bims-mitdis Biomed News
on Mitochondrial Disorders
Issue of 2021‒03‒21
forty-eight papers selected by
Catalina Vasilescu
University of Helsinki

  1. Mol Genet Metab Rep. 2021 Jun;27 100733
      Objective: To describe the clinical and molecular features of a group of Argentinian pediatric patients with mitochondrial DNA (mtDNA) disorders, and to evaluate the results of the implementation of a classical approach for the molecular diagnosis of mitochondrial diseases.Methods: Clinical data from 27 patients with confirmed mtDNA pathogenic variants were obtained from a database of 89 patients with suspected mitochondrial disease, registered from 2014 to 2020. Clinical data, biochemical analysis, neuroimaging findings, muscle biopsy and molecular studies were analyzed.
    Results: Patients were 18 females and 9 males, with ages at onset ranging from 1 week to 14 years (median = 4 years). The clinical phenotypes were: mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome (n = 11), Leigh syndrome (n = 5), Kearns-Sayre syndrome (n = 3), Chronic Progressive External Ophthalmoplegia (n = 2), Leber hereditary optic neuropathy (n = 2), myoclonic epilepsy associated with ragged-red fibers (n = 1) and reversible infantile myopathy with cytochrome-C oxidase deficiency (n = 3). Most of the patients harbored pathogenic single nucleotide variants, mainly involving mt-tRNA genes, such as MT-TL1, MT-TE and MT-TK. Other point variants were found in complex I subunits, like MT-ND6, MT-ND4, MT-ND5; or in MT-ATP6. The m.13513G > A variant in MT-ND5 and the m.9185 T > C variant in MT-ATP6 were apparently de novo. The rest of the patients presented large scale-rearrangements, either the "common" deletion or a larger deletion.
    Conclusions: This study highlights the clinical and genetic heterogeneity of pediatric mtDNA disorders. All the cases presented with classical phenotypes, being MELAS the most frequent. Applying classical molecular methods, it was possible to achieve a genetic diagnosis in 30% of the cases, suggesting that this is an effective first approach, especially for those centers from low-middle income countries, leaving NGS studies for those patients with inconclusive results.
    Keywords:  Leigh syndrome; MELAS; Mitochondrial DNA; Mitochondrial diseases; Molecular diagnosis; Pediatrics
  2. Hum Mutat. 2021 Mar 14.
      Isolated biochemical deficiency of mitochondrial complex I is the most frequent signature amongst mitochondrial diseases and is associated with a wide variety of clinical symptoms. Leigh syndrome represents the most frequent neuroradiological finding in patients with complex I defect and >80 monogenic causes have been involved in the disease. In this report, we describe 7 patients from four unrelated families harbouring novel NDUFA12 variants, 6 of them presenting with Leigh syndrome. Molecular genetic characterization was performed using next generation sequencing combined with the Sanger method. Biochemical and protein studies were achieved by enzymatic activities, blue native gel electrophoresis and Western blotting. All patients displayed novel homozygous mutations in the NDUFA12 gene leading to the virtual absence of the corresponding protein. Surprisingly, despite in none of the analyzed patients NDUFA12 protein was detected, they present a different onset and clinical course of the disease. Our report expands the array of genetic alterations in NDUFA12 and underlines phenotype variability associated with NDUFA12 defect. This article is protected by copyright. All rights reserved.
    Keywords:  Leigh syndrome; NADH ubiquinone oxidoreductase; NDUFA12; mitochondrial disease
  3. Redox Biol. 2021 Mar 02. pii: S2213-2317(21)00071-9. [Epub ahead of print]41 101923
      Mutations in nuclear genes encoding for mitochondrial proteins very long-chain acyl-CoA dehydrogenase (VLCAD) and trifunctional protein (TFP) cause rare autosomal recessive disorders. Studies in fibroblasts derived from patients with mutations in VLCAD and TFP exhibit mitochondrial defects. To gain insights on pathological changes that account for the mitochondrial deficits we performed quantitative proteomic, biochemical, and morphometric analyses in fibroblasts derived from subjects with three different VLCAD and three different TFP mutations. Proteomic data that was corroborated by antibody-based detection, indicated reduced levels of VLCAD and TFP protein in cells with VLCAD and TFP mutations respectively, which in part accounted for the diminished fatty acid oxidation capacity. Decreased mitochondrial respiratory capacity in cells with VLCAD and TFP mutations was quantified after glucose removal and cells with TFP mutations had lower levels of glycogen. Despite these energetic deficiencies, the cells with VLCAD and TFP mutations did not exhibit changes in mitochondria morphology, distribution, fusion and fission, quantified by either confocal or transmission electron microscopy and corroborated by proteomic and antibody-based protein analysis. Fibroblasts with VLCAD and to a lesser extend cells with TFP mutations had increased levels of mitochondrial respiratory chain proteins and proteins that facilitate the assembly of respiratory complexes. With the exception of reduced levels of catalase and glutathione S-transferase theta-1 in cells with TFP mutations, the levels of 45 proteins across all major intracellular antioxidant networks were similar between cells with VLCAD and TFP mutations and non-disease controls. Collectively the data indicate that despite the metabolic deficits, cells with VLCAD and TFP mutations maintain their proteomic integrity to preserve cellular and mitochondria architecture, support energy production and protect against oxidative stress.
