bims-mitdis Biomed News
on Mitochondrial disorders
Issue of 2023‒05‒28
68 papers selected by
Catalina Vasilescu
Helmholz Munich


  1. J Vis Exp. 2023 05 05.
      The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.
    DOI:  https://doi.org/10.3791/65236
  2. EMBO Mol Med. 2023 May 24. e16951
      Mitochondrial diseases are a heterogeneous group of monogenic disorders that result from impaired oxidative phosphorylation (OXPHOS). As neuromuscular tissues are highly energy-dependent, mitochondrial diseases often affect skeletal muscle. Although genetic and bioenergetic causes of OXPHOS impairment in human mitochondrial myopathies are well established, there is a limited understanding of metabolic drivers of muscle degeneration. This knowledge gap contributes to the lack of effective treatments for these disorders. Here, we discovered fundamental muscle metabolic remodeling mechanisms shared by mitochondrial disease patients and a mouse model of mitochondrial myopathy. This metabolic remodeling is triggered by a starvation-like response that evokes accelerated oxidation of amino acids through a truncated Krebs cycle. While initially adaptive, this response evolves in an integrated multiorgan catabolic signaling, lipid store mobilization, and intramuscular lipid accumulation. We show that this multiorgan feed-forward metabolic response involves leptin and glucocorticoid signaling. This study elucidates systemic metabolic dyshomeostasis mechanisms that underlie human mitochondrial myopathies and identifies potential new targets for metabolic intervention.
    Keywords:  amino acid metabolism; glucocorticoids; leptin; mitochondrial myopathy; muscle wasting
    DOI:  https://doi.org/10.15252/emmm.202216951
  3. Int J Mol Sci. 2023 May 09. pii: 8505. [Epub ahead of print]24(10):
      Congenital myasthenic syndromes (CMS) are a group of rare, neuromuscular disorders that usually present in childhood or infancy. While the phenotypic presentation of these disorders is diverse, the unifying feature is a pathomechanism that disrupts neuromuscular transmission. Recently, two mitochondrial genes-SLC25A1 and TEFM-have been reported in patients with suspected CMS, prompting a discussion about the role of mitochondria at the neuromuscular junction (NMJ). Mitochondrial disease and CMS can present with similar symptoms, and potentially one in four patients with mitochondrial myopathy exhibit NMJ defects. This review highlights research indicating the prominent roles of mitochondria at both the pre- and postsynapse, demonstrating the potential for mitochondrial involvement in neuromuscular transmission defects. We propose the establishment of a novel subcategorization for CMS-mitochondrial CMS, due to unifying clinical features and the potential for mitochondrial defects to impede transmission at the pre- and postsynapse. Finally, we highlight the potential of targeting the neuromuscular transmission in mitochondrial disease to improve patient outcomes.
    Keywords:  SLC25A1; TEFM; congenital myasthenic syndrome; mitochondria; mitochondrial disease; neuromuscular; neuromuscular junction
    DOI:  https://doi.org/10.3390/ijms24108505
  4. Curr Issues Mol Biol. 2023 May 19. 45(5): 4451-4479
      Mitochondria have made a long evolutionary path from ancient bacteria immigrants within the eukaryotic cell to become key players for the cell, assuming crucial multitasking skills critical for human health and disease. Traditionally identified as the powerhouses of eukaryotic cells due to their central role in energy metabolism, these chemiosmotic machines that synthesize ATP are known as the only maternally inherited organelles with their own genome, where mutations can cause diseases, opening up the field of mitochondrial medicine. More recently, the omics era has highlighted mitochondria as biosynthetic and signaling organelles influencing the behaviors of cells and organisms, making mitochondria the most studied organelles in the biomedical sciences. In this review, we will especially focus on certain 'novelties' in mitochondrial biology "left in the shadows" because, although they have been discovered for some time, they are still not taken with due consideration. We will focus on certain particularities of these organelles, for example, those relating to their metabolism and energy efficiency. In particular, some of their functions that reflect the type of cell in which they reside will be critically discussed, for example, the role of some carriers that are strictly functional to the typical metabolism of the cell or to the tissue specialization. Furthermore, some diseases in whose pathogenesis, surprisingly, mitochondria are involved will be mentioned.
    Keywords:  ancient bacteria; cardiolipin; cell death; mitochondria; mitochondria-related disorders; mitochondrial dysfunction; mitochondrial energy metabolism
    DOI:  https://doi.org/10.3390/cimb45050283
  5. Antioxidants (Basel). 2023 Apr 26. pii: 1001. [Epub ahead of print]12(5):
      Mitochondrial oxidative stress has been implicated in aging and several cardiovascular diseases, including heart failure and cardiomyopathy, ventricular tachycardia, and atrial fibrillation. The role of mitochondrial oxidative stress in bradyarrhythmia is less clear. Mice with a germline deletion of Ndufs4 subunit respiratory complex I develop severe mitochondrial encephalomyopathy resembling Leigh Syndrome (LS). Several types of cardiac bradyarrhythmia are present in LS mice, including a frequent sinus node dysfunction and episodic atrioventricular (AV) block. Treatment with the mitochondrial antioxidant Mitotempo or mitochondrial protective peptide SS31 significantly ameliorated the bradyarrhythmia and extended the lifespan of LS mice. Using an ex vivo Langendorff perfused heart with live confocal imaging of mitochondrial and total cellular reactive oxygen species (ROS), we showed increased ROS in the LS heart, which was potentiated by ischemia-reperfusion. A simultaneous ECG recording showed a sinus node dysfunction and AV block concurrent with the severity of the oxidative stress. Treatment with Mitotempo abolished ROS and restored the sinus rhythm. Our study reveals robust evidence of the direct mechanistic roles of mitochondrial and total ROS in bradyarrhythmia in the setting of LS mitochondrial cardiomyopathy. Our study also supports the potential clinical application of mitochondrial-targeted antioxidants or SS31 for the treatment of LS patients.
    Keywords:  Leigh Syndrome; arrhythmia; bradycardia; cardiomyopathy; mitochondria; oxidative stress
    DOI:  https://doi.org/10.3390/antiox12051001
  6. Nat Biotechnol. 2023 May 22.
      A number of mitochondrial diseases in humans are caused by point mutations that could be corrected by base editors, but delivery of CRISPR guide RNAs into the mitochondria is difficult. In this study, we present mitochondrial DNA base editors (mitoBEs), which combine a transcription activator-like effector (TALE)-fused nickase and a deaminase for precise base editing in mitochondrial DNA. Combining mitochondria-localized, programmable TALE binding proteins with the nickase MutH or Nt.BspD6I(C) and either the single-stranded DNA-specific adenine deaminase TadA8e or the cytosine deaminase ABOBEC1 and UGI, we achieve A-to-G or C-to-T base editing with up to 77% efficiency and high specificity. We find that mitoBEs are DNA strand-selective mitochondrial base editors, with editing results more likely to be retained on the nonnicked DNA strand. Furthermore, we correct pathogenic mitochondrial DNA mutations in patient-derived cells by delivering mitoBEs encoded in circular RNAs. mitoBEs offer a precise, efficient DNA editing tool with broad applicability for therapy in mitochondrial genetic diseases.
    DOI:  https://doi.org/10.1038/s41587-023-01791-y
  7. Biochim Biophys Acta Mol Basis Dis. 2023 May 19. pii: S0925-4439(23)00124-2. [Epub ahead of print]1869(6): 166758
      The mitochondrial citrate carrier (CIC) is a member of the mitochondrial carrier family and is responsible for the transit of tricarboxylates and dicarboxylates across the inner membrane. By modulating the flux of these molecules, it represents the molecular link between catabolic and anabolic reactions that take place in distinct cellular sub-compartments. Therefore, this transport protein represents an important element of investigation both in physiology and in pathology. In this review we critically analyze the involvement of the mitochondrial CIC in several human pathologies, which can be divided into two subgroups, one characterized by a decrease and the other by an increase in the flux of citrate across the inner mitochondrial membrane. In particular, a decrease in the activity of the mitochondrial CIC is responsible for several congenital diseases of different severity, which are also characterized by the increase in urinary levels of L-2- and D-2-hydroxyglutaric acids. On the other hand, an increase in the activity of the mitochondrial CIC is involved, in various ways, in the onset of inflammation, autoimmune diseases, and cancer. Then, understanding the role of CIC and the mechanisms driving the flux of metabolic intermediates between mitochondria and cytosol would potentially allow for manipulation and control of metabolism in pathological conditions.
    Keywords:  Autoimmune diseases; Cancer; Congenital diseases; Inflammation; Metabolic alterations; Mitochondrial carrier family
    DOI:  https://doi.org/10.1016/j.bbadis.2023.166758
  8. Int J Mol Sci. 2023 May 22. pii: 9108. [Epub ahead of print]24(10):
      Cardiomyopathies are mostly determined by genetic mutations affecting either cardiac muscle cell structure or function. Nevertheless, cardiomyopathies may also be part of complex clinical phenotypes in the spectrum of neuromuscular (NMD) or mitochondrial diseases (MD). The aim of this study is to describe the clinical, molecular, and histological characteristics of a consecutive cohort of patients with cardiomyopathy associated with NMDs or MDs referred to a tertiary cardiomyopathy clinic. Consecutive patients with a definitive diagnosis of NMDs and MDs presenting with a cardiomyopathy phenotype were described. Seven patients were identified: two patients with ACAD9 deficiency (Patient 1 carried the c.1240C>T (p.Arg414Cys) homozygous variant in ACAD9; Patient 2 carried the c.1240C>T (p.Arg414Cys) and the c.1646G>A (p.Ar549Gln) variants in ACAD9); two patients with MYH7-related myopathy (Patient 3 carried the c.1325G>A (p.Arg442His) variant in MYH7; Patient 4 carried the c.1357C>T (p.Arg453Cys) variant in MYH7); one patient with desminopathy (Patient 5 carried the c.46C>T (p.Arg16Cys) variant in DES); two patients with mitochondrial myopathy (Patient 6 carried the m.3243A>G variant in MT-TL1; Patient 7 carried the c.253G>A (p.Gly85Arg) and the c.1055C>T (p.Thr352Met) variants in MTO1). All patients underwent a comprehensive cardiovascular and neuromuscular evaluation, including muscle biopsy and genetic testing. This study described the clinical phenotype of rare NMDs and MDs presenting as cardiomyopathies. A multidisciplinary evaluation, combined with genetic testing, plays a main role in the diagnosis of these rare diseases, and provides information about clinical expectations, and guides management.
    Keywords:  cardiomyopathy; genetic testing; neuromuscular disease
    DOI:  https://doi.org/10.3390/ijms24109108
  9. Metabolites. 2023 May 19. pii: 671. [Epub ahead of print]13(5):
      Mitochondrial health declines with age, and older patients can demonstrate dysfunction in mitochondrial-rich tissues, such as cardiac and skeletal muscle. Aged mitochondria may make older adults more susceptible to adverse drug reactions (ADRs). We assessed mitochondrial metabolic function by measuring two metabolites, l-carnitine and acetylcarnitine, to determine their effectiveness as candidate clinical biomarkers for age-related, drug-induced alterations in mitochondrial metabolism. To study age- and medication-related changes in mitochondrial metabolism, we administered the FDA-approved mitochondriotropic drug, clofazimine (CFZ), or vehicle for 8 weeks to young (4-week-old) and old (61-week-old) male C57BL/6J mice. At the end of treatment, whole blood and cardiac and skeletal muscle were analyzed for l-carnitine, acetylcarnitine, and CFZ levels; muscle function was measured via a treadmill test. No differences were found in blood or cardiac carnitine levels of CFZ-treated mice, but CFZ-treated mice displayed lost body mass and alterations in endurance and levels of skeletal muscle mitochondrial metabolites. These findings demonstrate the age-related susceptibility of the skeletal muscle to mitochondria drug toxicity. Since drug-induced alterations in mitochondrial metabolism in skeletal muscle were not reflected in the blood by l-carnitine or acetylcarnitine levels, drug-induced catabolism and changes in muscle function appear more relevant to stratifying individuals at increased risk for ADRs.
