA) replisome. In vitro, mtSSB stimulates the activity of enzymatic components of the replisome, namely mtDNA helicase and DNA polymerase gamma (Pol γ). We have demonstrated that the stimulatory properties of mtSSB result from its ability to organize the single-stranded DNA template in a specific manner. Here we present methods employing electron microscopy and enzymatic assays to characterize and classify the mtSSB-DNA complexes and their effects on the activity of Pol γ.
    Keywords:  DNA polymerase γ; Electron microscopy; Mitochondrial DNA replication; Mitochondrial single-stranded DNA-binding protein; Nucleoprotein complexes
  7. J Cell Biol. 2021 Jun 07. pii: e202006043. [Epub ahead of print]220(6):
      Here, we report that acute reduction in mitochondrial translation fidelity (MTF) causes ubiquitination of the inner mitochondrial membrane (IMM) proteins, including TRAP1 and CPOX, which occurs selectively in mitochondria with a severed outer mitochondrial membrane (OMM). Ubiquitinated IMM recruits the autophagy machinery. Inhibiting autophagy leads to increased accumulation of mitochondria with severed OMM and ubiquitinated IMM. This process occurs downstream of the accumulation of cytochrome c/CPOX in a subset of mitochondria heterogeneously distributed throughout the cell ("mosaic distribution"). Formation of mosaic mitochondria, OMM severing, and IMM ubiquitination require active mitochondrial translation and mitochondrial fission, but not the proapoptotic proteins Bax and Bak. In contrast, in Parkin-overexpressing cells, MTF reduction does not lead to the severing of the OMM or IMM ubiquitination, but it does induce Drp1-independent ubiquitination of the OMM. Furthermore, high-cytochrome c/CPOX mitochondria are preferentially targeted by Parkin, indicating that in the context of reduced MTF, they are mitophagy intermediates regardless of Parkin expression. In sum, Parkin-deficient cells adapt to mitochondrial proteotoxicity through a Drp1-mediated mechanism that involves the severing of the OMM and autophagy targeting ubiquitinated IMM proteins.
  8. Front Neurol. 2021 ;12 602979
      Advances in next-generation sequencing (NGS) facilitate the diagnosis of genetic disorders. To evaluate its use for the molecular diagnosis of inherited optic neuropathy (ION), a blinding disease caused by the degeneration of retinal ganglion cells, we performed genetic analysis using targeted NGS of 22 already known and candidate genes in a cohort of 1,102 affected individuals. The panel design, library preparation, and sequencing reactions were performed using the Ion AmpliSeq technology. Pathogenic variants were detected in 16 genes in 245 patients (22%), including 186 (17%) and 59 (5%) dominant and recessive cases, respectively. Results confirmed that OPA1 variants are responsible for the majority of dominant IONs, whereas ACO2 and WFS1 variants are also frequently involved in both dominant and recessive forms of ION. All pathogenic variants were found in genes encoding proteins involved in the mitochondrial function, highlighting the importance of mitochondria in the survival of retinal ganglion cells.
    Keywords:  inherited optic neuropathies; mitochondrial disorders; molecular diagnosis; next generation sequencing; retinal ganglia cells
  9. Cell Rep. 2021 Apr 13. pii: S2211-1247(21)00277-1. [Epub ahead of print]35(2): 108963
      The assembly pathways of mitochondrial respirasome (supercomplex I+III2+IV) are not fully understood. Here, we show that an early sub-complex I assembly, rather than holo-complex I, is sufficient to initiate mitochondrial respirasome assembly. We find that a distal part of the membrane arm of complex I (PD-a module) is a scaffold for the incorporation of complexes III and IV to form a respirasome subcomplex. Depletion of PD-a, rather than other complex I modules, decreases the steady-state levels of complexes III and IV. Both HEK293T cells lacking TIMMDC1 and patient-derived cells with disease-causing mutations in TIMMDC1 showed accumulation of this respirasome subcomplex. This suggests that TIMMDC1, previously known as a complex-I assembly factor, may function as a respirasome assembly factor. Collectively, we provide a detailed, cooperative assembly model in which most complex-I subunits are added to the respirasome subcomplex in the lateral stages of respirasome assembly.
    Keywords:  Leigh syndrome; TIMMDC1; cooperative assembly; mitochondrial respirasome; oxidative phosphorylation
  10. Adv Exp Med Biol. 2021 ;1256 237-264
      Strong experimental evidence from studies in human donor retinas and animal models supports the idea that the retinal pathology associated with age-related macular degeneration (AMD) involves mitochondrial dysfunction and consequent altered retinal metabolism. This chapter provides a brief overview of mitochondrial structure and function, summarizes evidence for mitochondrial defects in AMD, and highlights the potential ramifications of these defects on retinal health and function. Discussion of mitochondrial haplogroups and their association with AMD brings to light how mitochondrial genetics can influence disease outcome. As one of the most metabolically active tissues in the human body, there is strong evidence that disruption in key metabolic pathways contributes to AMD pathology. The section on retinal metabolism reviews cell-specific metabolic differences and how the metabolic interdependence of each retinal cell type creates a unique ecosystem that is disrupted in the diseased retina. The final discussion includes strategies for therapeutic interventions that target key mitochondrial pathways as a treatment for AMD.
    Keywords:  Age-related macular degeneration; Ecosystem model; Metabolism; Mitochondria; Proteomics; mtDNA
  11. Methods Mol Biol. 2021 ;2281 313-322
      Defects in mitochondrial DNA (mtDNA) maintenance may lead to disturbances in mitochondrial homeostasis and energy production in eukaryotic cells, causing diseases. During mtDNA replication, the mitochondrial single-stranded DNA-binding protein (mtSSB) stabilizes and protects the exposed single-stranded mtDNA from nucleolysis; perhaps more importantly, it appears to coordinate the actions of both the replicative mtDNA helicase Twinkle and DNA polymerase gamma at the replication fork. Here, we describe a helicase stimulation protocol to test in vitro the functional interaction between mtSSB and variant forms of Twinkle. We show for the first time that the C-terminal tail of Twinkle is important for such an interaction, and that it negatively regulates helicase unwinding activity in a salt-dependent manner.
    Keywords:  Mitochondrial DNA replication; P66; Twinkle; dsDNA unwinding assay; mtSSB
  12. Neurol Clin Pract. 2021 Apr;11(2): 97-104
      Objective: To determine the prevalence of neuromuscular junction (NMJ) abnormalities in patients with mitochondrial disease.Methods: Eighty patients with genetically proven mitochondrial disease were recruited from a national center for mitochondrial disease in the United Kingdom. Participants underwent detailed clinical and neurophysiologic testing including single-fiber electromyography.
    Results: The overall prevalence of neuromuscular transmission defects was 25.6%. The highest prevalence was in patients with pathogenic dominant RRM2B variants (50%), but abnormalities were found in a wide range of mitochondrial genotypes. The presence of NMJ abnormalities was strongly associated with coexistent myopathy, but not with neuropathy. Furthermore, 15% of patients with NMJ abnormality had no evidence of either myopathy or neuropathy.
    Conclusions: NMJ transmission defects are common in mitochondrial disease. In some patients, NMJ dysfunction occurs in the absence of obvious pre- or post-synaptic pathology, suggesting that the NMJ may be specifically affected.
  13. Anal Methods. 2021 Apr 14. 13(14): 1715-1719
      Mitochondria are important organelles in cells, which play an important role in metabolism and many other vital biological events. Mitochondrial membrane potential (MMP) is a significant biological parameter participating in various procedures. However, fluorescent probes for monitoring MMP are rarely reported, which greatly limited the related studies. Herein, we present the rational design, synthesis, and living cell imaging studies of a fluorescent probe REP for monitoring MMP changes based on organic cationic fluorophores. In live cells with high MMP levels, REP can exclusively light up mitochondria with intense fluorescence. Upon the loss of MMP, the emission of intracellular REP evidently decreased. The reversible changes in MMP have been successfully monitored by REP, and the oxidative damages to live cells have been detected with the probe. The probe is expected to serve as a desired tool in studying MMP and related areas.