    Keywords:  Beta-oxidation; Long-chain fatty acids (LCFA); Mitochondria; Proteomics; Trifunctional protein (TFP); Very long-chain acyl-CoA dehydrogenase (VLCAD)
  4. Cell Rep. 2021 Mar 16. pii: S2211-1247(21)00183-2. [Epub ahead of print]34(11): 108869
      Mitochondrial carriers (MCs) mediate the passage of small molecules across the inner mitochondrial membrane (IMM), enabling regulated crosstalk between compartmentalized reactions. Despite MCs representing the largest family of solute carriers in mammals, most have not been subjected to a comprehensive investigation, limiting our understanding of their metabolic contributions. Here, we functionally characterize SFXN1, a member of the non-canonical, sideroflexin family. We find that SFXN1, an integral IMM protein with an uneven number of transmembrane domains, is a TIM22 complex substrate. SFXN1 deficiency leads to mitochondrial respiratory chain impairments, most detrimental to complex III (CIII) biogenesis, activity, and assembly, compromising coenzyme Q levels. The CIII dysfunction is independent of one-carbon metabolism, the known primary role for SFXN1 as a mitochondrial serine transporter. Instead, SFXN1 supports CIII function by participating in heme and α-ketoglutarate metabolism. Our findings highlight the multiple ways that SFXN1-based amino acid transport impacts mitochondrial and cellular metabolic efficiency.
    Keywords:  Complex III; OXPHOS; SFXN1; TIM22 complex; amino acid; heme; mitochondria; mitochondrial carrier; serine; sideroflexin
  5. FEBS Lett. 2021 Mar 20.
      In animals, mitochondria are mainly organised into an interconnected tubular network extending across the cell along a cytoskeletal scaffold. Mitochondrial fission and fusion, as well as distribution along cytoskeletal tracks, are counterbalancing mechanisms acting in concert to maintain a mitochondrial network tuned to cellular function. Balanced mitochondrial dynamics permits quality control of the network including biogenesis and turnover, distribution of mtDNA, and are tuned to metabolic status. Cellular and organismal health relies on a delicate balance between fission and fusion and large rearrangements in the mitochondrial network can be seen in response to cellular insults and disease. Indeed, dysfunction in the major components of the fission and fusion machineries including Dynamin-related protein 1 (DRP1), Mitofusins 1 and 2 (MFN1, MFN2) and Optic atrophy protein 1 (OPA1) and ensuing imbalance of mitochondrial dynamics can lead to neurodegenerative disease. Altered mitochondrial dynamics is also seen in more common diseases. In this review, the machinery involved in mitochondrial dynamics and their dysfunction in disease will be discussed.
    Keywords:  membrane dynamics; mitochondria; mitochondrial disease; mitochondrial fission; mitochondrial fusion; organelles; oxidative phosphorylation
  6. Mol Biol Rep. 2021 Mar 19.
      Mutations in nuclear-encoded protein subunits of the mitochondrial ribosome are an increasingly recognised cause of oxidative phosphorylation system (OXPHOS) disorders. Among them, mutations in the MRPL44 gene, encoding a structural protein of the large subunit of the mitochondrial ribosome, have been identified in four patients with OXPHOS defects and early-onset hypertrophic cardiomyopathy with or without additional clinical features. A 23-year-old individual with cardiac and skeletal myopathy, neurological involvement, and combined deficiency of OXPHOS complexes in skeletal muscle was clinically and genetically investigated. Analysis of whole-exome sequencing data revealed a homozygous mutation in MRPL44 (c.467 T > G), which was not present in the biological father, and a region of homozygosity involving most of chromosome 2, raising the possibility of uniparental disomy. Short-tandem repeat and genome-wide SNP microarray analyses of the family trio confirmed complete maternal uniparental isodisomy of chromosome 2. Mitochondrial ribosome assembly and mitochondrial translation were assessed in patient derived-fibroblasts. These studies confirmed that c.467 T > G affects the stability or assembly of the large subunit of the mitochondrial ribosome, leading to impaired mitochondrial protein synthesis and decreased levels of multiple OXPHOS components. This study provides evidence of complete maternal uniparental isodisomy of chromosome 2 in a patient with MRPL44-related disease, and confirms that MRLP44 mutations cause a mitochondrial translation defect that may present as a multisystem disorder with neurological involvement.