    Keywords:  acetylcarnitine; adverse drug reactions; cardiac muscle; l-carnitine; mitochondrial metabolism
    DOI:  https://doi.org/10.3390/metabo13050671
  10. J Mol Med (Berl). 2023 May 20.
      With advancing age, the skeletal muscle phenotype is characterized by a progressive loss of mass, strength, and quality. This phenomenon, known as sarcopenia, has a negative impact on quality of life and increases the risk of morbidity and mortality in older adults. Accumulating evidence suggests that damaged and dysfunctional mitochondria play a critical role in the pathogenesis of sarcopenia. Lifestyle modifications, such as physical activity, exercise, and nutrition, as well as medical interventions with therapeutic agents, are effective in the management of sarcopenia and offer solutions to maintain and improve skeletal muscle health. Although a great deal of effort has been devoted to the identification of the best treatment option, these strategies are not sufficient to overcome sarcopenia. Recently, it has been reported that mitochondrial transplantation may be a possible therapeutic approach for the treatment of mitochondria-related pathological conditions such as ischemia, liver toxicity, kidney injury, cancer, and non-alcoholic fatty liver disease. Given the role of mitochondria in the function and metabolism of skeletal muscle, mitochondrial transplantation may be a possible option for the treatment of sarcopenia. In this review, we summarize the definition and characteristics of sarcopenia and molecular mechanisms associated with mitochondria that are known to contribute to sarcopenia. We also discuss mitochondrial transplantation as a possible option. Despite the progress made in the field of mitochondrial transplantation, further studies are needed to elucidate the role of mitochondrial transplantation in sarcopenia. KEY MESSAGES: Sarcopenia is the progressive loss of skeletal muscle mass, strength, and quality. Although the specific mechanisms that lead to sarcopenia are not fully understood, mitochondria have been identified as a key factor in the development of sarcopenia. Damaged and dysfunctional mitochondria initiate various cellular mediators and signaling pathways, which largely contribute to the age-related loss of skeletal muscle mass and strength. Mitochondrial transplantation has been reported to be a possible option for the treatment/prevention of several diseases. Mitochondrial transplantation may be a possible therapeutic option for improving skeletal muscle health and treating sarcopenia. Mitochondrial transplantation as a possible treatment option for sarcopenia.
    Keywords:  Mitochondrial dysfunction; Mitochondrial transplantation; Sarcopenia; Skeletal muscle
    DOI:  https://doi.org/10.1007/s00109-023-02326-3
  11. Antioxidants (Basel). 2023 Apr 25. pii: 992. [Epub ahead of print]12(5):
      The mitochondrion is the primary energy generator of a cell and is a central player in cellular redox regulation. Mitochondrial reactive oxygen species (mtROS) are the natural byproducts of cellular respiration that are critical for the redox signaling events that regulate a cell's metabolism. These redox signaling pathways primarily rely on the reversible oxidation of the cysteine residues on mitochondrial proteins. Several key sites of this cysteine oxidation on mitochondrial proteins have been identified and shown to modulate downstream signaling pathways. To further our understanding of mitochondrial cysteine oxidation and to identify uncharacterized redox-sensitive cysteines, we coupled mitochondrial enrichment with redox proteomics. Briefly, differential centrifugation methods were used to enrich for mitochondria. These purified mitochondria were subjected to both exogenous and endogenous ROS treatments and analyzed by two redox proteomics methods. A competitive cysteine-reactive profiling strategy, termed isoTOP-ABPP, enabled the ranking of the cysteines by their redox sensitivity, due to a loss of reactivity induced by cysteine oxidation. A modified OxICAT method enabled a quantification of the percentage of reversible cysteine oxidation. Initially, we assessed the cysteine oxidation upon treatment with a range of exogenous hydrogen peroxide concentrations, which allowed us to differentiate the mitochondrial cysteines by their susceptibility to oxidation. We then analyzed the cysteine oxidation upon inducing reactive oxygen species generation via the inhibition of the electron transport chain. Together, these methods identified the mitochondrial cysteines that were sensitive to endogenous and exogenous ROS, including several previously known redox-regulated cysteines and uncharacterized cysteines on diverse mitochondrial proteins.
    Keywords:  OxICAT; ROS; cysteine; isoTOP-ABPP; mass spectrometry; mitochondria; oxidation
    DOI:  https://doi.org/10.3390/antiox12050992
  12. Cell Calcium. 2023 May 22. pii: S0143-4160(23)00075-1. [Epub ahead of print]113 102763
      Mitochondria-associated membranes (MAMs) are signaling domains formed at points of contact between the endoplasmic reticulum and mitochondria that are essential for mitochondrial Ca2+ signaling, energy metabolism and cell survival. Thoudam et al. now show that MAMs are dynamically regulated by pyruvate dehydrogenase kinase 4 in alcohol-associate liver disease, adding one more piece to the ever more complex puzzle of ER-mitochondria interactions in health and disease.
    DOI:  https://doi.org/10.1016/j.ceca.2023.102763
  13. Genes (Basel). 2023 May 15. pii: 1087. [Epub ahead of print]14(5):
      BACKGROUND: Next-generation sequencing (NGS) technology is revolutionizing diagnostic screening for mitochondrial diseases (MDs). Moreover, an investigation by NGS still requires analyzing the mitochondrial genome and nuclear genes separately, with limitations in terms of time and costs. We describe the validation and implementation of a custom blended MITOchondrial-NUCLEAR (MITO-NUCLEAR) assay for the simultaneous identification of genetic variants both in whole mtDNA and in nuclear genes included in a clinic exome panel. Furthermore, the MITO-NUCLEAR assay, implemented in our diagnostic process, has allowed us to arrive at a molecular diagnosis in a young patient.METHODS: Massive sequencing strategy was applied for the validation experiments, performed using multiple tissues (blood, buccal swab, fresh tissue, tissue from slide, and formalin-fixed paraffin-embedded tissue section) and two different blend-in ratios of the mitochondrial probes: nuclear probes; 1:900 and 1:300.
    RESULTS: Data suggested that 1:300 was the optimal probe dilution, where 100% of the mtDNA was covered at least 3000×, the median coverage was >5000×, and 93.84% of nuclear regions were covered at least 100×.
    CONCLUSIONS: Our custom Agilent SureSelect MITO-NUCLEAR panel provides a potential "one-step" investigation that may be applied to both research and genetic diagnosis of MDs, allowing the simultaneous discovery of nuclear and mitochondrial mutations.
    Keywords:  MITO-NUCLEAR; combined sequencing of mitochondrial and nuclear DNA; mitochondrial and nuclear disease panel; mitochondrial diseases; mtDNA; next-generation sequencing (NGS); nuclear genes
    DOI:  https://doi.org/10.3390/genes14051087
  14. Mult Scler. 2023 May 25. 13524585231172950
      We report two children with molecularly confirmed mitochondrial disease mimicking Neuromyelitis Optica Spectrum Disorder (NMOSD). The first patient presented at the age of 15 months with acute deterioration following a pyrexial illness with clinical features localising to the brainstem and spinal cord. The second patient presented at 5 years with acute bilateral visual loss. In both cases, MOG and AQP4 antibodies were negative. Both patients died within a year of symptoms onset from respiratory failure. Arriving at an early genetic diagnosis is important for redirection of care and avoiding potentially harmful immunosuppressant therapies.
    Keywords:  Neuromyelitis optica spectrum disorder; acquired demyelinating syndromes; mitochondrial diseases; neurogenetics
    DOI:  https://doi.org/10.1177/13524585231172950
  15. Biomolecules. 2023 Apr 27. pii: 753. [Epub ahead of print]13(5):
      Nuclear-mitochondrial DNA segments (NUMTs) are mitochondrial DNA (mtDNA) fragments that have been inserted into the nuclear genome. Some NUMTs are common within the human population but most NUMTs are rare and specific to individuals. NUMTs range in size from 24 base pairs to encompassing nearly the entire mtDNA and are found throughout the nuclear genome. Emerging evidence suggests that the formation of NUMTs is an ongoing process in humans. NUMTs contaminate sequencing results of the mtDNA by introducing false positive variants, particularly heteroplasmic variants present at a low variant allele frequency (VAF). In our review, we discuss the prevalence of NUMTs in the human population, the potential mechanisms of de novo NUMT insertion via DNA repair mechanisms, and provide an overview of the existing approaches for minimizing NUMT contamination. Apart from filtering known NUMTs, both wet lab-based and computational methods can be used to minimize the contamination of NUMTs in analyses of human mtDNA. Current approaches include: (1) isolating mitochondria to enrich for mtDNA; (2) applying basic local alignment to identify NUMTs for subsequent filtering; (3) bioinformatic pipelines for NUMT detection; (4) k-mer-based NUMT detection; and (5) filtering candidate false positive variants by mtDNA copy number, VAF, or sequence quality score. Multiple approaches must be applied in order to effectively identify NUMTs in samples. Although next-generation sequencing is revolutionizing our understanding of heteroplasmic mtDNA, it also raises new challenges with the high prevalence and individual-specific NUMTs that need to be handled with care in studies of mitochondrial genetics.
    Keywords:  bioinformatics; mitochondrial DNA; nuclear-mitochondrial DNA segments  mitochondrial genome
    DOI:  https://doi.org/10.3390/biom13050753
  16. J Peripher Nerv Syst. 2023 May 23.
      BACKGROUND AND AIMS: Mitofusin 1 (MFN1) and MFN2 are outer mitochondrial membrane fusogenic proteins regulating mitochondrial network morphology. MFN2 mutations cause Charcot-Marie-Tooth type 2A (CMT2A), an axonal neuropathy characterized by mitochondrial fusion defects, which in the case of a GTPase domain mutant, were rescued following wild type MFN1/2 (MFN1/2WT ) overexpression. In this study, we compared the therapeutic efficiency between MFN1WT and MFN2WT overexpression in correcting mitochondrial defects induced by the novel MFN2K357T mutation located in the highly conserved R3 region.METHODS: Constructs expressing either MFN2K357T , MFN2WT , or MFN1WT under the ubiquitous chicken β-actin hybrid (CBh) promoter were generated. Flag or myc tag was used for their detection. Differentiated SH-SY5Y cells were single transfected with MFN1WT , MFN2WT , or MFN2K357T , as well as double transfected with MFN2K357T /MFN2WT or MFN2K357T /MFN1WT .
    RESULTS: SH-SY5Y cells transfected with MFN2K357T exhibited severe perinuclear mitochondrial clustering with axon-like processes devoid of mitochondria. Single transfection with MFN1WT resulted in a more interconnected mitochondrial network than transfection with MFN2WT , accompanied by mitochondrial clusters. Double transfection of MFN2K357T with either MFN1WT or MFN2WT resolved the mutant-induced mitochondrial clusters and led to detectable mitochondria throughout the axon-like processes. MFN1WT showed higher efficacy than MFN2WT in rescuing these defects.
    INTERPRETATION: These results further demonstrate the higher potential of MFN1WT over MFN2WT overexpression to rescue CMT2A-induced mitochondrial network abnormalities due to mutations outside the GTPase domain. This higher phenotypic rescue conferred by MFN1WT , possibly due to its higher mitochondrial fusogenic ability, may be applied to different CMT2A cases regardless of the MFN2 mutation type. This article is protected by copyright. All rights reserved.