  14. J Biochem. 2021 Apr 12. pii: mvab031. [Epub ahead of print]
      The translocator protein (TSPO) is a five-helix transmembrane protein localized to the outer mitochondria membrane. Radioligand binding assays and chemical crosslinking showed TSPO to be a high affinity cholesterol-binding protein. In this report, we show that TSPO in mitochondrial fractions from MA-10 mouse tumour Leydig cells can interact directly and competitively with the clickable photoreactive cholesterol analogue. PhotoClick cholesterol showed saturable photoaffinity labelling of TSPO that could be specifically immunoprecipitated with anti-TSPO antibody, following the click reaction with the fluorescent-azide probe, tetramethylrhodamine (TAMRA)-azide. Moreover, excess cholesterol reduced the photolabelling of both total mitochondrial proteins and TSPO. Together, the results of this study demonstrated direct binding of PhotoClick cholesterol to TSPO and that this interaction occurs at physiologically relevant site(s).
    Keywords:  cholesterol; click chemistry; photoaffinity labelling; translocator protein (TSPO)
  15. Mitochondrion. 2021 Apr 08. pii: S1567-7249(21)00048-9. [Epub ahead of print]
      Cell-free mitochondrial DNA (cf-mtDNA) is a marker of inflammatory disease and a predictor of mortality, but little is known about cf-mtDNA in relation to psychobiology. A systematic review of the literature reveals that blood cf-mtDNA varies in relation to common real-world stressors including psychopathology, acute psychological stress, and exercise. Moreover, cf-mtDNA is inducible within minutes and exhibits high intra-individual day-to-day variation, highlighting the dynamic regulation of cf-mtDNA levels. We discuss current knowledge on the mechanisms of cf-mtDNA release, its forms of transport ("cell-free" does not mean "membrane-free"), potential physiological functions, putative cellular and neuroendocrine triggers, and factors that may contribute to cf-mtDNA removal from the circulation. A review of in vitro, pre-clinical, and clinical studies shows conflicting results around the dogma that physiological forms of cf-mtDNA are pro-inflammatory, opening the possibility of other physiological functions, including the cell-to-cell transfer of whole mitochondria. Finally, to enhance the reproducibility and biological interpretation of human cf-mtDNA research, we propose guidelines for blood collection, cf-mtDNA isolation, quantification, and reporting standards, which can promote concerted advances by the community. Defining the mechanistic basis for cf-mtDNA signaling is an opportunity to elucidate the role of mitochondria in brain-body interactions and psychopathology.
    Keywords:  cell-free DNA; mitochondria; mtDNA; non-inflammatory effects; psychosocial stress; standard protocol
  16. Genet Med. 2021 Apr 12.
      PURPOSE: Reports have questioned the dogma of exclusive maternal transmission of human mitochondrial DNA (mtDNA), including the recent report of an admixture of two mtDNA haplogroups in individuals from three multigeneration families. This was interpreted as being consistent with biparental transmission of mtDNA in an autosomal dominant-like mode. The authenticity and frequency of these findings are debated.METHODS: We retrospectively analyzed individuals with two mtDNA haplogroups from 2017 to 2019 and selected four families for further study.
    RESULTS: We identified this phenomenon in 104/27,388 (approximately 1/263) unrelated individuals. Further study revealed (1) a male with two mitochondrial haplogroups transmits only one haplogroup to some of his offspring, consistent with nuclear transmission; (2) the heteroplasmy level of paternally transmitted variants is highest in blood, lower in buccal, and absent in muscle or urine of the same individual, indicating it is inversely correlated with mtDNA content; and (3) paternally transmitted apparent large-scale mtDNA deletions/duplications are not associated with a disease phenotype.
    CONCLUSION: These findings strongly suggest that the observed mitochondrial haplogroup of paternal origin resulted from coamplification of rare, concatenated nuclear mtDNA segments with genuine mtDNA during testing. Evaluation of additional specimen types can help clarify the clinical significance of the observed results.
  17. PLoS Pathog. 2021 Apr 15. 17(4): e1009506
      Regulation of mitochondrial structure and function is a central component of infection with viruses, including human cytomegalovirus (HCMV), as a virus means to modulate cellular metabolism and immune responses. Here, we link the activity of the mitochondrial deacetylase SIRT3 and global mitochondrial acetylation status to host antiviral responses via regulation of both mitochondrial structural integrity and metabolism during HCMV infection. We establish that SIRT3 deacetylase activity is necessary for suppressing virus production, and that SIRT3 maintains mitochondrial pH and membrane potential during infection. By defining the temporal dynamics of SIRT3-substrate interactions during infection, and overlaying acetylome and proteome information, we find altered SIRT3 associations with the mitochondrial fusion factor OPA1 and acetyl-CoA acyltransferase 2 (ACAA2), concomitant with changes in their acetylation levels. Using mutagenesis, microscopy, and virology assays, we determine OPA1 regulates mitochondrial morphology of infected cells and inhibits HCMV production. OPA1 acetylation status modulates these functions, and we establish K834 as a site regulated by SIRT3. Control of SIRT3 protein levels or enzymatic activity is sufficient for regulating mitochondrial filamentous structure. Lastly, we establish a virus restriction function for ACAA2, an enzyme involved in fatty acid beta-oxidation. Altogether, we highlight SIRT3 activity as a regulatory hub for mitochondrial acetylation and morphology during HCMV infection and point to global acetylation as a reflection of mitochondrial health.
  18. Clin Rheumatol. 2021 Apr 13.
      We analyzed the prevalence of anti-mitochondrial autoantibodies (AMA) in adult- and juvenile-onset myositis longitudinal cohorts and investigated phenotypic differences in myositis patients with AMA. We screened sera from myositis patients including 619 adult- and 371 juvenile-onset dermatomyositis (DM, JDM), polymyositis (PM, JPM), inclusion body myositis (IBM), or amyopathic DM patients and from healthy controls, including 164 adults and 92 children, for AMA by ELISA. Clinical characteristics were compared between myositis patients with and without AMA. AMA were present in 5% of adult myositis patients (16 of 216 DM, 10 of 222 PM, 4 of 140 IBM, 1 of 19 amyopathic DM), 1% of juvenile myositis patients (3 of 302 JDM, 1 of 25 JPM), and 1% of both adult and juvenile healthy controls. In patients with adult-onset myositis, AMA were associated with persistent muscle weakness, Raynaud's phenomenon, dysphagia, and cardiomyopathy. Adult myositis patients with AMA may have more severe or treatment refractory disease, as they more frequently received glucocorticoids and intravenous immunoglobulin. In juvenile myositis, children with AMA often had falling episodes and dysphagia, but no other clinical features or medications were significantly associated with AMA. AMA are present in 5% of adult myositis patients and associated with cardiomyopathy, dysphagia, and other signs of severe disease. The prevalence of AMA is not increased in patients with juvenile myositis compared to age-matched healthy controls. Our data suggest that the presence of AMA in adult myositis patients should prompt screening for cardiac and swallowing involvement. Key Points • Approximately 5% of a large North American cohort of adult myositis patients have anti-mitochondrial autoantibodies. • Adults with anti-mitochondrial autoantibodies often have chronic weakness, Raynaud's, dysphagia, cardiomyopathy, and more severe disease. • Anti-mitochondrial autoantibodies are rare in juvenile myositis and not associated with a specific clinical phenotype.
    Keywords:  Anti-mitochondrial autoantibodies; Juvenile myositis; Myositis
  19. Biochimie. 2021 Apr 12. pii: S0300-9084(21)00098-5. [Epub ahead of print]
      Glycogen storage disease type IV (GSD IV) is caused by mutations in the glycogen branching enzyme gene (GBE1) that lead to the accumulation of aberrant glycogen in affected tissues, mostly in the liver. To determine whether dysfunctional glycogen metabolism in GSD IV affects other components of cellular bioenergetics, we studied mitochondrial function in heterozygous Gbe1 knockout (Gbe1+/-) mice. Mitochondria isolated from the livers of Gbe1+/- mice showed elevated respiratory complex I activity and increased reactive oxygen species production, particularly by respiratory chain complex III. These observations indicate that GBE1 deficiency leads to broader rearrangements in energy metabolism and that the mechanisms underlying GSD IV pathogenesis may include more than merely mechanical cell damage caused by the presence of glycogen aggregates.