    Keywords:  MRPL44; Mitochondrial disease; Oxidative phosphorylation; Uniparental disomy; Whole-exome sequencing
  7. Open Biol. 2021 Mar;11(3): 210002
      The mitochondrial intermembrane space (IMS) is the most constricted sub-mitochondrial compartment, housing only about 5% of the mitochondrial proteome, and yet is endowed with the largest variability of protein import mechanisms. In this review, we summarize our current knowledge of the major IMS import pathway based on the oxidative protein folding pathway and discuss the stunning variability of other IMS protein import pathways. As IMS-localized proteins only have to cross the outer mitochondrial membrane, they do not require energy sources like ATP hydrolysis in the mitochondrial matrix or the inner membrane electrochemical potential which are critical for import into the matrix or insertion into the inner membrane. We also explore several atypical IMS import pathways that are still not very well understood and are guided by poorly defined or completely unknown targeting peptides. Importantly, many of the IMS proteins are linked to several human diseases, and it is therefore crucial to understand how they reach their normal site of function in the IMS. In the final part of this review, we discuss current understanding of how such IMS protein underpin a large spectrum of human disorders.
    Keywords:  intermembrane space; mitochondria; oxidative protein folding; protein import
  8. Aging Cell. 2021 Mar 18. e13329
      Senescence phenotypes and mitochondrial dysfunction are implicated in aging and in premature aging diseases, including ataxia telangiectasia (A-T). Loss of mitochondrial function can drive age-related decline in the brain, but little is known about whether improving mitochondrial homeostasis alleviates senescence phenotypes. We demonstrate here that mitochondrial dysfunction and cellular senescence with a senescence-associated secretory phenotype (SASP) occur in A-T patient fibroblasts, and in ATM-deficient cells and mice. Senescence is mediated by stimulator of interferon genes (STING) and involves ectopic cytoplasmic DNA. We further show that boosting intracellular NAD+ levels with nicotinamide riboside (NR) prevents senescence and SASP by promoting mitophagy in a PINK1-dependent manner. NR treatment also prevents neurodegeneration, suppresses senescence and neuroinflammation, and improves motor function in Atm-/- mice. Our findings suggest a central role for mitochondrial dysfunction-induced senescence in A-T pathogenesis, and that enhancing mitophagy as a potential therapeutic intervention.
    Keywords:  Ataxia Telangiectasia; Nicotinamide riboside; SASP; mitophagy; senescence
  9. Elife. 2021 Mar 18. pii: e61230. [Epub ahead of print]10
      Mitochondrial import deficiency causes cellular toxicity due to the accumulation of non-imported mitochondrial precursor proteins, termed mitoprotein-induced stress. Despite the burden mis-localized mitochondrial precursors place on cells, our understanding of the systems that dispose of these proteins is incomplete. Here, we cataloged the location and steady-state abundance of mitochondrial precursor proteins during mitochondrial impairment in S. cerevisiae. We found that a number of non-imported mitochondrial proteins localize to the nucleus, where they are subjected to proteasome-dependent degradation through a process we term nuclear-associated mitoprotein degradation (mitoNUC). Recognition and destruction of mitochondrial precursors by the mitoNUC pathway requires the presence of an N-terminal mitochondrial targeting sequence (MTS) and is mediated by combined action of the E3 ubiquitin ligases San1, Ubr1, and Doa10. Impaired breakdown of precursors leads to alternative sequestration in nuclear-associated foci. These results identify the nucleus as an important destination for the disposal of non-imported mitochondrial precursors.
    Keywords:  S. cerevisiae; cell biology
  10. Autophagy. 2021 Mar 17.
      CREG1 (cellular repressor of E1A-stimulated genes 1) is involved in tissue homeostasis and influences macroautophagy/autophagy to protect cardiovascular function. However, the physiological and pathological role of CREG1 in the skeletal muscle is not clear. Here, we established a skeletal muscle-specific creg1 knockout mouse model (creg1;Ckm-Cre) by crossing the Creg1-floxed mice (Creg1fl/fl) with a transgenic line expressing Cre recombinase under the muscle-specific Ckm (creatine kinase, muscle) promoter. In creg1;Ckm-Cre mice, the exercise time to exhaustion and running distance were significantly reduced compared to Creg1fl/fl mice at the age of 9 months. In addition, the administration of recombinant (re)CREG1 protein improved the motor function of 9-month-old creg1;Ckm-Cre mice. Moreover, electron microscopy images of 9-month-old creg1;Ckm-Cre mice showed that the mitochondrial quality and quantity were abnormal and associated with increased levels of PINK1 (PTEN induced putative kinase 1) and PRKN/PARKIN (parkin RBR E3 ubiquitin protein ligase) but reduced levels of the mitochondrial proteins PTGS2/COX2, COX4I1/COX4, and TOMM20. These results suggested that CREG1 deficiency accelerated the induction of mitophagy in the skeletal muscle. Mechanistically, gain-and loss-of-function mutations of Creg1 altered mitochondrial morphology and function, impairing mitophagy in C2C12 cells. Furthermore, HSPD1/HSP60 (heat shock protein 1) (401-573 aa) interacted with CREG1 (130-220 aa) to antagonize the degradation of CREG1 and was involved in the regulation of mitophagy. To the best of our knowledge, this was the first time to demonstrate that CREG1 localized to the mitochondria and played an important role in mitophagy modulation that determined skeletal muscle wasting during the growth process or disease conditions.