    Keywords:  Charcot-Marie-Tooth type 2A; Mitofusin 1; Mitofusin 2
    DOI:  https://doi.org/10.1111/jns.12564
  17. Front Mol Biosci. 2023 ;10 1169109
      Collectively, rare genetic disorders affect a substantial portion of the world's population. In most cases, those affected face difficulties in receiving a clinical diagnosis and genetic characterization. The understanding of the molecular mechanisms of these diseases and the development of therapeutic treatments for patients are also challenging. However, the application of recent advancements in genome sequencing/analysis technologies and computer-aided tools for predicting phenotype-genotype associations can bring significant benefits to this field. In this review, we highlight the most relevant online resources and computational tools for genome interpretation that can enhance the diagnosis, clinical management, and development of treatments for rare disorders. Our focus is on resources for interpreting single nucleotide variants. Additionally, we present use cases for interpreting genetic variants in clinical settings and review the limitations of these results and prediction tools. Finally, we have compiled a curated set of core resources and tools for analyzing rare disease genomes. Such resources and tools can be utilized to develop standardized protocols that will enhance the accuracy and effectiveness of rare disease diagnosis.
    Keywords:  genetic disorder; genome interpretation; genotype-phenotype association; machine learning; precision medicine; rare disease; single nucleotide variant (SNV)
    DOI:  https://doi.org/10.3389/fmolb.2023.1169109
  18. Cell Biosci. 2023 May 23. 13(1): 96
      BACKGROUND: Patients suffered from severe traumatic brain injury (TBI) have twice the risk of developing into neurodegenerative diseases later in their life. Thus, early intervention is needed not only to treat TBI but also to reduce neurodegenerative diseases in the future. Physiological functions of neurons highly depend on mitochondria. Thus, when mitochondrial integrity is compromised by injury, neurons would initiate a cascade of events to maintain homeostasis of mitochondria. However, what protein senses mitochondrial dysfunction and how mitochondrial homeostasis is maintained during regeneration remains unclear.RESULTS: We found that TBI-increased transcription of a mitochondrial protein, phosphoglycerate mutase 5 (PGAM5), during acute phase was via topological remodeling of a novel enhancer-promoter interaction. This up-regulated PGAM5 correlated with mitophagy, whereas presenilins-associated rhomboid-like protein (PARL)-dependent PGAM5 cleavage at a later stage of TBI enhanced mitochondrial transcription factor A (TFAM) expression and mitochondrial mass. To test whether PGAM5 cleavage and TFAM expression were sufficient for functional recovery, mitochondrial oxidative phosphorylation uncoupler carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) was used to uncouple electron transport chain and reduce mitochondrial function. As a result, FCCP triggered PGAM5 cleavage, TFAM expression and recovery of motor function deficits of CCI mice.
    CONCLUSIONS: Findings from this study implicate that PGAM5 may act as a mitochondrial sensor for brain injury to activate its own transcription at acute phase, serving to remove damaged mitochondria through mitophagy. Subsequently, PGAM5 is cleaved by PARL, and TFAM expression is increased for mitochondrial biogenesis at a later stage after TBI. Taken together, this study concludes that timely regulation of PGAM5 expression and its own cleavage are required for neurite re-growth and functional recovery.
    Keywords:  Epigenetic regulation; Mitochondrial homeostasis; Neurite re-growth; PGAM5; Traumatic brain injury
    DOI:  https://doi.org/10.1186/s13578-023-01052-0
  19. Nat Cell Biol. 2023 May 22.
      Mitochondrial proteases are emerging as key regulators of mitochondrial plasticity and acting as both protein quality surveillance and regulatory enzymes by performing highly regulated proteolytic reactions. However, it remains unclear whether the regulated mitochondrial proteolysis is mechanistically linked to cell identity switching. Here we report that cold-responsive mitochondrial proteolysis is a prerequisite for white-to-beige adipocyte cell fate programming during adipocyte thermogenic remodelling. Thermogenic stimulation selectively promotes mitochondrial proteostasis in mature white adipocytes via the mitochondrial protease LONP1. Disruption of LONP1-dependent proteolysis substantially impairs cold- or β3 adrenergic agonist-induced white-to-beige identity switching of mature adipocytes. Mechanistically, LONP1 selectively degrades succinate dehydrogenase complex iron sulfur subunit B and ensures adequate intracellular succinate levels. This alters the histone methylation status on thermogenic genes and thereby enables adipocyte cell fate programming. Finally, augmented LONP1 expression raises succinate levels and corrects ageing-related impairments in white-to-beige adipocyte conversion and adipocyte thermogenic capacity. Together, these findings reveal that LONP1 links proteolytic surveillance to mitochondrial metabolic rewiring and directs cell identity conversion during adipocyte thermogenic remodelling.
    DOI:  https://doi.org/10.1038/s41556-023-01155-3
  20. J Mol Cell Cardiol. 2023 May 23. pii: S0022-2828(23)00093-7. [Epub ahead of print]
      Transport of Ca2+ into mitochondria is thought to stimulate the production of ATP, a critical process in the heart's fight or flight response, but excess Ca2+ can trigger cell death. The mitochondrial Ca2+ uniporter complex is the primary route of Ca2+ transport into mitochondria, in which the channel-forming protein MCU and the regulatory protein EMRE are essential for activity. In previous studies, chronic Mcu or Emre deletion differ from acute cardiac Mcu deletion in response to adrenergic stimulation and ischemia/reperfusion (I/R) injury, despite equivalent inactivation of rapid mitochondrial Ca2+ uptake. To explore this discrepancy between chronic and acute loss of uniporter activity, we compared short-term and long-term Emre deletion using a novel conditional cardiac-specific, tamoxifen-inducible mouse model. After short-term Emre deletion (3 weeks post-tamoxifen) in adult mice, cardiac mitochondria were unable to take up Ca2+, had lower basal mitochondrial Ca2+ levels, and displayed attenuated Ca2+-induced ATP production and mPTP opening. Moreover, short-term EMRE loss blunted cardiac response to adrenergic stimulation and improved maintenance of cardiac function in an ex vivo I/R model. We then tested whether the long-term absence of EMRE (3 months post-tamoxifen) in adulthood would lead to distinct outcomes. After long-term Emre deletion, mitochondrial Ca2+ handling and function, as well as cardiac response to adrenergic stimulation, were similarly impaired as in short-term deletion. Interestingly, however, protection from I/R injury was lost in the long-term. These data suggest that several months without uniporter function are insufficient to restore bioenergetic response but are sufficient to restore susceptibility to I/R.
    Keywords:  ATP; Calcium; Ischemia/reperfusion; Mitochondria; Mitochondrial calcium uniporter; Permeability transition pore
    DOI:  https://doi.org/10.1016/j.yjmcc.2023.05.007
  21. bioRxiv. 2023 May 08. pii: 2023.05.08.539795. [Epub ahead of print]
      Dopa-responsive dystonia (DRD) and Parkinson's disease (PD) are movement disorders caused by the dysfunction of nigrostriatal dopaminergic neurons. Identifying druggable pathways and biomarkers for guiding therapies is crucial due to the debilitating nature of these disorders. Recent genetic studies have identified variants of GTP cyclohydrolase-1 (GCH1), the rate-limiting enzyme in tetrahydrobiopterin (BH4) synthesis, as causative for these movement disorders. Here, we show that genetic and pharmacological inhibition of BH4 synthesis in mice and human midbrain-like organoids accurately recapitulates motor, behavioral and biochemical characteristics of these human diseases, with severity of the phenotype correlating with extent of BH4 deficiency. We also show that BH4 deficiency increases sensitivities to several PD-related stressors in mice and PD human cells, resulting in worse behavioral and physiological outcomes. Conversely, genetic and pharmacological augmentation of BH4 protects mice from genetically- and chemically induced PD-related stressors. Importantly, increasing BH4 levels also protects primary cells from PD-affected individuals and human midbrain-like organoids (hMLOs) from these stressors. Mechanistically, BH4 not only serves as an essential cofactor for dopamine synthesis, but also independently regulates tyrosine hydroxylase levels, protects against ferroptosis, scavenges mitochondrial ROS, maintains neuronal excitability and promotes mitochondrial ATP production, thereby enhancing mitochondrial fitness and cellular respiration in multiple preclinical PD animal models, human dopaminergic midbrain-like organoids and primary cells from PD-affected individuals. Our findings pinpoint the BH4 pathway as a key metabolic program at the intersection of multiple protective mechanisms for the health and function of midbrain dopaminergic neurons, identifying it as a potential therapeutic target for PD.
    DOI:  https://doi.org/10.1101/2023.05.08.539795
  22. Int J Mol Sci. 2023 May 20. pii: 9036. [Epub ahead of print]24(10):
      Mitochondria contain connexins, a family of proteins that is known to form gap junction channels. Connexins are synthesized in the endoplasmic reticulum and oligomerized in the Golgi to form hemichannels. Hemichannels from adjacent cells dock with one another to form gap junction channels that aggregate into plaques and allow cell-cell communication. Cell-cell communication was once thought to be the only function of connexins and their gap junction channels. In the mitochondria, however, connexins have been identified as monomers and assembled into hemichannels, thus questioning their role solely as cell-cell communication channels. Accordingly, mitochondrial connexins have been suggested to play critical roles in the regulation of mitochondrial functions, including potassium fluxes and respiration. However, while much is known about plasma membrane gap junction channel connexins, the presence and function of mitochondrial connexins remain poorly understood. In this review, the presence and role of mitochondrial connexins and mitochondrial/connexin-containing structure contact sites will be discussed. An understanding of the significance of mitochondrial connexins and their connexin contact sites is essential to our knowledge of connexins' functions in normal and pathological conditions, and this information may aid in the development of therapeutic interventions in diseases linked to mitochondria.
    Keywords:  annular gap junctions; connexin; contact sites; mitochondria
    DOI:  https://doi.org/10.3390/ijms24109036
  23. FASEB J. 2023 Jun;37(6): e22953
      Adenine nucleotide translocases (ANTs) are central to mitochondrial integrity and bioenergetic metabolism. This review aims to integrate the progresses and knowledge on ANTs over the last few years, contributing to a potential implication of ANTs for various diseases. Structures, functions, modifications, regulators and pathological implications of ANTs for human diseases are intensively demonstrated here. ANTs have four isoforms (ANT1-4), responsible for exchanging ATP/ADP, possibly composing of pro-apoptotic mPTP as a major component, and mediating FA-dependent uncoupling of proton efflux. ANT can be modified by methylation, nitrosylation and nitroalkylation, acetylation, glutathionylation, phosphorylation, carbonylation and hydroxynonenal-induced modifications. Compounds, including bongkrekic acid, atractyloside calcium, carbon monoxide, minocycline, 4-(N-(S-penicillaminylacetyl)amino) phenylarsonous acid, cardiolipin, free long-chain fatty acids, agaric acid, long chain acyl-coenzyme A esters, all have an ability to regulate ANT activities. ANT impairment leads to bioenergetic failure and mitochondrial dysfunction, contributing to pathogenesis of diseases, such as diabetes (deficiency), heart disease (deficiency), Parkinson's disease (reduction), Sengers Syndrome (decrease), cancer (isoform shifting), Alzheimer's Disease (coaggregation with Tau), Progressive External Opthalmoplegia (mutation), and Fascioscapulohumeral muscular dystrophy (overexpression). This review improves the understanding of the mechanism of ANT in pathogenesis of human diseases, and opens a window for novel therapeutic strategies targeted on ANT in diseases.