    Keywords:  Glycogen; Glycogen storage disease type IV; Mitochondria; Reactive oxygen species; Respiratory chain
  20. Biochim Biophys Acta Mol Cell Res. 2021 Apr 08. pii: S0167-4889(21)00092-6. [Epub ahead of print] 119038
      In addition to its uptake across the Ca2+ uniporter, intracellular calcium signals can stimulate mitochondrial metabolism activating metabolite exchangers of the inner mitochondrial membrane belonging to the mitochondrial carrier family (SLC25). One of these Ca2+-regulated mitochondrial carriers (CaMCs) are the reversible ATP-Mg2+/Pi transporters, or SCaMCs, required for maintaining optimal adenine nucleotide (AdN) levels in the mitochondrial matrix representing an alternative transporter to the ADP/ATP translocases (AAC). This CaMC has a distinctive Calmodulin-like (CaM-like) domain fused to the carrier domain that makes its transport activity strictly dependent on cytosolic Ca2+ signals. Here we investigate about its origin analysing its distribution and features in unicellular eukaryotes. Unexpectedly, we find two types of ATP-Mg2+/Pi carriers, the canonical ones and shortened variants lacking the CaM-like domain. Phylogenetic analysis shows that both SCaMC variants have a common origin, unrelated to AACs, suggesting in turn that recurrent losses of the regulatory module have occured in the different phyla. They are excluding variants that show a more limited distribution and less conservation than AACs. Interestingly, these truncated variants of SCaMC are found almost exclusively in parasitic protists, such as apicomplexans, kinetoplastides or animal-patogenic oomycetes, and in green algae, suggesting that its lost could be related to certain life-styles. In addition, we find an intricate structural diversity in these variants that may be associated with their pathogenicity. The consequences on SCaMC functions of these new SCaMC-b variants are discussed.
    Keywords:  ATP transport; Evolution; Mitochondria; Mitochondrial carrier; Protozoan; calcium
  21. Ophthalmic Genet. 2021 Apr 16. 1-6
      Background: Leber hereditary optic neuropathy (LHON) is a mitochondrial neurodegenerative disease. The majority (>90%) is related to three primary mitochondrial DNA (mtDNA) variants: ND1 m.3460G>A, ND4 m.11778G>A and ND6 m.14484T>C. The remaining 10% is associated with >40 secondary variants with variable penetrance and incidence between different ethnic backgrounds.Materials and methods: Five sisters underwent an extensive ophthalmic workup including psychophysical, electrophysiological, multimodal brain imaging, biochemical testing and molecular screening. MT-ND6 protein modelling was performed.Results: A 23-year-old woman presented with acute central visual loss to counting fingers in the right eye. She developed a central visual field scotoma, severe color vision deficiencies and impaired pattern visual evoked responses. Progressive optic atrophy ensued. The left eye was unremarkable, except for borderline thinning of the temporal retinal nerve fiber layer. Alcohol use and passive smoking were noted. MtDNA analysis revealed a rare variant, m.14502T>C in MT-ND6, exclusively known to cause optic neuropathy in an Asian population. Three sisters of the proband, two of whom reported tobacco and alcohol abuse, had bilateral temporal optic disc pallor without functional impact. A fourth non-smoker sister had a completely normal eye exam.Conclusions: The rare Asian m.14502T>C variant in the MT-ND6 gene was linked to a mild LHON phenotype in a Western European family. Penetrance in this family was likely triggered by alcohol and tobacco abuse. A full mtDNA sequencing is warranted in the case of high clinical suspicion of LHON when mutation analysis for the three common pathogenic variants is negative.
    Keywords:  LHON; Leber hereditary optic neuropathy; MT-ND6 gene; alcohol and tobacco abuse; m.14502T>C
  22. Front Neurol. 2021 ;12 652590
      Leber hereditary optic neuropathy (LHON) is a mitochondrial disease causing severe bilateral visual loss, typically in young adults. The disorder is commonly caused by one of three primary point mutations in mitochondrial DNA, but a number of other rare mutations causing or associated with the clinical syndrome of LHON have been reported. The mutations in LHON are almost exclusively located in genes encoding subunits of complex I in the mitochondrial respiratory chain. Here we report two patients, a mother and her son, with the typical LHON phenotype. Genetic investigations for the three common mutations were negative, instead we found a new and previously unreported mutation in mitochondrial DNA. This homoplasmic mutation, m.13345G>A, is located in the MT-ND5 gene, encoding a core subunit in complex I in the mitochondrial respiratory chain. Investigation of the patients mitochondrial respiratory chain in muscle found a mild defect in the combined activity of complex I+III. In the literature six other mutations in the MT-ND5 gene have been associated with LHON and by this report a new putative mutation in the MT-ND5 can be added.
    Keywords:  LHON; MT-ND5; case report; complex 1; leber hereditary optic neuropathy; mitochondrial DNA; optic neuropathy
  23. Mol Neurodegener. 2021 Apr 14. 16(1): 25
      BACKGROUND: Apoptosis-inducing factor (AIF), as a mitochondrial flavoprotein, plays a fundamental role in mitochondrial bioenergetics that is critical for cell survival and also mediates caspase-independent cell death once it is released from mitochondria and translocated to the nucleus under ischemic stroke or neurodegenerative diseases. Although alternative splicing regulation of AIF has been implicated, it remains unknown which AIF splicing isoform will be induced under pathological conditions and how it impacts mitochondrial functions and neurodegeneration in adult brain.METHODS: AIF splicing induction in brain was determined by multiple approaches including 5' RACE, Sanger sequencing, splicing-specific PCR assay and bottom-up proteomic analysis. The role of AIF splicing in mitochondria and neurodegeneration was determined by its biochemical properties, cell death analysis, morphological and functional alterations and animal behavior. Three animal models, including loss-of-function harlequin model, gain-of-function AIF3 knockin model and conditional inducible AIF splicing model established using either Cre-loxp recombination or CRISPR/Cas9 techniques, were applied to explore underlying mechanisms of AIF splicing-induced neurodegeneration.
    RESULTS: We identified a nature splicing AIF isoform lacking exons 2 and 3 named as AIF3. AIF3 was undetectable under physiological conditions but its expression was increased in mouse and human postmortem brain after stroke. AIF3 splicing in mouse brain caused enlarged ventricles and severe neurodegeneration in the forebrain regions. These AIF3 splicing mice died 2-4 months after birth. AIF3 splicing-triggered neurodegeneration involves both mitochondrial dysfunction and AIF3 nuclear translocation. We showed that AIF3 inhibited NADH oxidase activity, ATP production, oxygen consumption, and mitochondrial biogenesis. In addition, expression of AIF3 significantly increased chromatin condensation and nuclear shrinkage leading to neuronal cell death. However, loss-of-AIF alone in harlequin or gain-of-AIF3 alone in AIF3 knockin mice did not cause robust neurodegeneration as that observed in AIF3 splicing mice.
    CONCLUSIONS: We identified AIF3 as a disease-inducible isoform and established AIF3 splicing mouse model. The molecular mechanism underlying AIF3 splicing-induced neurodegeneration involves mitochondrial dysfunction and AIF3 nuclear translocation resulting from the synergistic effect of loss-of-AIF and gain-of-AIF3. Our study provides a valuable tool to understand the role of AIF3 splicing in brain and a potential therapeutic target to prevent/delay the progress of neurodegenerative diseases.