    Keywords:  CREG1; HSPD1; mitochondria; mitophagy; skeletal muscle
  11. Invest Ophthalmol Vis Sci. 2021 Mar 01. 62(3): 22
      Mitochondrial function is essential for the viability of aerobic eukaryotic cells, as mitochondria provide energy through the generation of adenosine triphosphate (ATP), regulate cellular metabolism, provide redox balancing, participate in immune signaling, and can initiate apoptosis. Mitochondria are dynamic organelles that participate in a cyclical and ongoing process of regeneration and autophagy (clearance), termed mitophagy specifically for mitochondrial (macro)autophagy. An imbalance in mitochondrial function toward mitochondrial dysfunction can be catastrophic for cells and has been characterized in several common ophthalmic diseases. In this article, we review mitochondrial homeostasis in detail, focusing on the balance of mitochondrial dynamics including the processes of fission and fusion, and provide a description of the mechanisms involved in mitophagy. Furthermore, this article reviews investigations of ocular diseases with impaired mitophagy, including Fuchs endothelial corneal dystrophy, primary open-angle glaucoma, diabetic retinopathy, and age-related macular degeneration, as well as several primary mitochondrial diseases with ocular phenotypes that display impaired mitophagy, including mitochondrial encephalopathy lactic acidosis stroke, Leber hereditary optic neuropathy, and chronic progressive external ophthalmoplegia. The results of various studies using cell culture, animal, and human tissue models are presented and reflect a growing awareness of mitophagy impairment as an important feature of ophthalmic disease pathology. As this review indicates, it is imperative that mitophagy be investigated as a targetable mechanism in developing therapies for ocular diseases characterized by oxidative stress and mitochondrial dysfunction.
  12. Adv Exp Med Biol. 2021 ;1286 65-76
      Mitochondrial bioenergetics is vital for the proper functioning of cellular compartments. Impairments in mitochondrial DNA encoding the respiratory chain complexes and other assisting proteins, accumulation of intracellular reactive oxygen species, an imbalance in cellular calcium transport, or the presence of organic pollutants, high fat-ketogenic diets or toxins, and advancing age can result in complex disorders, including cancer, metabolic disease, and neurodegenerative disorders. Such manifestations are distinctly exhibited in several age-related neurodegenerative diseases, such as in Parkinson's disease (PD). Defects in complex I along with perturbed signaling pathways is a common manifestation of PD. Impaired oxidative phosphorylation could increase the susceptibility to PD. Therefore, unraveling the mechanisms of mitochondrial complexes in clinical scenarios will assist in developing potential early biomarkers and standard tests for energy failure diagnosis and assist to pave a new path for targeted therapeutics against PD.
    Keywords:  Complex I; Genomic DNA; Mitochondria; Mitochondrial DNA; OxPhos; Parkinson’s disease
  13. iScience. 2021 Mar 19. 24(3): 102196
      Phosphatidylethanolamine (PE) made in mitochondria has long been recognized as an important precursor for phosphatidylcholine production that occurs in the endoplasmic reticulum (ER). Recently, the strict mitochondrial localization of the enzyme that makes PE in the mitochondrion, phosphatidylserine decarboxylase 1 (Psd1), was questioned. Since a dual localization of Psd1 to the ER would have far-reaching implications, we initiated our study to independently re-assess the subcellular distribution of Psd1. Our results support the unavoidable conclusion that the vast majority, if not all, of functional Psd1 resides in the mitochondrion. Through our efforts, we discovered that mutant forms of Psd1 that impair a self-processing step needed for it to become functional are dually localized to the ER when expressed in a PE-limiting environment. We conclude that severely impaired cellular PE metabolism provokes an ER-assisted adaptive response that is capable of identifying and resolving nonfunctional mitochondrial precursors.
    Keywords:  Cell Biology; Molecular Physiology; Proteomics
  14. Nat Chem Biol. 2021 Mar 15.
      The protein complexes of the mitochondrial electron transport chain exist in isolation and in higher order assemblies termed supercomplexes (SCs) or respirasomes (SC I+III2+IV). The association of complexes I, III and IV into the respirasome is regulated by unknown mechanisms. Here, we designed a nanoluciferase complementation reporter for complex III and IV proximity to determine in vivo respirasome levels. In a chemical screen, we found that inhibitors of the de novo pyrimidine synthesis enzyme dihydroorotate dehydrogenase (DHODH) potently increased respirasome assembly and activity. By-passing DHODH inhibition via uridine supplementation decreases SC assembly by altering mitochondrial phospholipid composition, specifically elevated peroxisomal-derived ether phospholipids. Cell growth rates upon DHODH inhibition depend on ether lipid synthesis and SC assembly. These data reveal that nucleotide pools signal to peroxisomes to modulate synthesis and transport of ether phospholipids to mitochondria for SC assembly, which are necessary for optimal cell growth in conditions of nucleotide limitation.