    Keywords:  ATP/ADP exchanging; Parkinson's disease; adenine nucleotide translocases; cancer; heart disease; human diseases; mitochondria; post-translational modifications
    DOI:  https://doi.org/10.1096/fj.202201855RR
  24. Sci Rep. 2023 May 24. 13(1): 8391
      Maintaining mitochondrial function is critical to an improved healthspan and lifespan. Introducing mild stress by inhibiting mitochondrial translation invokes the mitochondrial unfolded protein response (UPRmt) and increases lifespan in several animal models. Notably, lower mitochondrial ribosomal protein (MRP) expression also correlates with increased lifespan in a reference population of mice. In this study, we tested whether partially reducing the gene expression of a critical MRP, Mrpl54, reduced mitochondrial DNA-encoded protein content, induced the UPRmt, and affected lifespan or metabolic health using germline heterozygous Mrpl54 mice. Despite reduced Mrpl54 expression in multiple organs and a reduction in mitochondrial-encoded protein expression in myoblasts, we identified few significant differences between male or female Mrpl54+/- and wild type mice in initial body composition, respiratory parameters, energy intake and expenditure, or ambulatory motion. We also observed no differences in glucose or insulin tolerance, treadmill endurance, cold tolerance, heart rate, or blood pressure. There were no differences in median life expectancy or maximum lifespan. Overall, we demonstrate that genetic manipulation of Mrpl54 expression reduces mitochondrial-encoded protein content but is not sufficient to improve healthspan in otherwise healthy and unstressed mice.
    DOI:  https://doi.org/10.1038/s41598-023-35196-3
  25. J Transl Med. 2023 May 25. 21(1): 347
      Cardiovascular disease (CVD) is the leading cause of noncommunicable disease-related death worldwide, and effective therapeutic strategies against CVD are urgently needed. Mitochondria dysfunction involves in the onset and development of CVD. Nowadays, mitochondrial transplantation, an alternative treatment aimed at increasing mitochondrial number and improving mitochondrial function, has been emerged with great therapeutic potential. Substantial evidence indicates that mitochondrial transplantation improves cardiac function and outcomes in patients with CVD. Therefore, mitochondrial transplantation has profound implications in the prevention and treatment of CVD. Here, we review the mitochondrial abnormalities that occur in CVD and summarize the therapeutic strategies of mitochondrial transplantation for CVD.
    Keywords:  Cardiovascular diseases; Ischemia reperfusion injury; Mitochondrial dysfunction; Mitochondrial transplantation; Therapy
    DOI:  https://doi.org/10.1186/s12967-023-04203-6
  26. Biomolecules. 2023 May 20. pii: 869. [Epub ahead of print]13(5):
      The regulation of mitochondria structure and function is at the core of numerous viral infections. Acting in support of the host or of virus replication, mitochondria regulation facilitates control of energy metabolism, apoptosis, and immune signaling. Accumulating studies have pointed to post-translational modification (PTM) of mitochondrial proteins as a critical component of such regulatory mechanisms. Mitochondrial PTMs have been implicated in the pathology of several diseases and emerging evidence is starting to highlight essential roles in the context of viral infections. Here, we provide an overview of the growing arsenal of PTMs decorating mitochondrial proteins and their possible contribution to the infection-induced modulation of bioenergetics, apoptosis, and immune responses. We further consider links between PTM changes and mitochondrial structure remodeling, as well as the enzymatic and non-enzymatic mechanisms underlying mitochondrial PTM regulation. Finally, we highlight some of the methods, including mass spectrometry-based analyses, available for the identification, prioritization, and mechanistic interrogation of PTMs.
    Keywords:  acylation; mass spectrometry; mitochondria; phosphorylation; post-translational modifications; proteomics; virus–host interactions
    DOI:  https://doi.org/10.3390/biom13050869
  27. Int J Mol Sci. 2023 May 16. pii: 8848. [Epub ahead of print]24(10):
      Mitochondrial dysfunction is a hallmark of numerous diseases, including neurodegenerative disorders, metabolic disorders, and cancer. Mitochondrial transfer, the transfer of mitochondria from one cell to another, has recently emerged as a potential therapeutic approach for restoring mitochondrial function in diseased cells. In this review, we summarize the current understanding of mitochondrial transfer, including its mechanisms, potential therapeutic applications, and impact on cell death pathways. We also discuss the future directions and challenges in the field of mitochondrial transfer as a novel therapeutic approach in disease diagnosis and treatment.
    Keywords:  cancer; cell death; disease diagnosis; disease treatment; metabolic disorders; mitochondrial transfer; neurodegenerative diseases
    DOI:  https://doi.org/10.3390/ijms24108848
  28. Redox Biol. 2023 May 19. pii: S2213-2317(23)00156-8. [Epub ahead of print]63 102755
      During cardiac ischemia-reperfusion, excess reactive oxygen species can damage mitochondrial, cellular and organ function. Here we show that cysteine oxidation of the mitochondrial protein Opa1 contributes to mitochondrial damage and cell death caused by oxidative stress. Oxy-proteomics of ischemic-reperfused hearts reveal oxidation of the C-terminal C786 of Opa1 and treatment of perfused mouse hearts, adult cardiomyocytes, and fibroblasts with H2O2 leads to the formation of a reduction-sensitive ∼180 KDa Opa1 complex, distinct from the ∼270 KDa one antagonizing cristae remodeling. This Opa1 oxidation process is curtailed by mutation of C786 and of the other 3 Cys residues of its C-terminal domain (Opa1TetraCys). When reintroduced in Opa1-/- cells, Opa1TetraCys is not efficiently processed into short Opa1TetraCys and hence fails to fuse mitochondria. Unexpectedly, Opa1TetraCys restores mitochondrial ultrastructure in Opa1-/- cells and protects them from H2O2-induced mitochondrial depolarization, cristae remodeling, cytochrome c release and cell death. Thus, preventing the Opa1 oxidation occurring during cardiac ischemia-reperfusion reduces mitochondrial damage and cell death induced by oxidative stress independent of mitochondrial fusion.
    DOI:  https://doi.org/10.1016/j.redox.2023.102755
  29. Redox Biol. 2023 May 15. pii: S2213-2317(23)00141-6. [Epub ahead of print]63 102740
      Mitochondrial supercomplexes are observed in mammalian tissues with high energy demand and may influence metabolism and redox signaling. Nevertheless, the mechanisms that regulate supercomplex abundance remain unclear. In this study, we examined the composition of supercomplexes derived from murine cardiac mitochondria and determined how their abundance changes with substrate provision or by genetically induced changes to the cardiac glucose-fatty acid cycle. Protein complexes from digitonin-solubilized cardiac mitochondria were resolved by blue-native polyacrylamide gel electrophoresis and were identified by mass spectrometry and immunoblotting to contain constituents of Complexes I, III, IV, and V as well as accessory proteins involved in supercomplex assembly and stability, cristae architecture, carbohydrate and fat oxidation, and oxidant detoxification. Respiratory analysis of high molecular mass supercomplexes confirmed the presence of intact respirasomes, capable of transferring electrons from NADH to O2. Provision of respiratory substrates to isolated mitochondria augmented supercomplex abundance, with fatty acyl substrate (octanoylcarnitine) promoting higher supercomplex abundance than carbohydrate-derived substrate (pyruvate). Mitochondria isolated from transgenic hearts that express kinase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (GlycoLo), which decreases glucose utilization and increases reliance on fatty acid oxidation for energy, had higher mitochondrial supercomplex abundance and activity compared with mitochondria from wild-type or phosphatase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-expressing hearts (GlycoHi), the latter of which encourages reliance on glucose catabolism for energy. These findings indicate that high energetic reliance on fatty acid catabolism bolsters levels of mitochondrial supercomplexes, supporting the idea that the energetic state of the heart is regulatory factor in supercomplex assembly or stability.
    Keywords:  Glycolysis; Heart; Metabolism; Mitochondria; Respirasome; Supercomplex
    DOI:  https://doi.org/10.1016/j.redox.2023.102740
  30. Basic Res Cardiol. 2023 May 22. 118(1): 20
      SMYD1, a striated muscle-specific lysine methyltransferase, was originally shown to play a key role in embryonic cardiac development but more recently we demonstrated that loss of Smyd1 in the murine adult heart leads to cardiac hypertrophy and failure. However, the effects of SMYD1 overexpression in the heart and its molecular function in the cardiomyocyte in response to ischemic stress are unknown. In this study, we show that inducible, cardiomyocyte-specific overexpression of SMYD1a in mice protects the heart from ischemic injury as seen by a > 50% reduction in infarct size and decreased myocyte cell death. We also demonstrate that attenuated pathological remodeling is a result of enhanced mitochondrial respiration efficiency, which is driven by increased mitochondrial cristae formation and stabilization of respiratory chain supercomplexes within the cristae. These morphological changes occur concomitant with increased OPA1 expression, a known driver of cristae morphology and supercomplex formation. Together, these analyses identify OPA1 as a novel downstream target of SMYD1a whereby cardiomyocytes upregulate energy efficiency to dynamically adapt to the energy demands of the cell. In addition, these findings highlight a new epigenetic mechanism by which SMYD1a regulates mitochondrial energetics and functions to protect the heart from ischemic injury.
    Keywords:  Cristae; Ischemic heart failure; Methyltransferase; Mitochondrial respiration; SMYD1
    DOI:  https://doi.org/10.1007/s00395-023-00991-6
  31. Front Genet. 2023 ;14 1177204
      Like many other Arab countries, the United Arab Emirates (UAE) has a relatively high prevalence of genetic disorders. Here we present the first review and analysis of all genetic disorders and gene variants reported in Emirati nationals and hosted on the Catalogue for Transmission Genetics in Arabs (CTGA), an open-access database hosting bibliographic data on human gene variants associated with inherited or heritable phenotypes in Arabs. To date, CTGA hosts 665 distinct genetic conditions that have been described in Emiratis, 621 of which follow a clear Mendelian inheritance. Strikingly, over half of these are extremely rare according to global prevalence rates, predominantly with an autosomal recessive mode of inheritance. This is likely due to the relatively high consanguinity rates within the Emirati population. The 665 conditions include disorders that are unique to the Emirati population, as well as clearly monogenic disorders that have not yet been mapped to a causal genetic locus. We also describe 1,365 gene variants reported in Emiratis, most of which are substitutions and over half are classified as likely pathogenic or pathogenic. Of these, 235 had not been reported on the international databases dbSNP and Clinvar, as of December 2022. Further analysis of this Emirati variant dataset allows a comparison of clinical significance as reported by Clinvar and CTGA, where the latter is derived from the study cited. A total of 307 pathogenic/likely pathogenic variants from CTGA's Emirati dataset, were classified as benign, variants of uncertain significance, or were missing a clinical significance or had not been reported by Clinvar. In conclusion, we present here the spectrum of genetic disorders and gene variants reported in Emiratis. This review emphasizes the importance of ethnic databases such as CTGA in addressing the underrepresentation of Arab variant data in international databases and documenting population-specific discrepancies in variant interpretation, reiterating the value of such repositories for clinicians and researchers, especially when dealing with rare disorders.