    Keywords:  AIF; AIF3 splicing; Mitochondrial dysfunction; Neurodegeneration
  24. Sci Rep. 2021 Apr 13. 11(1): 8032
      Mammalian cytosine DNA methylation (5mC) is associated with the integrity of the genome and the transcriptional status of nuclear DNA. Due to technical limitations, it has been less clear if mitochondrial DNA (mtDNA) is methylated and whether 5mC has a regulatory role in this context. Here, we used bisulfite-independent single-molecule sequencing of native human and mouse DNA to study mitochondrial 5mC across different biological conditions. We first validated the ability of long-read nanopore sequencing to detect 5mC in CpG (5mCpG) and non-CpG (5mCpH) context in nuclear DNA at expected genomic locations (i.e. promoters, gene bodies, enhancers, and cell type-specific transcription factor binding sites). Next, using high coverage nanopore sequencing we found low levels of mtDNA CpG and CpH methylation (with several exceptions) and little variation across biological processes: differentiation, oxidative stress, and cancer. 5mCpG and 5mCpH were overall higher in tissues compared to cell lines, with small additional variation between cell lines of different origin. Despite general low levels, global and single-base differences were found in cancer tissues compared to their adjacent counterparts, in particular for 5mCpG. In conclusion, nanopore sequencing is a useful tool for the detection of modified DNA bases on mitochondria that avoid the biases introduced by bisulfite and PCR amplification. Enhanced nanopore basecalling models will provide further resolution on the small size effects detected here, as well as rule out the presence of other DNA modifications such as oxidized forms of 5mC.
  25. Neurobiol Dis. 2021 Apr 12. pii: S0969-9961(21)00110-8. [Epub ahead of print] 105361
      The classic view of organelle cell biology is undergoing a constant revision fueled by the new insights unraveled by fluorescence nanoscopy, which enable sensitive, faster and gentler observation of specific proteins in situ. The endoplasmic reticulum (ER) is one of the most challenging structure to capture due the rapid and constant restructuring of fine sheets and tubules across the full 3D cell volume. Here we apply STED and parallelized 2D and 3D RESOLFT live imaging to uncover the tubular ER organization in the fine processes of neuronal cells with focus on mitochondria-ER contacts, which recently gained medical attention due to their role in neurodegeneration. Multi-color STED nanoscopy enables the simultaneous visualization of small transversal ER tubules crossing and constricting mitochondria all along axons and dendrites. Parallelized RESOLFT allows for dynamic studies of multiple contact sites within seconds and minutes with prolonged time-lapse imaging at ~50 nm spatial resolution. When operated in 3D super resolution mode it enables a new isotropic visualization of such contacts extending our understanding of the three-dimensional architecture of these packed structures in axons and dendrites.
    Keywords:  Endoplasmic reticulum; Mitochondria-ER contacts; RESOLFT; STED; Super resolution microscopy
  26. Methods Mol Biol. 2021 ;2281 289-301
      Optical tweezers can monitor and control the activity of individual DNA polymerase molecules in real time, providing in this way unprecedented insight into the complex dynamics and mechanochemical processes that govern their operation. Here, we describe an optical tweezers-based assay to determine at the single-molecule level the effect of single-stranded DNA-binding proteins (SSB) on the real-time replication kinetics of the human mitochondrial DNA polymerase during the synthesis of the lagging strand.
    Keywords:  DNA replication; Optical tweezers; Protein-protein interactions; Real-time kinetics; Single-molecule manipulation
  27. Front Endocrinol (Lausanne). 2021 ;12 660095
      Mitochondrial fission protein 1 (Fis1) was identified in yeast as being essential for mitochondrial division or fission and subsequently determined to mediate human mitochondrial and peroxisomal fission. Yet, its exact functions in humans, especially in regard to mitochondrial fission, remains an enigma as genetic deletion of Fis1 elongates mitochondria in some cell types, but not others. Fis1 has also been identified as an important component of apoptotic and mitophagic pathways suggesting the protein may have multiple, essential roles. This review presents current perspectives on the emerging functions of Fis1 and their implications in human health and diseases, with an emphasis on Fis1's role in both endocrine and neurological disorders.
    Keywords:  FIS1; apoptosis; cancer; diabetes; mitochondria; mitochondrial dynamics; mitophagy; neurodegenerative diseases
  28. Front Immunol. 2021 ;12 630380
      Activation of the Nod-like receptor 3 (NLRP3) inflammasome is important for activation of innate immune responses, but improper and excessive activation can cause inflammatory disease. We previously showed that glycolysis, a metabolic pathway that converts glucose into pyruvate, is essential for NLRP3 inflammasome activation in macrophages. Here, we investigated the role of metabolic pathways downstream glycolysis - lactic acid fermentation and pyruvate oxidation-in activation of the NLRP3 inflammasome. Using pharmacological or genetic approaches, we show that decreasing lactic acid fermentation by inhibiting lactate dehydrogenase reduced caspase-1 activation and IL-1β maturation in response to various NLRP3 inflammasome agonists such as nigericin, ATP, monosodium urate (MSU) crystals, or alum, indicating that lactic acid fermentation is required for NLRP3 inflammasome activation. Inhibition of lactate dehydrogenase with GSK2837808A reduced lactate production and activity of the NLRP3 inflammasome regulator, phosphorylated protein kinase R (PKR), but did not reduce the common trigger of NLRP3 inflammasome, potassium efflux, or reactive oxygen species (ROS) production. By contrast, decreasing the activity of pyruvate oxidation by depletion of either mitochondrial pyruvate carrier 2 (MPC2) or pyruvate dehydrogenase E1 subunit alpha 1 (PDHA1) enhanced NLRP3 inflammasome activation, suggesting that inhibition of mitochondrial pyruvate transport enhanced lactic acid fermentation. Moreover, treatment with GSK2837808A reduced MSU-mediated peritonitis in mice, a disease model used for studying the consequences of NLRP3 inflammasome activation. Our results suggest that lactic acid fermentation is important for NLRP3 inflammasome activation, while pyruvate oxidation is not. Thus, reprograming pyruvate metabolism in mitochondria and in the cytoplasm should be considered as a novel strategy for the treatment of NLRP3 inflammasome-associated diseases.
    Keywords:  NLRP3 inflammasome; glycolysis; inflammation; lactic acid fermentation; pyruvate oxidation
  29. Antioxid Redox Signal. 2021 Apr 13.
      SIGNIFICANCE: Sepsis is a major public health concern, with high mortality and morbidity, especially among trauma patients. It is characterized by a systemic inflammatory response syndrome (SIRS) occurring in response to infection. Although classically associated with pathogens, many patients with SIRS do not have infection. The variability of the disease course cannot be fully explained by our current understanding of its pathogenesis. Thus other factors are likely to play key roles in the development and progression of SIRS/sepsis. Recent Advances: Circulating levels of damage-associated molecular patterns (DAMPs) seem to correlate with SIRS/sepsis morbidity and mortality. Of the known DAMPs, those of mitochondrial (mt) origin have been of particular interest, since their DNA (mtDNA) and formyl peptides (mtFPs) resemble bacterial DNA and peptides, and hence, when released, may be recognized as "danger signals".CRITICAL ISSUES: mtDAMPs released after tissue injury trigger immune responses similar to those induced by pathogens. Thus they can result in systemic inflammation and organ damage, like that observed in SIRS/sepsis. We will discuss recent findings on the roles of mtDAMPs, particularly concerning the less recognized mtFPs, in the activation of inflammatory responses and development of SIRS/sepsis.
    FUTURE DIRECTIONS: There are no established methods to predict the course of SIRS/sepsis, but clinical studies reveal that plasma levels of mtDAMPs may correlate with the outcome of the disease. We propose that non-pathogen-initiated, mtDAMPs-induced SIRS/sepsis events need further studies aimed at early clinical recognition and better treatment of this disease.
  30. Nat Genet. 2021 Apr 12.
      Genome editing has therapeutic potential for treating genetic diseases and cancer. However, the currently most practicable approaches rely on the generation of DNA double-strand breaks (DSBs), which can give rise to a poorly characterized spectrum of chromosome structural abnormalities. Here, using model cells and single-cell whole-genome sequencing, as well as by editing at a clinically relevant locus in clinically relevant cells, we show that CRISPR-Cas9 editing generates structural defects of the nucleus, micronuclei and chromosome bridges, which initiate a mutational process called chromothripsis. Chromothripsis is extensive chromosome rearrangement restricted to one or a few chromosomes that can cause human congenital disease and cancer. These results demonstrate that chromothripsis is a previously unappreciated on-target consequence of CRISPR-Cas9-generated DSBs. As genome editing is implemented in the clinic, the potential for extensive chromosomal rearrangements should be considered and monitored.