  15. Sci Rep. 2021 Mar 18. 11(1): 6363
      Abnormal mitochondrial function is a key process in the pathogenesis of Parkinson's disease (PD). The central pore-forming protein TOM40 of the mitochondria is encoded by the translocase of outer mitochondrial membrane 40 homologue gene (TOMM40). The highly variant '523' poly-T repeat is associated with age-related cognitive decline and age of onset in Alzheimer's disease, but whether it plays a role in modifying the risk or clinical course of PD it yet to be elucidated. The TOMM40 '523' allele length was determined in 634 people with PD and 422 healthy controls from an Australian cohort and the Parkinson's Progression Markers Initiative (PPMI) cohort, using polymerase chain reaction or whole genome sequencing analysis. Genotype and allele frequencies of TOMM40 '523' and APOE ε did not differ significantly between the cohorts. Analyses revealed TOMM40 '523' allele groups were not associated with disease risk, while considering APOE ε genotype. Regression analyses revealed the TOMM40 S/S genotype was associated with a significantly later age of symptom onset in the PPMI PD cohort, but not after correction for covariates, or in the Australian cohort. Whilst variation in the TOMM40 '523' polymorphism was not associated with PD risk, the possibility that it may be a modifying factor for age of symptom onset warrants further investigation in other PD populations.
  16. Child Neurol Open. 2021 Jan-Dec;8:8 2329048X21991382
      In the field of mitochondrial medicine, correlation of clinical phenotype with mutation heteroplasmy remains an outstanding question with few, if any, clear thresholds corresponding to a given phenotype. The m.8344A>G mutation is most commonly associated with myoclonus epilepsy and ragged red fiber syndrome (MERRF) at varying levels of heteroplasmy. However, a handful of cases been previously reported in which individuals homoplasmic or nearly homoplasmic for this mutation in the blood have presented with multiple bulbar palsies, respiratory failure, and progressive neurologic decline almost uniformly following a respiratory illness. MRI brain in all affected individuals revealed symmetric T2 hyperintense lesions of subcortical gray matter structures, consistent with Leigh syndrome. Here, we present 3 cases with clinical, biochemical, and neuro-imaging findings with the additional reporting of spinal lesions. This new phenotype supports a heteroplasmy-dependent phenotype model for this mutation and recognition of this can help clinicians with diagnosis and anticipatory clinical guidance.
    Keywords:  Leigh syndrome; MERRF; heteroplasmy; homoplasmy; mitochondria; phenotype
  17. Nat Commun. 2021 03 15. 12(1): 1680
      Branched-chain amino acids (BCAA) and their cognate α-ketoacids (BCKA) are elevated in an array of cardiometabolic diseases. Here we demonstrate that the major metabolic fate of uniformly-13C-labeled α-ketoisovalerate ([U-13C]KIV) in the heart is reamination to valine. Activation of cardiac branched-chain α-ketoacid dehydrogenase (BCKDH) by treatment with the BCKDH kinase inhibitor, BT2, does not impede the strong flux of [U-13C]KIV to valine. Sequestration of BCAA and BCKA away from mitochondrial oxidation is likely due to low levels of expression of the mitochondrial BCAA transporter SLC25A44 in the heart, as its overexpression significantly lowers accumulation of [13C]-labeled valine from [U-13C]KIV. Finally, exposure of perfused hearts to levels of BCKA found in obese rats increases phosphorylation of the translational repressor 4E-BP1 as well as multiple proteins in the MEK-ERK pathway, leading to a doubling of total protein synthesis. These data suggest that elevated BCKA levels found in obesity may contribute to pathologic cardiac hypertrophy via chronic activation of protein synthesis.