    Keywords:  Emirati; database; rare disease; variant; variant annotation
    DOI:  https://doi.org/10.3389/fgene.2023.1177204
  32. Biomolecules. 2023 May 10. pii: 808. [Epub ahead of print]13(5):
      The human mitochondrial carrier family (MCF) consists of 53 members. Approximately one-fifth of them are still orphans of a function. Most mitochondrial transporters have been functionally characterized by reconstituting the bacterially expressed protein into liposomes and transport assays with radiolabeled compounds. The efficacy of this experimental approach is constrained to the commercial availability of the radiolabeled substrate to be used in the transport assays. A striking example is that of N-acetylglutamate (NAG), an essential regulator of the carbamoyl synthetase I activity and the entire urea cycle. Mammals cannot modulate mitochondrial NAG synthesis but can regulate the levels of NAG in the matrix by exporting it to the cytosol, where it is degraded. The mitochondrial NAG transporter is still unknown. Here, we report the generation of a yeast cell model suitable for identifying the putative mammalian mitochondrial NAG transporter. In yeast, the arginine biosynthesis starts in the mitochondria from NAG which is converted to ornithine that, once transported into cytosol, is metabolized to arginine. The deletion of ARG8 makes yeast cells unable to grow in the absence of arginine since they cannot synthetize ornithine but can still produce NAG. To make yeast cells dependent on a mitochondrial NAG exporter, we moved most of the yeast mitochondrial biosynthetic pathway to the cytosol by expressing four E. coli enzymes, argB-E, able to convert cytosolic NAG to ornithine. Although argB-E rescued the arginine auxotrophy of arg8∆ strain very poorly, the expression of the bacterial NAG synthase (argA), which would mimic the function of a putative NAG transporter increasing the cytosolic levels of NAG, fully rescued the growth defect of arg8∆ strain in the absence of arginine, demonstrating the potential suitability of the model generated.
    Keywords:  N-acetylglutamate; mitochondrial carriers; urea cycle; yeast cell model
    DOI:  https://doi.org/10.3390/biom13050808
  33. Nat Rev Mol Cell Biol. 2023 May 24.
      Viewing metabolism through the lens of exercise biology has proven an accessible and practical strategy to gain new insights into local and systemic metabolic regulation. Recent methodological developments have advanced understanding of the central role of skeletal muscle in many exercise-associated health benefits and have uncovered the molecular underpinnings driving adaptive responses to training regimens. In this Review, we provide a contemporary view of the metabolic flexibility and functional plasticity of skeletal muscle in response to exercise. First, we provide background on the macrostructure and ultrastructure of skeletal muscle fibres, highlighting the current understanding of sarcomeric networks and mitochondrial subpopulations. Next, we discuss acute exercise skeletal muscle metabolism and the signalling, transcriptional and epigenetic regulation of adaptations to exercise training. We address knowledge gaps throughout and propose future directions for the field. This Review contextualizes recent research of skeletal muscle exercise metabolism, framing further advances and translation into practice.
    DOI:  https://doi.org/10.1038/s41580-023-00606-x
  34. J Inherit Metab Dis. 2023 May 27.
      Methylmalonic Acidemia (MMA) is a heterogenous group of inborn errors of metabolism caused by a defect in the methylmalonyl-CoA mutase (MMUT) enzyme or the synthesis and transport of its cofactor, 5'-deoxy- adenosylcobalamin. It is characterized by life-threatening episodes of ketoacidosis, chronic kidney disease, and other multiorgan complications. Liver transplantation can improve patient stability and survival and thus provides clinical and biochemical benchmarks for the development of hepatocyte-targeted genomic therapies. Data are presented from a US natural history protocol that evaluated subjects with different types of MMA including mut- (N=91), cblB- (15), and cblA-type MMA (17), as well as from an Italian cohort of mut- (N=19) and cblB-type MMA (N=2) subjects, including data before and after organ transplantation in both cohorts. Canonical metabolic markers, such as serum methylmalonic acid and propionylcarnitine, are variable and affected by dietary intake and renal function. We have therefore explored the use of the 1-13 C-propionate oxidation breath test (POBT) to measure metabolic capacity and the changes in circulating proteins to assess mitochondrial dysfunction (fibroblast growth factor-21, FGF-21 and growth differentiation factor-15, GDF-15) and kidney injury (lipocalin-2, LCN2). Biomarker concentrations are higher in patients with the severe mut0 - and cblB-type MMA, correlate with a decreased POBT, and show a significant response post-liver transplant. Additional circulating and imaging markers to assess disease burden are necessary to monitor disease progression. A combination of biomarkers reflecting disease severity and multisystem involvement will be needed to help stratify patients for clinical trials and assess the efficacy of new therapies for MMA. This article is protected by copyright. All rights reserved.
    Keywords:  FGF21; GDF15; biomarker; clinical trial endpoint; methylmalonic acidemia; propionate oxidation; stable isotope
    DOI:  https://doi.org/10.1002/jimd.12636
  35. Mult Scler. 2023 May 25. 13524585231172947
      Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune disease primarily affecting the optic nerves and spinal cord, which is usually associated with anti-aquaporin-4 antibodies. Here, we present two individuals who were negative for anti-aquaporin-4 antibodies and were initially diagnosed with seronegative NMOSD. Each patient's clinical course and radiographic features raised suspicion for an alternative disease process. Both individuals were found to have pathogenic variants of MT-ND5, encoding subunit 5 of mitochondrial complex I, ultimately leading to a revised diagnosis of a primary mitochondrial disorder. These cases illustrate the importance of biochemical and genetic testing in atypical cases of NMOSD.
    Keywords:  Neuromyelitis optica; genetics; mitochondrial disease; relapsing/remitting
    DOI:  https://doi.org/10.1177/13524585231172947
  36. EMBO J. 2023 May 23. e112542
      Lipid droplets (LDs) form inter-organelle contacts with the endoplasmic reticulum (ER) that promote their biogenesis, while LD contacts with mitochondria enhance β-oxidation of contained fatty acids. Viruses have been shown to take advantage of lipid droplets to promote viral production, but it remains unclear whether they also modulate the interactions between LDs and other organelles. Here, we showed that coronavirus ORF6 protein targets LDs and is localized to the mitochondria-LD and ER-LD contact sites, where it regulates LD biogenesis and lipolysis. At the molecular level, we find that ORF6 inserts into the LD lipid monolayer via its two amphipathic helices. ORF6 further interacts with ER membrane proteins BAP31 and USE1 to mediate ER-LDs contact formation. Additionally, ORF6 interacts with the SAM complex in the mitochondrial outer membrane to link mitochondria to LDs. In doing so, ORF6 promotes cellular lipolysis and LD biogenesis to reprogram host cell lipid flux and facilitate viral production.
    Keywords:  ORF6; endoplasmic reticulum; lipid droplets; mitochondria; organelle interaction
    DOI:  https://doi.org/10.15252/embj.2022112542
  37. Nat Commun. 2023 May 23. 14(1): 2978
      Mapping the subcellular organization of proteins is crucial for understanding their biological functions. Herein, we report a reactive oxygen species induced protein labeling and identification (RinID) method for profiling subcellular proteome in the context of living cells. Our method capitalizes on a genetically encoded photocatalyst, miniSOG, to locally generate singlet oxygen that reacts with proximal proteins. Labeled proteins are conjugated in situ with an exogenously supplied nucleophilic probe, which serves as a functional handle for subsequent affinity enrichment and mass spectrometry-based protein identification. From a panel of nucleophilic compounds, we identify biotin-conjugated aniline and propargyl amine as highly reactive probes. As a demonstration of the spatial specificity and depth of coverage in mammalian cells, we apply RinID in the mitochondrial matrix, capturing 477 mitochondrial proteins with 94% specificity. We further demonstrate the broad applicability of RinID in various subcellular compartments, including the nucleus and the endoplasmic reticulum (ER). The temporal control of RinID enables pulse-chase labeling of ER proteome in HeLa cells, which reveals substantially higher clearance rate for secreted proteins than ER resident proteins.
    DOI:  https://doi.org/10.1038/s41467-023-38565-8
  38. Circ Res. 2023 May 26.
      RATIONALE: Obesity induces cardiomyopathy characterized by hypertrophy and diastolic dysfunction. Whereas mitophagy mediated through an Atg7-dependent mechanism serves as an essential mechanism to maintain mitochondrial quality during the initial development of obesity cardiomyopathy, Rab9-dependent alternative mitophagy takes over the role during the chronic phase. Although it has been postulated that DRP1 (dynamin-related protein 1)-mediated mitochondrial fission and consequent separation of the damaged portions of mitochondria are essential for mitophagy, the involvement of DRP1 in mitophagy remains controversial.OBJECTIVE: We investigated whether endogenous DRP1 is essential in mediating the 2 forms of mitophagy during high-fat diet (HFD)-induced obesity cardiomyopathy and, if so, what the underlying mechanisms are.
    METHODS AND RESULTS: Mice were fed either a normal diet or an HFD (60 kcal %fat). Mitophagy, evaluated with Mito-Keima, was increased after 3 weeks of HFD consumption. The induction of mitophagy by HFD consumption was completely abolished in tamoxifen-inducible cardiac-specific Drp1knockout (Drp1 MCM) mouse hearts, in which both diastolic and systolic dysfunction were exacerbated. The increase in LC3-dependent general autophagy and colocalization between LC3 and mitochondrial proteins was abolished in Drp1 MCM mice. Activation of alternative mitophagy was also completely abolished in Drp1 MCM mice during the chronic phase of HFD consumption. DRP1 was phosphorylated at Ser616, localized at the mitochondria-associated membranes, and associated with Rab9 and Fis1 only during the chronic, but not acute, phase of HFD consumption.
    CONCLUSIONS: DRP1 is an essential factor in mitochondrial quality control during obesity cardiomyopathy that controls multiple forms of mitophagy. Although DRP1 regulates conventional mitophagy through a mitochondria-associated membrane-independent mechanism during the acute phase, it acts as a component of the mitophagy machinery at the mitochondria-associated membranes in alternative mitophagy during the chronic phase of HFD consumption.
    Keywords:  diet, high-fat; mice, knockout; mitochondria; mitophagy; obesity cardiomyopathy
    DOI:  https://doi.org/10.1161/CIRCRESAHA.123.322512
  39. Biomedicines. 2023 May 22. pii: 1500. [Epub ahead of print]11(5):
      Mitochondria are the main site of intracellular synthesis of ATP, which provides energy for various physiological activities of the cell. Cardiomyocytes have a high density of mitochondria and mitochondrial damage is present in a variety of cardiovascular diseases. In this paper, we describe mitochondrial damage in mitochondrial cardiomyopathy, congenital heart disease, coronary heart disease, myocardial ischemia-reperfusion injury, heart failure, and drug-induced cardiotoxicity, in the context of the key roles of mitochondria in cardiac development and homeostasis. Finally, we discuss the main current therapeutic strategies aimed at alleviating mitochondrial impairment-related cardiac dysfunction, including pharmacological strategies, gene therapy, mitochondrial replacement therapy, and mitochondrial transplantation. It is hoped that this will provide new ideas for the treatment of cardiovascular diseases.
    Keywords:  cardiomyocytes; cardiovascular diseases; mitochondria; mitochondrial dysfunction; therapeutic strategies targeting mitochondria
    DOI:  https://doi.org/10.3390/biomedicines11051500
  40. Curr Issues Mol Biol. 2023 May 02. 45(5): 3911-3932
      Based on the knowledge that many diseases are caused by defects in the metabolism of the cells and, in particular, in defects of the mitochondria, mitochondrial medicine starts precisely at this point. This new form of therapy is used in numerous fields of human medicine and has become a central focus within the field of medicine in recent years. With this form of therapy, the disturbed cellular energy metabolism and an out-of-balance antioxidant system of the patient are to be influenced to a greater extent. The most important tool here is mitotropic substances, with the help of which attempts are made to compensate for existing dysfunction. In this article, both mitotropic substances and accompanying studies showing their efficacy are summarized. It appears that the action of many mitotropic substances is based on two important properties. First, on the property of acting antioxidantly, both directly as antioxidants and via activation of downstream enzymes and signaling pathways of the antioxidant system, and second, via enhanced transport of electrons and protons in the mitochondrial respiratory chain.