  31. EMBO J. 2021 Apr 13. e106491
      Exercise can alter the skeletal muscle DNA methylome, yet little is known about the role of the DNA methylation machinery in exercise capacity. Here, we show that DNMT3A expression in oxidative red muscle increases greatly following a bout of endurance exercise. Muscle-specific Dnmt3a knockout mice have reduced tolerance to endurance exercise, accompanied by reduction in oxidative capacity and mitochondrial respiration. Moreover, Dnmt3a-deficient muscle overproduces reactive oxygen species (ROS), the major contributors to muscle dysfunction. Mechanistically, we show that DNMT3A suppresses the Aldh1l1 transcription by binding to its promoter region, altering its epigenetic profile. Forced expression of ALDH1L1 elevates NADPH levels, which results in overproduction of ROS by the action of NADPH oxidase complex, ultimately resulting in mitochondrial defects in myotubes. Thus, inhibition of ALDH1L1 pathway can rescue oxidative stress and mitochondrial dysfunction from Dnmt3a deficiency in myotubes. Finally, we show that in vivo knockdown of Aldh1l1 largely rescues exercise intolerance in Dnmt3a-deficient mice. Together, we establish that DNMT3A in skeletal muscle plays a pivotal role in endurance exercise by controlling intracellular oxidative stress.
    Keywords:  DNA methylation; exercise; oxidative stress
  32. Ir J Med Sci. 2021 Apr 11.
      BACKGROUND: Mutations in mitochondrial DNA (mtDNA) are found to be associated with type 2 diabetes mellitus (T2DM). However, the molecular pathogenesis of these mutations in T2DM is still poorly understood.METHODS: In this study, we report here the molecular features of two Han Chinese families with maternally transmitted T2DM. The matrilineal relatives are undergoing clinical, biochemical, genetic evaluations, and molecular analysis. Furthermore, the entire mitochondrial genomes of these matrilineal relatives are screened by PCR-Sanger sequencing.
    RESULTS: The age at onset of T2DM of these participants varies from 28 to 71 years, with an average of 43 years. Molecular analysis of mitochondrial genomes identifies the existence of ND1 T3394C mutation in both families, together with sets of variants belonging to mitochondrial haplogroup Y2 and M9a. The m.T3394C mutation is localized at very conserved tyrosine at position 30 of ND1, may result the failure in ND1 mRNA metabolism, and lead to mitochondrial dysfunction. Moreover, sequence analysis of matrilineal relatives in Family 1 identifies the m.A14693G mutation which occurs in the TΨC-loop of tRNAGlu (position 54), and is critical to the structural formation and stabilization of this tRNA. Thus, m.A14693G mutation may cause the impairment in tRNA metabolism, thereby worsens the mitochondrial dysfunction caused by ND1 T3394C mutation. However, no functional mtDNA variants are identified in Family 2 which suggest that mitochondrial haplogroup may not play an important role in diabetes expression.
    CONCLUSIONS: Our study indicates that mitochondrial ND1 T3394C mutation is involved in the pathogenesis of maternally inherited T2DM in these families.
    Keywords:  Chinese families; M.A14693G; M.T3394C; MtDNA mutations; T2DM
  33. Nat Genet. 2021 Apr 15.
      A central question in the post-genomic era is how genes interact to form biological pathways. Measurements of gene dependency across hundreds of cell lines have been used to cluster genes into 'co-essential' pathways, but this approach has been limited by ubiquitous false positives. In the present study, we develop a statistical method that enables robust identification of gene co-essentiality and yields a genome-wide set of functional modules. This atlas recapitulates diverse pathways and protein complexes, and predicts the functions of 108 uncharacterized genes. Validating top predictions, we show that TMEM189 encodes plasmanylethanolamine desaturase, a key enzyme for plasmalogen synthesis. We also show that C15orf57 encodes a protein that binds the AP2 complex, localizes to clathrin-coated pits and enables efficient transferrin uptake. Finally, we provide an interactive webtool for the community to explore our results, which establish co-essentiality profiling as a powerful resource for biological pathway identification and discovery of new gene functions.
  34. Clin Otolaryngol. 2021 Apr 13.
      OBJECTIVE: Identification of variations in tumor suppressor genes encoding the tetrameric succinate dehydrogenase (SDHx) mitochondrial enzyme complex may lead to personalized therapeutic concepts for the orphan disease, familial paraganglioma (PGL) type I. We undertook to determine the causative variation in a family suffering from idiopathic early-onset (22±2 years) head and neck PGL by PCR and Sanger sequencing.DESIGN, SETTING AND PARTICIPANTS: In a prospective genetic study, twelve members of the family were recruited at a tertiary referral otolaryngology centre.
    RESULTS: A novel heterozygous c.298delA frameshift variation in exon 3 of SDH subunit D (SDHD) was associated with a paternal transmission pattern of PGL in affected family members available to the study. Family history over five generations in adulthood indicated a variable penetrance for PGL inheritance in older generations. The c.298delA variant would cause translation of a 34-residue C-terminus distal to lysine residue 99 in the predicted transmembrane domain II of the full-length sequence p.(Thr100LeufsTer35) and would affect the translation products of all protein-coding SDHD isoforms containing transmembrane topologies required for positional integration in the inner mitochondrial membrane and complex formation.
    CONCLUSIONS: A novel c.298delA variation in SDHD that causes paternally inherited PGL1 with an atypically low penetrance was identified. These results underly the importance of genetic screening for PGL also in cases of unclear inheritance and variation carriers should benefit from screening and lifelong follow-up.
    Keywords:  1; Succinate dehydrogenase; familial; frameshift variation; head and neck neoplasms; orphan diseases; paragangliomas; paternal inheritance
  35. Bone. 2021 Apr 01. pii: S8756-3282(21)00103-4. [Epub ahead of print]148 115941
      Mitochondria are essential organelles that form highly complex, interconnected dynamic networks inside cells. The GTPase mitofusin 2 (MFN2) is a highly conserved outer mitochondrial membrane protein involved in the regulation of mitochondrial morphology, which can affect various metabolic and signaling functions. The role of mitochondria in bone formation remains unclear. Since MFN2 levels increase during osteoblast (OB) differentiation, we investigated the role of MFN2 in the osteolineage by crossing mice bearing floxed Mfn2 alleles with those bearing Prx-cre to generate cohorts of conditional knock out (cKO) animals. By ex vivo microCT, cKO female mice, but not males, display an increase in cortical thickness at 8, 18, and 30 weeks, compared to wild-type (WT) littermate controls. However, the cortical anabolic response to mechanical loading was not different between genotypes. To address how Mfn2 deficiency affects OB differentiation, bone marrow-derived mesenchymal stromal cells (MSCs) from both wild-type and cKO mice were cultured in osteogenic media with different levels of β-glycerophosphate. cKO MSCs show increased mineralization and expression of multiple markers of OB differentiation only at the lower dose. Interestingly, despite showing the expected mitochondrial rounding and fragmentation due to loss of MFN2, cKO MSCs have an increase in oxygen consumption during the first 7 days of OB differentiation. Thus, in the early phases of osteogenesis, MFN2 restrains oxygen consumption thereby limiting differentiation and cortical bone accrual during homeostasis in vivo.
    Keywords:  Bone formation; Mitochondria; Mitofusin; Osteoblast; Osteogenesis
  36. STAR Protoc. 2021 Jun 18. 2(2): 100408
      Here, we describe a protocol for comprehensive quantification of autophagosome recruitment to mitochondria as an early step in mitophagy. Data collected using this protocol can be useful in the study of neurodegenerative disease, cancer, and metabolism-related disorders using models in which co-expression of mito-GFP and mCherry-Atg8a is feasible. This protocol has the advantage of assessment in an in vivo model organism (Drosophila melanogaster), where tissue-specific mitophagy can be investigated. For complete details on the use and execution of this protocol, please refer to (Cackovic et al., 2018).