  18. Autophagy. 2021 Mar 14. 1-22
      Recent reports indicated that mitophagy protects against alcohol-induced liver injury, which helps remove damaged mitochondria to reduce the accumulation of reactive oxygen species (ROS). AMP-activated protein kinase (AMPK) has been recently used in ALD (alcoholic liver disease) and mitochondrial dysfunction research. However, the inner mechanism, whether AMPK can regulate mitophagy in ALD, remains unknown. Here we found that AMPK can significantly reduce alcohol-induced liver injury and enhances hepatocytes' mitophagy level. Next, we identified that AMPK rescued alcohol-induced low expression of UQCRC2 (ubiquinol-cytochrome c reductase core protein 2). Interestingly, UQCRC2 knockdown (KD) treatment causes impaired mitophagy, whereas UQCRC2 overexpression (OE) can significantly increase mitophagy to attenuate liver injury. Also, we identified that AMPK indirectly upregulates UQCRC2 protein level, and RNA-seq, chromatin immunoprecipitation (ChIP) assay, bioinformatics, and luciferase assays helped us understand that AMPK enhanced UQCRC2 gene transcription through activating NFE2L2/NRF2 (nuclear factor, erythroid 2 like 2). Our results demonstrate that AMPK regulating UQCRC2 is a significant mitochondrial event in mitophagy. It identifies a new signaling axis, AMPK-NFE2L2-UQCRC2, in the regulation of mitophagy levels in the liver, suggesting a possible therapeutic strategy to treat ALD.Abbreviations: AAV: AENO-associated virus; ALD: alcoholic liver disease; AMPK: AMP-activated protein kinase; BUN: blood urea nitrogen; H&E: hematoxylin and eosin; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; ChIP: chromatin immunoprecipitation assay; CO-IP: co-immunoprecipitation; COPD: chronic obstructive pulmonary disease; EM: electron microscope; GOT1/AST: glutamic-oxaloacetic transaminase 1; GPT/ALT: glutamic-pyruvic transaminase; IF: immunofluorescence; IHC: immunohistochemistry; KD: knockdown; MAP1LC3/LC3: microtubule associated protein 1 light chain protein 3; MTDR: MitoTracker Deep Red; NFE2L2/NRF2: nuclear factor, erythroid 2 like 2; mtDNA: mitochondrial DNA; MTRC: MitoTracker Red CMXRos; OCR: Oxygen consumption rate; OE: overexpress; PINK1: PTEN induced kinase 1; qRT-PCR: quantitative real-time PCR; ROS: reactive oxygen species; SD: standard deviation; SOD2: superoxide dismutase 2; UQCRC2: ubiquinol-cytochrome c reductase core protein 2; WB: western blot; ΔΨ: mitochondrial membrane potential.
    Keywords:  AMPK; bioinformatics; mitophagy; rna-seq; transcription factor; uqcrc2
  19. Blood Adv. 2021 Mar 23. 5(6): 1706-1718
      Blood platelets are essential for controlling hemostasis. They are released by megakaryocytes (MKs) located in the bone marrow, upon extension of cytoplasmic protrusions into the lumen of bone marrow sinusoids. Their number increases in postpulmonary capillaries, suggesting a role for oxygen gradient in thrombopoiesis (ie, platelet biogenesis). In this study, we show that initiation of thrombopoiesis from human mature MKs was enhanced under hyperoxia or during pro-oxidant treatments, whereas antioxidants dampened it. Quenching mitochondrial reactive oxygen species (mtROS) with MitoTEMPO decreased thrombopoiesis, whereas genetically enhancing mtROS by deacetylation-null sirtuin-3 expression increased it. Blocking cytosolic ROS production by NOX inhibitors had no impact. Classification according to the cell roundness index delineated 3 stages of thrombopoiesis in mature MKs. Early-stage round MKs exhibited the highest index, which correlated with low mtROS levels, a mitochondrial tubular network, and the mitochondrial recruitment of the fission activator Drp1. Intermediate MKs at the onset of thrombopoiesis showed high mtROS levels and small, well-delineated mitochondria. Terminal MKs showed the lowest roundness index and long proplatelet extensions. Inhibiting Drp1-dependent mitochondrial fission of mature MKs by Mdivi-1 favored a tubular mitochondrial network and lowered both mtROS levels and intermediate MKs proportion, whereas enhancing Drp1 activity genetically had opposite effects. Reciprocally, quenching mtROS limited mitochondrial fission in round MKs. These data demonstrate a functional coupling between ROS and mitochondrial fission in MKs, which is crucial for the onset of thrombopoiesis. They provide new molecular cues that control initiation of platelet biogenesis and may help elucidate some unexplained thrombocytopenia.
  20. Cytotherapy. 2021 Mar 13. pii: S1465-3249(20)30848-3. [Epub ahead of print]
      With the redefinition of osteoarthritis (OA) and the understanding that the joint behaves as an organ, OA is now considered a systemic illness with a low grade of chronic inflammation. Mitochondrial dysfunction is well documented in OA and has the capacity to alter chondrocyte and synoviocyte function. Transmitochondrial cybrids are suggested as a useful cellular model to study mitochondrial biology in vitro, as they carry different mitochondrial variants with the same nuclear background. The aim of this work was to study mitochondrial and metabolic function of cybrids with mitochondrial DNA from healthy (N) and OA donors. In this work, the authors demonstrate that cybrids from OA patients behave differently from cybrids from N donors in several mitochondrial parameters. Furthermore, OA cybrids behave similarly to OA chondrocytes. These results enhance our understanding of the role of mitochondria in the degeneration process of OA and present cybrids as a useful model to study OA pathogenesis.