    Keywords:  antioxidants; dysfunctions; mitochondria; mitotropic; respiratory chain
    DOI:  https://doi.org/10.3390/cimb45050250
  41. J Cell Sci. 2023 May 26. pii: jcs.260819. [Epub ahead of print]
      Mitochondrial dynamics regulate the quality and morphology of mitochondria. Calcium (Ca2+) plays an important role in regulating mitochondrial function. Here, we investigated the effects of optogenetically engineered Ca2+ signaling on mitochondrial dynamics. More specifically, customized illumination conditions could trigger unique Ca2+ oscillation waves to trigger specific signaling pathways. In this study, we found that modulating Ca2+ oscillations by increasing the light frequency, intensity, and exposure time could drive mitochondria toward the fission state, mitochondrial dysfunction, autophagy, and cell death. Moreover, illumination triggered phosphorylation at the Ser616 residue, but not the Ser637 residue of the mitochondrial fission protein, dynamin-related protein 1 (DRP1), via the activation of Ca2+-dependent kinases, CaMKII, ERK, and CDK1. However, optogenetically engineered Ca2+ signaling did not activate calcineurin phosphatase to dephosphorylate DRP1 at Ser637. In addition, light illumination had no effect on the expression levels of the mitochondrial fusion proteins, mitofusin (MFN)-1 and MFN2.Taken together, this study provides an effective and innovative approach to altering Ca2+ signaling for controlling mitochondrial fission with a more precise resolution than pharmacological approaches in the temporal dimension.
    Keywords:  Calcium oscillation; DRP1; Mitochondrial fission; Optogenetics
    DOI:  https://doi.org/10.1242/jcs.260819
  42. Int J Mol Sci. 2023 May 19. pii: 9027. [Epub ahead of print]24(10):
      The loss of the E3 ligase Parkin, in a familial form of Parkinson's disease, is thought to cause the failure of both the polyubiquitination of abnormal mitochondria and the consequent induction of mitophagy, resulting in abnormal mitochondrial accumulation. However, this has not been confirmed in patient autopsy cases or animal models. More recently, the function of Parkin as a redox molecule that directly scavenges hydrogen peroxide has attracted much attention. To determine the role of Parkin as a redox molecule in the mitochondria, we overexpressed various combinations of Parkin, along with its substrates FAF1, PINK1, and ubiquitin in cell culture systems. Here, we observed that the E3 Parkin monomer was surprisingly not recruited to abnormal mitochondria but self-aggregated with or without self-ubiquitination into the inner and outer membranes, becoming insoluble. Parkin overexpression alone generated aggregates without self-ubiquitination, but it activated autophagy. These results suggest that for damaged mitochondria, the polyubiquitination of Parkin substrates on the mitochondria is not indispensable for mitophagy.
    Keywords:  Parkin; Parkinson’s disease; neurodegeneration; ubiquitin
    DOI:  https://doi.org/10.3390/ijms24109027
  43. Genes (Basel). 2023 May 05. pii: 1043. [Epub ahead of print]14(5):
      Mitochondrial diseases are a group of heterogeneous disorders caused by dysfunctional mitochondria. Interestingly, a large proportion of mitochondrial diseases are caused by defects in genes associated with tRNA metabolism. We recently discovered that partial loss-of-function mutations in tRNA Nucleotidyl Transferase 1 (TRNT1), the nuclear gene encoding the CCA-adding enzyme essential for modifying both nuclear and mitochondrial tRNAs, causes a multisystemic and clinically heterogenous disease termed SIFD (sideroblastic anemia with B-cell immunodeficiency, periodic fevers, and developmental delay; SIFD). However, it is not clear how mutations in a general and essential protein like TRNT1 cause disease with such clinically broad but unique symptomatology and tissue involvement. Using biochemical, cell, and mass spectrometry approaches, we demonstrate that TRNT1 deficiency is associated with sensitivity to oxidative stress, which is due to exacerbated, angiogenin-dependent cleavage of tRNAs. Furthermore, reduced levels of TRNT1 lead to phosphorylation of Eukaryotic Translation Initiation Factor 2 Subunit Alpha (eIF2α), increased reactive oxygen species (ROS) production, and changes in the abundance of distinct proteins. Our data suggest that the observed variable SIFD phenotypes are likely due to dysregulation of tRNA maturation and abundance, which in turn negatively affects the translation of distinct proteins.
    Keywords:  CCA adding; SIFD; SILAC; TRNT1; oxidative stress; tiRNA
    DOI:  https://doi.org/10.3390/genes14051043
  44. Nat Commun. 2023 May 24. 14(1): 2996
      Neuronal function is highly energy demanding and thus requires efficient and constant metabolite delivery by glia. Drosophila glia are highly glycolytic and provide lactate to fuel neuronal metabolism. Flies are able to survive for several weeks in the absence of glial glycolysis. Here, we study how Drosophila glial cells maintain sufficient nutrient supply to neurons under conditions of impaired glycolysis. We show that glycolytically impaired glia rely on mitochondrial fatty acid breakdown and ketone body production to nourish neurons, suggesting that ketone bodies serve as an alternate neuronal fuel to prevent neurodegeneration. We show that in times of long-term starvation, glial degradation of absorbed fatty acids is essential to ensure survival of the fly. Further, we show that Drosophila glial cells act as a metabolic sensor and can induce mobilization of peripheral lipid stores to preserve brain metabolic homeostasis. Our study gives evidence of the importance of glial fatty acid degradation for brain function, and survival, under adverse conditions in Drosophila.
    DOI:  https://doi.org/10.1038/s41467-023-38813-x
  45. Ann Clin Transl Neurol. 2023 May 23.
      OBJECTIVE: To explore the clinical characteristics of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) caused by mitochondrial DNA-encoded complex I subunit (mt-ND) variants.METHODS: In this retrospective study, the clinical, myopathological and brain MRI features of patients with MELAS caused by mt-ND variants (MELAS-mtND) were collected and compared with those of MELAS patients carrying the m.3243A > G variant (MELAS-A3243G).
    RESULT: A total of 18 MELAS-mtND patients (female: 7; median age: 24.5 years) represented 15.9% (n = 113) of all patients with MELAS caused by mtDNA variants in our neuromuscular center from January 2012 to June 2022. In this MELAS-mtND cohort, the two most common variants were m.10191 T > C (4/18, 22.2%) and m.13513 G > A (3/18, 16.7%). The most frequent symptoms were seizures (14/18, 77.8%) and muscle weakness (11/18, 61.1%). Compared with 87 MELAS-A3243G patients, MELAS-mtND patients were significantly more likely to have a variant that was absent in blood cells (40% vs. 1.4%). Furthermore, MELAS-mtND patients had a significantly lower MDC score (7.8 ± 2.7 vs. 9.8 ± 1.9); less hearing loss (27.8% vs. 54.0%), diabetes (11.1% vs. 37.9%), and migraine (33.3% vs. 62.1%); less short stature (males ≤ 165 cm; females ≤ 155 cm; 23.1% vs. 60.8%) and higher body mass index (20.4 ± 2.5 vs. 17.8 ± 2.7). MELAS-mtND patients had significantly more normal muscle pathology (31.3% vs. 4.1%) and fewer RRFs/RBFs (62.5% vs. 91.9%), COX-deficient fibers/blue fibers (25.0% vs. 85.1%) and SSVs (50.0% vs. 81.1%). Moreover, brain MRI evaluated at the first stroke-like episode showed significantly more small cortical lesions in MELAS-mtND patients (66.7% vs. 12.2%).
    INTERPRETATION: Our results suggested that MELAS-mtND patients have distinct clinical, myopathological and brain MRI features compared with MELAS-A3243G patients.
    DOI:  https://doi.org/10.1002/acn3.51787
  46. Basic Res Cardiol. 2023 May 25. 118(1): 21
      Iron overload associated cardiac dysfunction remains a significant clinical challenge whose underlying mechanism(s) have yet to be defined. We aim to evaluate the involvement of the mitochondrial Ca2+ uniporter (MCU) in cardiac dysfunction and determine its role in the occurrence of ferroptosis. Iron overload was established in control (MCUfl/fl) and conditional MCU knockout (MCUfl/fl-MCM) mice. LV function was reduced by chronic iron loading in MCUfl/fl mice, but not in MCUfl/fl-MCM mice. The level of mitochondrial iron and reactive oxygen species were increased and mitochondrial membrane potential and spare respiratory capacity (SRC) were reduced in MCUfl/fl cardiomyocytes, but not in MCUfl/fl-MCM cardiomyocytes. After iron loading, lipid oxidation levels were increased in MCUfl/fl, but not in MCUfl/fl-MCM hearts. Ferrostatin-1, a selective ferroptosis inhibitor, reduced lipid peroxidation and maintained LV function in vivo after chronic iron treatment in MCUfl/fl hearts. Isolated cardiomyocytes from MCUfl/fl mice demonstrated ferroptosis after acute iron treatment. Moreover, Ca2+ transient amplitude and cell contractility were both significantly reduced in isolated cardiomyocytes from chronically Fe treated MCUfl/fl hearts. However, ferroptosis was not induced in cardiomyocytes from MCUfl/fl-MCM hearts nor was there a reduction in Ca2+ transient amplitude or cardiomyocyte contractility. We conclude that mitochondrial iron uptake is dependent on MCU, which plays an essential role in causing mitochondrial dysfunction and ferroptosis under iron overload conditions in the heart. Cardiac-specific deficiency of MCU prevents the development of ferroptosis and iron overload-induced cardiac dysfunction.
    Keywords:  Cardiac dysfunction; Ferroptosis; Iron overload cardiac dysfunction; Mitochondrial calcium uniporter; Myocardial energetics; Reactive oxygen species
    DOI:  https://doi.org/10.1007/s00395-023-00990-7
  47. Biophys Physicobiol. 2023 ;20(1): e200004
      Mitochondria play an important role in energy conversion as well as in intracellular calcium (Ca2+) storage. Ca2+ uptake from the cytosol to the mitochondria is mediated by the calcium uniporter, which functions as a Ca2+ ion channel. However, the molecular composition of this uniporter has remained unclear until recently. The Ca2+ ion channel consists of seven subunits. The yeast reconstitution technique revealed that the mitochondrial calcium uniporter (MCU) and essential MCU regulatory element (EMRE) are the core subunits of the complex. Furthermore, detailed structure-function analyses of the core subunits (MCU and EMRE) were performed. In this review, the regulatory mechanism of mitochondrial Ca2+ uptake is discussed.
    Keywords:  calcium; ion channel; mitochondria; yeast
    DOI:  https://doi.org/10.2142/biophysico.bppb-v20.0004
  48. Antioxid Redox Signal. 2023 May 22.
      SIGNIFICANCE: The architecture of the mitochondrial network and cristae critically impact cell differentiation and identity. Cells undergoing metabolic reprogramming to aerobic glycolysis (Warburg effect), such as immune cells, stem cells, and cancer cells, go through controlled modifications in mitochondrial architecture, which is critical for achieving the resulting cellular phenotype.RECENT ADVANCES: Recent studies in immunometabolism have shown that the manipulation of mitochondrial network dynamics and cristae shape directly affects T cell phenotype and macrophage polarization through altering energy metabolism. Similar manipulations also alter the specific metabolic phenotypes that accompany somatic reprogramming, stem cell differentiation, and cancer cells. The modulation of OXPHOS activity, accompanied by changes in metabolite signaling, ROS generation, and ATP levels is the shared underlying mechanism.