    Keywords:  Cell Biology; Microscopy; Model Organisms; Neuroscience
  37. Front Cell Dev Biol. 2021 ;9 626117
      Mammalian mitochondria are emerging as a critical stress-responsive contributor to cellular life/death and developmental outcomes. Maintained as an organellar network distributed throughout the cell, mitochondria respond to cellular stimuli and stresses through highly sensitive structural dynamics, particularly in energetically demanding cell settings such as cardiac and muscle tissues. Fusion allows individual mitochondria to form an interconnected reticular network, while fission divides the network into a collection of vesicular organelles. Crucially, optic atrophy-1 (OPA1) directly links mitochondrial structure and bioenergetic function: when the transmembrane potential across the inner membrane (ΔΨm) is intact, long L-OPA1 isoforms carry out fusion of the mitochondrial inner membrane. When ΔΨm is lost, L-OPA1 is cleaved to short, fusion-inactive S-OPA1 isoforms by the stress-sensitive OMA1 metalloprotease, causing the mitochondrial network to collapse to a fragmented population of organelles. This proteolytic mechanism provides sensitive regulation of organellar structure/function but also engages directly with apoptotic factors as a major mechanism of mitochondrial participation in cellular stress response. Furthermore, emerging evidence suggests that this proteolytic mechanism may have critical importance for cell developmental programs, particularly in cardiac, neuronal, and stem cell settings. OMA1's role as a key mitochondrial stress-sensitive protease motivates exciting new questions regarding its mechanistic regulation and interactions, as well as its broader importance through involvement in apoptotic, stress response, and developmental pathways.
    Keywords:  OMA1; OPA1; apoptosis; development; mitochondria
  38. Front Genet. 2021 ;12 630359
      Plasma lipids have been at the center stage of the prediction and prevention strategies for cardiovascular diseases (CVDs), and novel lipidomic traits have been recognized as reliable biomarkers for CVD risk prediction. The mitochondria serve as energy supply sites for cells and can synthesize a variety of lipids autonomously. Therefore, investigating the relationships between mitochondrial single nucleotide polymorphism (SNPs) and plasma lipidomic traits is meaningful. Here, we enrolled a total of 1,409 Han Chinese patients with coronary artery disease from three centers and performed linear regression analyses on the SNPs of mitochondrial DNA (mtDNA) and lipidomic traits in two independent groups. Sex, age, aspartate aminotransferase, estimated glomerular filtration rate, antihypertensive drugs, hypertension, and diabetes were adjusted. We identified three associations, namely, D-loop m.16089T>C with TG(50:4) NL-16:0, D-loop m.16145G>A with TG(54:5) NL-18:0, and D-loop m.16089T>C with PC(16:0_16:1) at the statistically significant threshold of FDR < 0.05. Then, we explored the relationships between mitochondrial genetic variants and traditional lipids, including triglyceride, total cholesterol (TC), low-density lipoprotein cholesterol (LDLC), and high-density lipoprotein cholesterol. Two significant associations were found, namely MT-ND6 m.14178T>C with TC and D-loop m.215A>G with LDLC. Furthermore, we performed linear regression analysis to determine on the SNPs of mtDNA and left ventricular ejection fraction (LVEF) and found that the SNP D-loop m.16145G>A was nominally significantly associated with LVEF (P = 0.047). Our findings provide insights into the lipidomic context of mtDNA variations and highlight the importance of studying mitochondrial genetic variants related to lipid species.
    Keywords:  association analyses; coronary artery disease; lipidomic; mitochondrial DNA; polymorphisms
  39. Bioessays. 2021 Apr 14. e2100014
      Since their discovery over two decades ago, the molecular and cellular functions of the NIPSNAP family of proteins (NIPSNAPs) have remained elusive until recently. NIPSNAPs interact with a variety of mitochondrial and cytoplasmic proteins. They have been implicated in multiple cellular processes and associated with different physiologic and pathologic conditions, including pain transmission, Parkinson's disease, and cancer. Recent evidence demonstrated a direct role for NIPSNAP1 and NIPSNAP2 proteins in regulation of mitophagy, a process that is critical for cellular health and maintenance. Importantly, NIPSNAPs contain a 110 amino acid domain that is evolutionary conserved from mammals to bacteria. However, the molecular function of the conserved NIPSNAP domain and its potential role in mitophagy have not been explored. It stands to reason that the highly conserved NIPSNAP domain interacts with a substrate that is ubiquitously present across all species and can perhaps act as a sensor for mitochondrial health.
    Keywords:  Alzheimer's disease; Parkinson's disease; Sirtuin3; metabolism; mitophagy
  40. J Mol Cell Cardiol. 2021 Apr 12. pii: S0022-2828(21)00078-X. [Epub ahead of print]
      Sudden cardiac death due to ventricular tachyarrhythmias remains the major cause of mortality in the world. Heart failure, diabetic cardiomyopathy, old age-related cardiac dysfunction and inherited disorders are associated with enhanced propensity to malignant cardiac arrhythmias. Both defective mitochondrial function and abnormal intracellular Ca2+ homeostasis have been established as the key contributing factors in the pathophysiology and arrhythmogenesis in these conditions. This article reviews current advances in understanding of bidirectional control of ryanodine receptor-mediated sarcoplasmic reticulum Ca2+ release and mitochondrial function, and how defects in crosstalk between these two organelles increase arrhythmic risk in cardiac disease.
    Keywords:  Calcium signaling; Cardiac arrhythmia; Mitochondria; Sarcoplasmic reticulum
  41. iScience. 2021 Apr 23. 24(4): 102302
      It has been shown that histone deacetylase (HDAC) inhibitors hold considerable therapeutic potentials for treating neurodegeneration-related diseases including Parkinson disease (PD). Here, we synthesized an HDAC inhibitor named as HGC and examined its neuroprotective roles in PD models. Our results showed that HGC protects dopaminergic neurons from 1-methyl-4-phenylpyridinium (MPP+)-induced insults. Furthermore, in 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced PD model mice, HGC application rectifies behavioral defects, improves tyrosine hydroxylase-positive neurons in the midbrain, and maintains mitochondrial integrity and functions. Mechanistically, mass spectrometry data revealed that HGC stimulates acetylation modification at lysine 28 of NDUFV1. Inhibition of HDAC6 by HGC is responsible for this acetylation modification. Functional tests showed that, as well as HGC, NDUFV1 exhibits beneficial roles against MPP+ injuries. Moreover, knockdown of NDUFV1 abolishes the neuroprotective roles of HGC. Taken together, our data indicate that HGC has a great therapeutic potential for treating PD and NDUFV1 might be a target for developing drugs against PD.
    Keywords:  Behavioral Neuroscience; Biological Sciences; Cellular Neuroscience; Molecular Neuroscience; Neuroscience; Systems Neuroscience
  42. J Inherit Metab Dis. 2021 Apr 11.
      The current diagnostic work-up of inborn errors of metabolism (IEM) is rapidly moving towards integrative analytical approaches. We aimed to develop an innovative, targeted urine metabolomics (TUM) screening procedure to accelerate the diagnosis of patients with IEM. Urinary samples, spiked with 3 stable isotope-labelled internal standards, were analysed for 258 diagnostic metabolites with an UHPLC-QTOF-MS configuration run in positive and negative ESI modes. The software automatically annotated peaks, corrected for peak overloading, and reported peak quality and shifting. Robustness and reproducibility were satisfactory for most metabolites. Z-scores were calculated against 4 age-group matched control cohorts. Disease phenotypes were scored based on database metabolite matching. Graphical reports comprised a needle plot, annotating abnormal metabolites, and a heatmap showing the prioritised disease phenotypes. In the clinical validation we analysed samples of 289 patients covering 78 OMIM phenotypes from 12 of the 15 SSIEM disease groups. The disease groups include disorders in the metabolism of amino acids, fatty acids, ketones, purines and pyrimidines, carbohydrates, porphyrias, neurotransmitters, vitamins, cofactors, and creatine. The reporting tool easily and correctly diagnosed most samples. Even subtle aberrant metabolite patterns as seen in mild multiple acyl-CoA dehydrogenase deficiency (, GAII) and maple syrup urine disease (MSUD) were correctly called without difficulty. Others, like creatine transporter deficiency, are illustrative of IEM that remain difficult to diagnose. We present TUM as a powerful diagnostic screening tool that that merges most urinary diagnostic assays expediting the diagnostics for patients suspected of an IEM. This article is protected by copyright. All rights reserved.