    Keywords:  chondrocytes; cybrids; metabolism; mitochondria; mtDNA; osteoarthritis
  21. Genetics. 2021 Mar 15. pii: iyab044. [Epub ahead of print]
      Traditional Hardy-Weinberg equilibrium (HWE) tests (the χ2 test and the exact test) have long been used as a metric for evaluating genotype quality, as technical artifacts leading to incorrect genotype calls often can be identified as deviations from HWE. However, in datasets comprised of individuals from diverse ancestries, HWE can be violated even without genotyping error, complicating the use of HWE testing to assess genotype data quality. In this manuscript, we present the Robust Unified Test for HWE (RUTH) to test for HWE while accounting for population structure and genotype uncertainty, and evaluate the impact of population heterogeneity and genotype uncertainty on the standard HWE tests and alternative methods using simulated and real sequence datasets. Our results demonstrate that ignoring population structure or genotype uncertainty in HWE tests can inflate false positive rates by many orders of magnitude. Our evaluations demonstrate different tradeoffs between false positives and statistical power across the methods, with RUTH consistently amongst the best across all evaluations. RUTH is implemented as a practical and scalable software tool to rapidly perform HWE tests across millions of markers and hundreds of thousands of individuals while supporting standard VCF/BCF formats. RUTH is publicly available at
    Keywords:  genotype likelihoods; next-generation sequencing; population structure; principal components analysis
  22. Science. 2021 Mar 19. 371(6535): 1249-1253
      Although cell lineage information is fundamental to understanding organismal development, very little direct information is available for humans. We performed high-depth (250×) whole-genome sequencing of multiple tissues from three individuals to identify hundreds of somatic single-nucleotide variants (sSNVs). Using these variants as "endogenous barcodes" in single cells, we reconstructed early embryonic cell divisions. Targeted sequencing of clonal sSNVs in different organs (about 25,000×) and in more than 1000 cortical single cells, as well as single-nucleus RNA sequencing and single-nucleus assay for transposase-accessible chromatin sequencing of ~100,000 cortical single cells, demonstrated asymmetric contributions of early progenitors to extraembryonic tissues, distinct germ layers, and organs. Our data suggest onset of gastrulation at an effective progenitor pool of about 170 cells and about 50 to 100 founders for the forebrain. Thus, mosaic mutations provide a permanent record of human embryonic development at very high resolution.
  23. Biochim Biophys Acta Bioenerg. 2021 Mar 13. pii: S0005-2728(21)00047-5. [Epub ahead of print] 148414
      The study of the mitochondrial respiratory chain (MRC) function in relation with its structural organization is of great interest due to the central role of this system in eukaryotic cell metabolism. The complexome profiling technique has provided invaluable information for our understanding of the composition and assembly of the individual MRC complexes, and also of their association into larger supercomplexes (SCs) and respirasomes. The formation of the SCs has been highly debated, and their assembly and regulation mechanisms are still unclear. Previous studies demonstrated a prominent role for COX7A2L (SCAFI) as a structural protein bridging the association of individual MRC complexes III and IV in the minor SC III2 + IV, although its relevance for respirasome formation and function remains controversial. In this work, we have used SILAC-based complexome profiling to dissect the structural organization of the human MRC in HEK293T cells depleted of SCAFI (SCAFIKO) by CRISPR-Cas9 genome editing. SCAFI ablation led to a preferential loss of SC III2 + IV and of a minor subset of respirasomes without affecting OXPHOS function. Our data suggest that the loss of SCAFI-dependent respirasomes in SCAFIKO cells is mainly due to alterations on early stages of CI assembly, without impacting the biogenesis of complexes III and IV. Contrary to the idea of SCAFI being the main player in respirasome formation, SILAC-complexome profiling showed that, in wild-type cells, the majority of respirasomes (ca. 70%) contained COX7A2 and that these species were present at roughly the same levels when SCAFI was knocked-out. We thus demonstrate the co-existence of structurally distinct respirasomes defined by the preferential binding of complex IV via COX7A2, rather than SCAFI, in human cultured cells.
    Keywords:  COX7A2; COX7A2L/SCAFI; Mitochondria; oxidative phosphorylation; respiratory chain; respiratory supercomplexes
  24. Neurol Genet. 2021 Apr;7(2): e566
      Objective: To investigate the pathogenicity of a novel MT-ND3 mutation identified in a patient with adult-onset sensorimotor axonal polyneuropathy and report the clinical, morphologic, and biochemical findings.Methods: Clinical assessments and morphologic and biochemical investigations of skeletal muscle and cultured myoblasts from the patient were performed. Whole-genome sequencing (WGS) of DNA from skeletal muscle and Sanger sequencing of mitochondrial DNA (mtDNA) from both skeletal muscle and cultured myoblasts were performed. Heteroplasmic levels of mutated mtDNA in different tissues were quantified by last-cycle hot PCR.
    Results: Muscle showed ragged red fibers, paracrystalline inclusions, a significant reduction in complex I (CI) respiratory chain (RC) activity, and decreased adenosine triphosphate (ATP) production for all substrates used by CI. Sanger sequencing of DNA from skeletal muscle detected a unique previously unreported heteroplasmic mutation in mtDNA encoded MT-ND3, coding for a subunit in CI. WGS confirmed the mtDNA mutation but did not detect any other mutation explaining the disease. Cultured myoblasts, however, did not carry the mutation, and RC activity measurements in myoblasts were normal.