    CRITICAL ISSUES: The plasticity of mitochondrial architecture is particularly vital for metabolic reprogramming. Consequently, failure to adapt the appropriate mitochondrial morphology often compromises the differentiation and identity of the cell. Immune, stem, and tumor cells exhibit striking similarities in their coordination of mitochondrial morphology with metabolic pathways. However, although many general unifying principles can be observed, their validity is not absolute, and the mechanistic links thus need to be further explored.
    FUTURE DIRECTIONS: Better knowledge of the molecular mechanisms involved and their relationships to both mitochondrial network and cristae morphology will not only further deepen our understanding of energy metabolism but may also contribute to improved therapeutic manipulation of cell viability, differentiation, proliferation, and identity in many different cell types.
    DOI:  https://doi.org/10.1089/ars.2023.0268
  49. Nat Commun. 2023 May 24. 14(1): 2847
      Phospholipase D3 (PLD3) polymorphisms are linked to late-onset Alzheimer's disease (LOAD). Being a lysosomal 5'-3' exonuclease, its neuronal substrates remained unknown as well as how a defective lysosomal nucleotide catabolism connects to AD-proteinopathy. We identified mitochondrial DNA (mtDNA) as a major physiological substrate and show its manifest build-up in lysosomes of PLD3-defective cells. mtDNA accretion creates a degradative (proteolytic) bottleneck that presents at the ultrastructural level as a marked abundance of multilamellar bodies, often containing mitochondrial remnants, which correlates with increased PINK1-dependent mitophagy. Lysosomal leakage of mtDNA to the cytosol activates cGAS-STING signaling that upregulates autophagy and induces amyloid precursor C-terminal fragment (APP-CTF) and cholesterol accumulation. STING inhibition largely normalizes APP-CTF levels, whereas an APP knockout in PLD3-deficient backgrounds lowers STING activation and normalizes cholesterol biosynthesis. Collectively, we demonstrate molecular cross-talks through feedforward loops between lysosomal nucleotide turnover, cGAS-STING and APP metabolism that, when dysregulated, result in neuronal endolysosomal demise as observed in LOAD.
    DOI:  https://doi.org/10.1038/s41467-023-38501-w
  50. Int J Mol Sci. 2023 May 18. pii: 8950. [Epub ahead of print]24(10):
      In human spermatozoa and oocytes (and their surrounding granulosa cells), mitochondria carry out important functions relating to human fertility and infertility. Sperm mitochondria are not transmitted to the future embryo, but are closely related to the generation of energy needed for sperm movement, capacitation, and acrosome reactions, as well as for sperm-oocyte fusion. On the other hand, oocyte mitochondria produce energy required for oocyte meiotic division and their abnormalities can thus cause oocyte and embryo aneuploidy. In addition, they play a role in oocyte calcium metabolism and in essential epigenetic events during the oocyte-to-embryo transition. They are transmitted to the future embryos and may thus cause hereditary diseases in the offspring. Due to the long life span of the female germ cells, the accumulation of mitochondrial DNA abnormalities often causes ovarian aging. Mitochondrial substitution therapy is the only way of dealing with these issues nowadays. New therapies based on mitochondrial DNA editing are under investigation.
    Keywords:  fertility; infertility; mitochondria; mitochondrial therapy; oocyte; sperm
    DOI:  https://doi.org/10.3390/ijms24108950
  51. Antioxidants (Basel). 2023 May 12. pii: 1087. [Epub ahead of print]12(5):
      Mitochondrial DNA (mtDNA) is particularly vulnerable to somatic mutagenesis. Potential mechanisms include DNA polymerase γ (POLG) errors and the effects of mutagens, such as reactive oxygen species. Here, we studied the effects of transient hydrogen peroxide (H2O2 pulse) on mtDNA integrity in cultured HEK 293 cells, applying Southern blotting, ultra-deep short-read and long-read sequencing. In wild-type cells, 30 min after the H2O2 pulse, linear mtDNA fragments appear, representing double-strand breaks (DSB) with ends characterized by short GC stretches. Intact supercoiled mtDNA species reappear within 2-6 h after treatment and are almost completely recovered after 24 h. BrdU incorporation is lower in H2O2-treated cells compared to non-treated cells, suggesting that fast recovery is not associated with mtDNA replication, but is driven by rapid repair of single-strand breaks (SSBs) and degradation of DSB-generated linear fragments. Genetic inactivation of mtDNA degradation in exonuclease deficient POLG p.D274A mutant cells results in the persistence of linear mtDNA fragments with no impact on the repair of SSBs. In conclusion, our data highlight the interplay between the rapid processes of SSB repair and DSB degradation and the much slower mtDNA re-synthesis after oxidative damage, which has important implications for mtDNA quality control and the potential generation of somatic mtDNA deletions.
    Keywords:  mitochondrial DNA; mtDNA degradation; mtDNA double-strand breaks mtDNA single-strand breaks; oxidative damage
    DOI:  https://doi.org/10.3390/antiox12051087
  52. bioRxiv. 2023 May 11. pii: 2023.05.11.540429. [Epub ahead of print]
      Cancer cells reprogram their metabolism to support cell growth and proliferation in harsh environments. While many studies have documented the importance of mitochondrial oxidative phosphorylation (OXPHOS) in tumor growth, some cancer cells experience conditions of reduced OXPHOS in vivo and induce alternative metabolic pathways to compensate. To assess how human cells respond to mitochondrial dysfunction, we performed metabolomics in fibroblasts and plasma from patients with inborn errors of mitochondrial metabolism, and in cancer cells subjected to inhibition of the electron transport chain (ETC). All these analyses revealed extensive perturbations in purine-related metabolites; in non-small cell lung cancer (NSCLC) cells, ETC blockade led to purine metabolite accumulation arising from a reduced cytosolic NAD + /NADH ratio (NADH reductive stress). Stable isotope tracing demonstrated that ETC deficiency suppressed de novo purine nucleotide synthesis while enhancing purine salvage. Analysis of NSCLC patients infused with [U- 13 C]glucose revealed that tumors with markers of low oxidative mitochondrial metabolism exhibited high expression of the purine salvage enzyme HPRT1 and abundant levels of the HPRT1 product inosine monophosphate (IMP). ETC blockade also induced production of ribose-5' phosphate (R5P) by the pentose phosphate pathway (PPP) and import of purine nucleobases. Blocking either HPRT1 or nucleoside transporters sensitized cancer cells to ETC inhibition, and overexpressing nucleoside transporters was sufficient to drive growth of NSCLC xenografts. Collectively, this study mechanistically delineates how cells compensate for suppressed purine metabolism in response to ETC blockade, and uncovers a new metabolic vulnerability in tumors experiencing NADH excess.
    DOI:  https://doi.org/10.1101/2023.05.11.540429
  53. Genes (Basel). 2023 Apr 22. pii: 954. [Epub ahead of print]14(5):
      Metabolic myopathies are rare inherited disorders that deserve more attention from neurologists and pediatricians. Pompe disease and McArdle disease represent some of the most common diseases in clinical practice; however, other less common diseases are now better-known. In general the pathophysiology of metabolic myopathies needs to be better understood. Thanks to the advent of next-generation sequencing (NGS), genetic testing has replaced more invasive investigations and sophisticated enzymatic assays to reach a final diagnosis in many cases. The current diagnostic algorithms for metabolic myopathies have integrated this paradigm shift and restrict invasive investigations for complicated cases. Moreover, NGS contributes to the discovery of novel genes and proteins, providing new insights into muscle metabolism and pathophysiology. More importantly, a growing number of these conditions are amenable to therapeutic approaches such as diets of different kinds, exercise training protocols, and enzyme replacement therapy or gene therapy. Prevention and management-notably of rhabdomyolysis-are key to avoiding serious and potentially life-threatening complications and improving patients' quality of life. Although not devoid of limitations, the newborn screening programs that are currently mushrooming across the globe show that early intervention in metabolic myopathies is a key factor for better therapeutic efficacy and long-term prognosis. As a whole NGS has largely increased the diagnostic yield of metabolic myopathies, but more invasive but classical investigations are still critical when the genetic diagnosis is unclear or when it comes to optimizing the follow-up and care of these muscular disorders.
    Keywords:  Cori–Forbes disease; McArdle disease; Pompe disease; TK2 myopathy; Tarui disease; glycogen storage disorders; lipid storage diseases; metabolic myopathies; mitochondrial myopathies; muscle glycogenoses; muscle metabolism; polyglucosan body myopathies
    DOI:  https://doi.org/10.3390/genes14050954
  54. Acta Pharmacol Sin. 2023 May 22.
      Renal fibrosis relies on multiple proteins and cofactors in its gradual development. Copper is a cofactor of many enzymes involved in renal microenvironment homeostasis. We previously reported that intracellular copper imbalance occurred during renal fibrosis development and was correlated with fibrosis intensity. In this study, we investigated the molecular mechanisms of how copper affected renal fibrosis development. Unilateral ureteral obstruction (UUO) mice were used for in vivo study; rat renal tubular epithelial cells (NRK-52E) treated with TGF-β1 were adapted as an in vitro fibrotic model. We revealed that the accumulation of copper in mitochondria, rather than cytosol, was responsible for mitochondrial dysfunction, cell apoptosis and renal fibrosis in both in vivo and in vitro fibrotic models. Furthermore, we showed that mitochondrial copper overload directly disrupted the activity of respiratory chain complex IV (cytochrome c oxidase), but not complex I, II and III, which hampered respiratory chain and disrupted mitochondrial functions, eventually leading to fibrosis development. Meanwhile, we showed that COX17, the copper chaperone protein, was significantly upregulated in the mitochondria of fibrotic kidneys and NRK-52E cells. Knockdown of COX17 aggravated mitochondrial copper accumulation, inhibited complex IV activity, augmented mitochondrial dysfunction and led to cell apoptosis and renal fibrosis, whereas overexpression of COX17 could discharge copper from mitochondria and protect mitochondrial function, alleviating renal fibrosis. In conclusion, copper accumulation in mitochondria blocks complex IV activity and induces mitochondrial dysfunction. COX17 plays a pivotal role in maintaining mitochondrial copper homeostasis, restoring complex IV activity, and ameliorating renal fibrosis.
    Keywords:  COX17; NRK-52E cells; copper; cytochrome c oxidase; mitochondria; renal fibrosis
    DOI:  https://doi.org/10.1038/s41401-023-01098-3
  55. J Intern Med. 2023 May 21.
      Molecular diagnostics is a cornerstone of modern precision medicine, broadly understood as tailoring an individual's treatment, follow-up, and care based on molecular data. In rare diseases (RDs), molecular diagnoses reveal valuable information about the cause of symptoms, disease progression, familial risk, and in certain cases, unlock access to targeted therapies. Due to decreasing DNA sequencing costs, genome sequencing (GS) is emerging as the primary method for precision diagnostics in RDs. Several ongoing European initiatives for precision medicine have chosen GS as their method of choice. Recent research supports the role for GS as first-line genetic investigation in individuals with suspected RD, due to its improved diagnostic yield compared to other methods. Moreover, GS can detect a broad range of genetic aberrations including those in non-coding regions, producing comprehensive data that can be periodically reanalyzed for years to come when further evidence emerges. Indeed, targeted drug development and repurposing of medicines can be accelerated as more individuals with RDs receive a molecular diagnosis. Multidisciplinary teams in which clinical specialists collaborate with geneticists, genomics education of professionals and the public, and dialogue with patient advocacy groups are essential elements for the integration of precision medicine into clinical practice worldwide. It is also paramount that large research projects share genetic data and leverage novel technologies to fully diagnose individuals with RDs. In conclusion, GS increases diagnostic yields and is a crucial step towards precision medicine for RDs. Its clinical implementation will enable better patient management, unlock targeted therapies, and guide the development of innovative treatments. This article is protected by copyright. All rights reserved.