  43. Cell. 2021 Apr 07. pii: S0092-8674(21)00353-6. [Epub ahead of print]
      A general approach for heritably altering gene expression has the potential to enable many discovery and therapeutic efforts. Here, we present CRISPRoff-a programmable epigenetic memory writer consisting of a single dead Cas9 fusion protein that establishes DNA methylation and repressive histone modifications. Transient CRISPRoff expression initiates highly specific DNA methylation and gene repression that is maintained through cell division and differentiation of stem cells to neurons. Pairing CRISPRoff with genome-wide screens and analysis of chromatin marks establishes rules for heritable gene silencing. We identify single guide RNAs (sgRNAs) capable of silencing the large majority of genes including those lacking canonical CpG islands (CGIs) and reveal a wide targeting window extending beyond annotated CGIs. The broad ability of CRISPRoff to initiate heritable gene silencing even outside of CGIs expands the canonical model of methylation-based silencing and enables diverse applications including genome-wide screens, multiplexed cell engineering, enhancer silencing, and mechanistic exploration of epigenetic inheritance.
    Keywords:  CRISPR; DNA methylation; cell therapy; dCas9; epigenetics
  44. Cell Rep. 2021 Apr 13. pii: S2211-1247(21)00299-0. [Epub ahead of print]35(2): 108985
      Decreased cognitive performance is a hallmark of brain aging, but the underlying mechanisms and potential therapeutic avenues remain poorly understood. Recent studies have revealed health-protective and lifespan-extending effects of dietary spermidine, a natural autophagy-promoting polyamine. Here, we show that dietary spermidine passes the blood-brain barrier in mice and increases hippocampal eIF5A hypusination and mitochondrial function. Spermidine feeding in aged mice affects behavior in homecage environment tasks, improves spatial learning, and increases hippocampal respiratory competence. In a Drosophila aging model, spermidine boosts mitochondrial respiratory capacity, an effect that requires the autophagy regulator Atg7 and the mitophagy mediators Parkin and Pink1. Neuron-specific Pink1 knockdown abolishes spermidine-induced improvement of olfactory associative learning. This suggests that the maintenance of mitochondrial and autophagic function is essential for enhanced cognition by spermidine feeding. Finally, we show large-scale prospective data linking higher dietary spermidine intake with a reduced risk for cognitive impairment in humans.
    Keywords:  Pink1; aging; autophagy; cognitive function; dietary spermidine; memory; mitochondria; mitophagy
  45. Circ Res. 2021 Apr 16. 128(8): 1205-1207
    Keywords:  Editorials; animal models; calcium; cardiac myocyte; heart failure; mitochondria
  46. Nat Commun. 2021 Apr 16. 12(1): 2280
      The tumor suppressor p53 is mutated in approximately half of all human cancers. p53 can induce apoptosis through mitochondrial membrane permeabilization by interacting with and antagonizing the anti-apoptotic proteins BCL-xL and BCL-2. However, the mechanisms by which p53 induces mitochondrial apoptosis remain elusive. Here, we report a 2.5 Å crystal structure of human p53/BCL-xL complex. In this structure, two p53 molecules interact as a homodimer, and bind one BCL-xL molecule to form a ternary complex with a 2:1 stoichiometry. Mutations at the p53 dimer interface or p53/BCL-xL interface disrupt p53/BCL-xL interaction and p53-mediated apoptosis. Overall, our current findings of the bona fide structure of p53/BCL-xL complex reveal the molecular basis of the interaction between p53 and BCL-xL, and provide insight into p53-mediated mitochondrial apoptosis.
  47. Mol Cell. 2021 Apr 10. pii: S1097-2765(21)00143-X. [Epub ahead of print]
      Oxidative phosphorylation (OXPHOS) and glycolysis are the two major pathways for ATP production. The reliance on each varies across tissues and cell states, and can influence susceptibility to disease. At present, the full set of molecular mechanisms governing the relative expression and balance of these two pathways is unknown. Here, we focus on genes whose loss leads to an increase in OXPHOS activity. Unexpectedly, this class of genes is enriched for components of the pre-mRNA splicing machinery, in particular for subunits of the U1 snRNP. Among them, we show that LUC7L2 represses OXPHOS and promotes glycolysis by multiple mechanisms, including (1) splicing of the glycolytic enzyme PFKM to suppress glycogen synthesis, (2) splicing of the cystine/glutamate antiporter SLC7A11 (xCT) to suppress glutamate oxidation, and (3) secondary repression of mitochondrial respiratory supercomplex formation. Our results connect LUC7L2 expression and, more generally, the U1 snRNP to cellular energy metabolism.
    Keywords:  7q-; LUC7; MDS; Tarui disease; cancer; ferroptosis; myelodysplastic syndrome; phosphofructokinase; spliceosome; system X(c)(−)
  48. Neuromuscul Disord. 2021 Mar 03. pii: S0960-8966(21)00064-X. [Epub ahead of print]
      Both mitochondrial and nuclear gene mutations can cause cytochrome c oxidase (COX, complex Ⅳ) dysfunction, leading to mitochondrial diseases. Although numerous diseases caused by defects of the COX subunits or COX assembly factors have been documented, clinical cases directly related to mitochondrial cytochrome c oxidase subunit 3 gene (MT-CO3) mutations are relatively rare. Here, we report a 47-year-old female patient presented with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome. Muscle pathology revealed ragged-red fibres and remarkable COX-deficient muscle fibres. Muscle mitochondrial DNA sequencing analysis identified a novel MT-CO3 variant (m.9553G>A) that changed a highly conserved amino acid to a stop codon (p.Trp116*). This variant was heteroplasmic in multiple tissues, where the mutation load was 13% in oral epithelial cells, 89% in muscle samples, and not detectable in the peripheral blood lymphocytes. Single muscle fiber PCR analysis showed clear segregation of the mutation load with COX deficient fibres. Western blot analysis of the muscle samples revealed a significant decrease in the levels of COX1, COX2, COX3, COX4 and UQCRC2. COX respiration activity was remarkably reduced (58.84%) relative to the controls according to spectrophotometric assays. Taken together, our results indicated that this m.9553G>A variant may be responsible for the MELAS symdrome in the proband by affecting the stability and function of COX. The study expands the clinical and molecular spectrum of COX3-specific mitochondrial diseases.
    Keywords:  Cytochrome c oxidase; Heteroplasmy; MELAS; MT-COX3; m.9553G>A
  49. ACS Pharmacol Transl Sci. 2021 Apr 09. 4(2): 624-646
      Metabolic reprogramming is a key hallmark of cancer and shifts cellular metabolism to meet the demands of biomass production necessary for abnormal cell reproduction. One-carbon metabolism (1CM) contributes to many biosynthetic pathways that fuel growth and is comprised of a complex network of enzymes. Methotrexate and 5-fluorouracil were pioneering drugs in this field and are still widely used today as anticancer agents as well as for other diseases such as arthritis. Besides dihydrofolate reductase and thymidylate synthase, two other enzymes of the folate cycle arm of 1CM have not been targeted clinically: serine hydroxymethyltransferase (SHMT) and methylenetetrahydrofolate dehydrogenase (MTHFD). An increasing body of literature suggests that the mitochondrial isoforms of these enzymes (SHMT2 and MTHFD2) are clinically relevant in the context of cancer. In this review, we focused on the 1CM pathway as a target for cancer therapy and, in particular, SHMT2 and MTHFD2. The function, regulation, and clinical relevance of SHMT2 and MTHFD2 are all discussed. We expand on previous clinical studies and evaluate the prognostic significance of these critical enzymes by performing a pan-cancer analysis of patient data from the The Cancer Genome Atlas and a transcriptional coexpression network enrichment analysis. We also provide an overview of preclinical and clinical inhibitors targeting the folate pathway, the methionine cycle, and folate-dependent purine biosynthesis enzymes.
  50. Cell Rep. 2021 Apr 13. pii: S2211-1247(21)00261-8. [Epub ahead of print]35(2): 108947
      During mitochondrial fission, key molecular and cellular factors assemble on the outer mitochondrial membrane, where they coordinate to generate constriction. Constriction sites can eventually divide or reverse upon disassembly of the machinery. However, a role for membrane tension in mitochondrial fission, although speculated, has remained undefined. We capture the dynamics of constricting mitochondria in mammalian cells using live-cell structured illumination microscopy (SIM). By analyzing the diameters of tubules that emerge from mitochondria and implementing a fluorescence lifetime-based mitochondrial membrane tension sensor, we discover that mitochondria are indeed under tension. Under perturbations that reduce mitochondrial tension, constrictions initiate at the same rate, but are less likely to divide. We propose a model based on our estimates of mitochondrial membrane tension and bending energy in living cells which accounts for the observed probability distribution for mitochondrial constrictions to divide.
    Keywords:  fluorescence lifetime; fluorescent tension sensor; membrane tension; microtubules; mitochondrial division; mitochondrial dynamics; super-resolution microscopy
  51. Brain. 2021 Apr 15. pii: awab056. [Epub ahead of print]
      Abnormal gut motility is a feature of several mitochondrial encephalomyopathies, and mutations in genes such as TYMP and POLG, have been linked to these rare diseases. The human genome encodes three DNA ligases, of which only one, ligase III (LIG3), has a mitochondrial splice variant and is crucial for mitochondrial health. We investigated the effect of reduced LIG3 activity and resulting mitochondrial dysfunction in seven patients from three independent families, who showed the common occurrence of gut dysmotility and neurological manifestations reminiscent of mitochondrial neurogastrointestinal encephalomyopathy. DNA from these patients was subjected to whole exome sequencing. In all patients, compound heterozygous variants in a new disease gene, LIG3, were identified. All variants were predicted to have a damaging effect on the protein. The LIG3 gene encodes the only mitochondrial DNA (mtDNA) ligase and therefore plays a pivotal role in mtDNA repair and replication. In vitro assays in patient-derived cells showed a decrease in LIG3 protein levels and ligase activity. We demonstrated that the LIG3 gene defects affect mtDNA maintenance, leading to mtDNA depletion without the accumulation of multiple deletions as observed in other mitochondrial disorders. This mitochondrial dysfunction is likely to cause the phenotypes observed in these patients. The most prominent and consistent clinical signs were severe gut dysmotility and neurological abnormalities, including leukoencephalopathy, epilepsy, migraine, stroke-like episodes, and neurogenic bladder. A decrease in the number of myenteric neurons, and increased fibrosis and elastin levels were the most prominent changes in the gut. Cytochrome c oxidase (COX) deficient fibres in skeletal muscle were also observed. Disruption of lig3 in zebrafish reproduced the brain alterations and impaired gut transit in vivo. In conclusion, we identified variants in the LIG3 gene that result in a mitochondrial disease characterized by predominant gut dysmotility, encephalopathy, and neuromuscular abnormalities. Bonora et al. identify a new mitochondrial recessive disorder caused by biallelic variants in the LIG3 gene encoding DNA ligase III, which is responsible for mitochondrial DNA repair. Clinical signs include gut dysmotility and neurological features such as leucoencephalopathy, epilepsy and stroke-like episodes.
    Keywords:   LIG3 ; CIPO; MNGIE; mtDNA repair; mtDNA replication
  52. Cell Rep. 2021 Apr 13. pii: S2211-1247(21)00255-2. [Epub ahead of print]35(2): 108941
      Mitochondrial function declines during brain aging and is suspected to play a key role in age-induced cognitive decline and neurodegeneration. Supplementing levels of spermidine, a body-endogenous metabolite, has been shown to promote mitochondrial respiration and delay aspects of brain aging. Spermidine serves as the amino-butyl group donor for the synthesis of hypusine (Nε-[4-amino-2-hydroxybutyl]-lysine) at a specific lysine residue of the eukaryotic translation initiation factor 5A (eIF5A). Here, we show that in the Drosophila brain, hypusinated eIF5A levels decline with age but can be boosted by dietary spermidine. Several genetic regimes of attenuating eIF5A hypusination all similarly affect brain mitochondrial respiration resembling age-typical mitochondrial decay and also provoke a premature aging of locomotion and memory formation in adult Drosophilae. eIF5A hypusination, conserved through all eukaryotes as an obviously critical effector of spermidine, might thus be an important diagnostic and therapeutic avenue in aspects of brain aging provoked by mitochondrial decline.
    Keywords:  CG8005; brain aging; deoxyhypusine synthase; eIF5A; eIF5A hypusination; learning and memory; locomotion; longevity; mitochondrial respiration; spermidine
  53. Theranostics. 2021 ;11(11): 5077-5091
      An increasing number of commonly prescribed drugs are known to interfere with mitochondrial function, causing cellular toxicity, but the underlying mechanisms are largely unknown. Although often not considered, mitochondrial transport proteins form a significant class of potential mitochondrial off-targets. So far, most drug interactions have been reported for the mitochondrial ADP/ATP carrier (AAC), which exchanges cytosolic ADP for mitochondrial ATP. Here, we show inhibition of cellular respiratory capacity by only a subset of the 18 published AAC inhibitors, which questions whether all compound do indeed inhibit such a central metabolic process. This could be explained by the lack of a simple, direct model system to evaluate and compare drug-induced AAC inhibition. Methods: For its development, we have expressed and purified human AAC1 (hAAC1) and applied two approaches. In the first, thermostability shift assays were carried out to investigate the binding of these compounds to human AAC1. In the second, the effect of these compounds on transport was assessed in proteoliposomes with reconstituted human AAC1, enabling characterization of their inhibition kinetics. Results: Of the proposed inhibitors, chebulinic acid, CD-437 and suramin are the most potent with IC50-values in the low micromolar range, whereas another six are effective at a concentration of 100 μM. Remarkably, half of all previously published AAC inhibitors do not show significant inhibition in our assays, indicating that they are false positives. Finally, we show that inhibitor strength correlates with a negatively charged surface area of the inhibitor, matching the positively charged surface of the substrate binding site. Conclusion: Consequently, we have provided a straightforward model system to investigate AAC inhibition and have gained new insights into the chemical compound features important for inhibition. Better evaluation methods of drug-induced inhibition of mitochondrial transport proteins will contribute to the development of drugs with an enhanced safety profile.
    Keywords:  adenine nucleotide translocase; drug-induced mitochondrial dysfunction; mitochondrial transport proteins; thermostability shift; transport inhibition kinetics.
  54. Nat Metab. 2021 Apr 12.
      Brown adipose tissue can expend large amounts of energy, and therefore increasing its size or activity is a promising therapeutic approach to combat metabolic disease. In humans, major deposits of brown fat cells are found intimately associated with large blood vessels, corresponding to perivascular adipose tissue (PVAT). However, the cellular origins of PVAT are poorly understood. Here, we determine the identity of perivascular adipocyte progenitors in mice and humans. In mice, thoracic PVAT develops from a fibroblastic lineage, consisting of progenitor cells (Pdgfra+, Ly6a+ and Pparg-) and preadipocytes (Pdgfra+, Ly6a+ and Pparg+), which share transcriptional similarity with analogous cell types in white adipose tissue. Interestingly, the aortic adventitia of adult animals contains a population of adipogenic smooth muscle cells (Myh11+, Pdgfra- and Pparg+) that contribute to perivascular adipocyte formation. Similarly, human PVAT contains presumptive fibroblastic and smooth muscle-like adipocyte progenitor cells, as revealed by single-nucleus RNA sequencing. Together, these studies define distinct populations of progenitor cells for thermogenic PVAT, providing a foundation for developing strategies to augment brown fat activity.