    Keywords:  data sharing; gene therapy; genome sequencing; molecular diagnosis; precision medicine; rare diseases
    DOI:  https://doi.org/10.1111/joim.13655
  56. Muscle Nerve. 2023 May 25.
      Metabolic myopathies are a set of rare inborn errors of metabolism leading to disruption in energy production. Relevant to skeletal muscle, glycogen storage disease and fatty acid oxidation defects can lead to exercise intolerance, rhabdomyolysis, and weakness in children and adults, distinct from the severe forms that involve multiple-organ systems. These nonspecific, dynamic symptoms along with conditions that mimic metabolic myopathies can make diagnosis challenging. Clinicians can shorten the time to diagnosis by recognizing the typical clinical phenotypes and performing next generation sequencing. With improved access and affordability of molecular testing, clinicians need to be well-versed in resolving variants of uncertain significance relevant to metabolic myopathies. Once identified, patients can improve quality of life, safely engage in exercise, and reduce episodes of rhabdomyolysis by modifying diet and lifestyle habits.
    Keywords:  fatty acid oxidation defects; glycogen storage disease; lipid storage myopathy; metabolic myopathy; muscle metabolism; rhabdomyolysis
    DOI:  https://doi.org/10.1002/mus.27840
  57. bioRxiv. 2023 May 09. pii: 2023.05.08.539908. [Epub ahead of print]
      Crosstalk between metabolism and stress-responsive signaling is essential to maintaining cellular homeostasis. One way this crosstalk is achieved is through the covalent modification of proteins by endogenous, reactive metabolites that regulate the activity of key stress-responsive transcription factors such as NRF2. Several metabolites including methylglyoxal, glyceraldehyde 3-phosphate, fumarate, and itaconate covalently modify sensor cysteines of the NRF2 regulatory protein KEAP1, resulting in stabilization of NRF2 and activation of its cytoprotective transcriptional program. Here, we employed a shRNA-based screen targeting the enzymes of central carbon metabolism to identify additional regulatory nodes bridging metabolic pathways to NRF2 activation. We found that succinic anhydride, increased by genetic depletion of the TCA cycle enzyme succinyl-CoA synthetase or by direct administration, results in N-succinylation of lysine 131 of KEAP1 to activate NRF2 transcriptional signaling. This study identifies KEAP1 as capable of sensing reactive metabolites not only by several cysteine residues but also by a conserved lysine residue, indicating its potential to sense an expanded repertoire of reactive metabolic messengers.
    DOI:  https://doi.org/10.1101/2023.05.08.539908
  58. Antioxidants (Basel). 2023 May 08. pii: 1065. [Epub ahead of print]12(5):
      Together with the global rise in obesity and metabolic syndrome, the prevalence of individuals who suffer from nonalcoholic fatty liver disease (NAFLD) has risen dramatically. NAFLD is currently the most common chronic liver disease and includes a continuum of liver disorders from initial fat accumulation to nonalcoholic steatohepatitis (NASH), considered the more severe forms, which can evolve in, cirrhosis, and hepatocellular carcinoma. Common features of NAFLD includes altered lipid metabolism mainly linked to mitochondrial dysfunction, which, as a vicious cycle, aggravates oxidative stress and promotes inflammation and, as a consequence, the progressive death of hepatocytes and the severe form of NAFLD. A ketogenic diet (KD), i.e., a diet very low in carbohydrates (<30 g/die) that induces "physiological ketosis", has been demonstrated to alleviate oxidative stress and restore mitochondrial function. Based on this, the aim of the present review is to analyze the body of evidence regarding the potential therapeutic role of KD in NAFLD, focusing on the interplay between mitochondria and the liver, the effects of ketosis on oxidative stress pathways, and the impact of KD on liver and mitochondrial function.
    Keywords:  ketogenic diet; ketone bodies; liver; mitochondria; nonalcoholic fatty liver disease (NAFLD); oxidative stress
    DOI:  https://doi.org/10.3390/antiox12051065
  59. J Invest Dermatol. 2023 May 19. pii: S0022-202X(23)02075-4. [Epub ahead of print]
      An effective healing response is critical to healthy aging. In particular, energy homeostasis has become increasingly recognized as a factor in effective skin regeneration. Adenine nucleotide translocase 2 (ANT2) is a mediator of ATP import into mitochondria for energy homeostasis. Although energy homeostasis and mitochondrial integrity are critical for wound healing, the role played by ANT2 in the repair process had not been elucidated to date. In our study, we found that ANT2 expression decreased in aged skin and cellular senescence. Interestingly, overexpression of ANT2 in aged mouse skin accelerated the healing of full-thickness cutaneous wounds. In addition, upregulation of ANT2 in replicative senescent human diploid dermal fibroblasts (HDFs) induced their proliferation and migration, which are critical processes in wound healing. Regarding energy homeostasis, ANT2 overexpression increased the ATP production rate by activating glycolysis and induced mitophagy. Notably, ANT2-mediated upregulation of HSPA6 in aged HDFs downregulated proinflammatory genes that mediate cellular senescence and mitochondrial damage. This study demonstrates a previously uncharacterized physiological role of ANT2 in skin wound healing via regulating cell proliferation, energy homeostasis and inflammation. Thus, our study links energy metabolism to skin homeostasis and reports to our knowledge previously unreported genetic factor that enhances wound healing in an aging model.
    Keywords:  ANT2; HSP70; SASP; aged skin; cellular senescence; energy homeostasis; glycolysis; mitophagy; wound healing
    DOI:  https://doi.org/10.1016/j.jid.2023.05.002
  60. Antioxidants (Basel). 2023 May 10. pii: 1072. [Epub ahead of print]12(5):
      Theragnostics is a promising approach that integrates diagnostics and therapeutics into a single personalized strategy. To conduct effective theragnostic studies, it is essential to create an in vitro environment that accurately reflects the in vivo conditions. In this review, we discuss the importance of redox homeostasis and mitochondrial function in the context of personalized theragnostic approaches. Cells have several ways to respond to metabolic stress, including changes in protein localization, density, and degradation, which can promote cell survival. However, disruption of redox homeostasis can lead to oxidative stress and cellular damage, which are implicated in various diseases. Models of oxidative stress and mitochondrial dysfunction should be developed in metabolically conditioned cells to explore the underlying mechanisms of diseases and develop new therapies. By choosing an appropriate cellular model, adjusting cell culture conditions and validating the cellular model, it is possible to identify the most promising therapeutic options and tailor treatments to individual patients. Overall, we highlight the importance of precise and individualized approaches in theragnostics and the need to develop accurate in vitro models that reflect the in vivo conditions.
    Keywords:  media composition; metabolic priming; mitochondria; oxygen levels; redox homeostasis; theragnostics
    DOI:  https://doi.org/10.3390/antiox12051072
  61. Nat Commun. 2023 May 24. 14(1): 2994
      Autophagy maintains cellular homeostasis during low energy states. According to the current understanding, glucose-depleted cells induce autophagy through AMPK, the primary energy-sensing kinase, to acquire energy for survival. However, contrary to the prevailing concept, our study demonstrates that AMPK inhibits ULK1, the kinase responsible for autophagy initiation, thereby suppressing autophagy. We found that glucose starvation suppresses amino acid starvation-induced stimulation of ULK1-Atg14-Vps34 signaling via AMPK activation. During an energy crisis caused by mitochondrial dysfunction, the LKB1-AMPK axis inhibits ULK1 activation and autophagy induction, even under amino acid starvation. Despite its inhibitory effect, AMPK protects the ULK1-associated autophagy machinery from caspase-mediated degradation during energy deficiency, preserving the cellular ability to initiate autophagy and restore homeostasis once the stress subsides. Our findings reveal that dual functions of AMPK, restraining abrupt induction of autophagy upon energy shortage while preserving essential autophagy components, are crucial to maintain cellular homeostasis and survival during energy stress.
    DOI:  https://doi.org/10.1038/s41467-023-38401-z
  62. Nat Rev Cardiol. 2023 May 26.
      Cardiac metabolism is vital for heart function. Given that cardiac contraction requires a continuous supply of ATP in large quantities, the role of fuel metabolism in the heart has been mostly considered from the perspective of energy production. However, the consequence of metabolic remodelling in the failing heart is not limited to a compromised energy supply. The rewired metabolic network generates metabolites that can directly regulate signalling cascades, protein function, gene transcription and epigenetic modifications, thereby affecting the overall stress response of the heart. In addition, metabolic changes in both cardiomyocytes and non-cardiomyocytes contribute to the development of cardiac pathologies. In this Review, we first summarize how energy metabolism is altered in cardiac hypertrophy and heart failure of different aetiologies, followed by a discussion of emerging concepts in cardiac metabolic remodelling, that is, the non-energy-generating function of metabolism. We highlight challenges and open questions in these areas and finish with a brief perspective on how mechanistic research can be translated into therapies for heart failure.
    DOI:  https://doi.org/10.1038/s41569-023-00887-x
  63. iScience. 2023 Jun 16. 26(6): 106777
      The retina is a notable tissue with high metabolic needs which relies on specialized vascular networks to protect the neural retina while maintaining constant supplies of oxygen, nutrients, and dietary essential fatty acids. Here we analyzed the lipidome of the mouse retina under healthy and pathological angiogenesis using the oxygen-induced retinopathy model. By matching lipid profiles to changes in mRNA transcriptome, we identified a lipid signature showing that pathological angiogenesis leads to intense lipid remodeling favoring pathways for neutral lipid synthesis, cholesterol import/export, and lipid droplet formation. Noteworthy, it also shows profound changes in pathways for long-chain fatty acid production, vital for retina homeostasis. The net result is accumulation of large quantities of mead acid, a marker of essential fatty acid deficiency, and a potential marker for retinopathy severity. Thus, our lipid signature might contribute to better understand diseases of the retina that lead to vision impairment or blindness.
    Keywords:  Metabolomics; Molecular mechanism of behavior
    DOI:  https://doi.org/10.1016/j.isci.2023.106777
  64. Biochim Biophys Acta Mol Basis Dis. 2023 May 23. pii: S0925-4439(23)00126-6. [Epub ahead of print] 166760
      The alternative oxidase, AOX, provides a by-pass of the cytochrome segment of the mitochondrial respiratory chain when the chain is unavailable. AOX is absent from mammals, but AOX from Ciona intestinalis is benign when expressed in mice. Although non-protonmotive, so does not contribute directly to ATP production, it has been shown to modify and in some cases rescue phenotypes of respiratory-chain disease models. Here we studied the effect of C. intestinalis AOX on mice engineered to express a disease-equivalent mutant of Uqcrh, encoding the hinge subunit of mitochondrial respiratory complex III, which results in a complex metabolic phenotype beginning at 4-5 weeks, rapidly progressing to lethality within a further 6-7 weeks. AOX expression delayed the onset of this phenotype by several weeks, but provided no long-term benefit. We discuss the significance of this finding in light of the known and hypothesized effects of AOX on metabolism, redox homeostasis, oxidative stress and cell signaling. Although not a panacea, the ability of AOX to mitigate disease onset and progression means it could be useful in treatment.
    Keywords:  Alternative oxidase; Complex III; Growth impairment; Hyperglycemia; Insulin sensitivity; Mitochondrial disease
    DOI:  https://doi.org/10.1016/j.bbadis.2023.166760