    Conclusions: We report a case with adult-onset sensorimotor axonal polyneuropathy caused by a novel mtDNA mutation in MT-ND3. Loss of heteroplasmy in blood, cultured fibroblasts and myoblasts from the patient, and normal measurement of RC activity of the myoblasts support pathogenicity of the mutation. These findings highlight the importance of mitochondrial investigations in patients presenting with seemingly idiopathic polyneuropathy, especially if muscle also is affected.
  25. J Cell Biol. 2021 Apr 05. pii: e201909139. [Epub ahead of print]220(4):
      Acute heat stress (aHS) can induce strong developmental defects in Caenorhabditis elegans larva but not lethality or sterility. This stress results in transitory fragmentation of mitochondria, formation of aggregates in the matrix, and decrease of mitochondrial respiration. Moreover, active autophagic flux associated with mitophagy events enables the rebuilding of the mitochondrial network and developmental recovery, showing that the autophagic response is protective. This adaptation to aHS does not require Pink1/Parkin or the mitophagy receptors DCT-1/NIX and FUNDC1. We also find that mitochondria are a major site for autophagosome biogenesis in the epidermis in both standard and heat stress conditions. In addition, we report that the depletion of the dynamin-related protein 1 (DRP-1) affects autophagic processes and the adaptation to aHS. In drp-1 animals, the abnormal mitochondria tend to modify their shape upon aHS but are unable to achieve fragmentation. Autophagy is induced, but autophagosomes are abnormally elongated and clustered on mitochondria. Our data support a role for DRP-1 in coordinating mitochondrial fission and autophagosome biogenesis in stress conditions.
  26. iScience. 2021 Mar 19. 24(3): 102181
      Perturbation of mitochondrial proteostasis provokes cell autonomous and cell non-autonomous responses that contribute to homeostatic adaptation. Here, we demonstrate distinct metabolic effects of hepatic metabokines as cell non-autonomous factors in mice with mitochondrial OxPhos dysfunction. Liver-specific mitochondrial stress induced by a loss-of-function mutation in Crif1 (LKO) leads to aberrant oxidative phosphorylation and promotes the mitochondrial unfolded protein response. LKO mice are highly insulin sensitive and resistant to diet-induced obesity. The hepatocytes of LKO mice secrete large quantities of metabokines, including GDF15 and FGF21, which confer metabolic benefits. We evaluated the metabolic phenotypes of LKO mice with global deficiency of GDF15 or FGF21 and show that GDF15 regulates body and fat mass and prevents diet-induced hepatic steatosis, whereas FGF21 upregulates insulin sensitivity, energy expenditure, and thermogenesis in white adipose tissue. This study reveals that the mitochondrial integrated stress response (ISRmt) in liver mediates metabolic adaptation through hepatic metabokines.
    Keywords:  Cell Biology; Physiology; Systems Biology
  27. Bioorg Med Chem Lett. 2021 Mar 10. pii: S0960-894X(21)00129-3. [Epub ahead of print] 127903
      Folate and related derivatives are essential small molecules required for survival. Of significant interest is the biological role and necessity of folate in the crosstalk between commensal organisms and their respective hosts, including the tremendously complex human distal gut microbiome. Here, we designed a folate-based probe consisting of a photo-crosslinker to detect and quantitate folate-binding proteins from proteomic samples. We demonstrate the selectivity of our probe for the well-established human folate-binding protein dihydrofolate reductase and show no promiscuous labeling occurs with human caspase-3 or bovine serum albumin, which served as negative controls. Affinity-based enrichment of folate-binding proteins from an E. coli lysate in combination with mass spectrometry proteomics verified the ability of our probe to isolate low-abundance folate-dependent proteins. We envision that our probe will serve as a tool to elucidate the roles of commensal microbial folate-binding proteins in health and microbiome-related diseases.
    Keywords:  affinity-based photo-activatable probe; folate; folic acid; mass spectrometry protein profiling; proteomics
  28. Cell Metab. 2021 Mar 13. pii: S1550-4131(21)00102-9. [Epub ahead of print]
      Exercise training positively affects metabolic health through increased mitochondrial oxidative capacity and improved glucose regulation and is the first line of treatment in several metabolic diseases. However, the upper limit of the amount of exercise associated with beneficial therapeutic effects has not been clearly identified. Here, we used a training model with a progressively increasing exercise load during an intervention over 4 weeks. We closely followed changes in glucose tolerance, mitochondrial function and dynamics, physical exercise capacity, and whole-body metabolism. Following the week with the highest exercise load, we found a striking reduction in intrinsic mitochondrial function that coincided with a disturbance in glucose tolerance and insulin secretion. We also assessed continuous blood glucose profiles in world-class endurance athletes and found that they had impaired glucose control compared with a matched control group.
    Keywords:  athletes; continuous glucose monitoring; exercise; exercise adaptations; glucose tolerance; high-intensity interval training; insulin resistance; metabolic dysfunction; mitochondria; mitochondrial dynamics; mitochondrial dysfunction
  29. Crit Rev Biochem Mol Biol. 2021 Mar 15. 1-15
      Overproduction of reactive oxygen species and compromised antioxidant defenses perturb intracellular redox homeostasis and is associated with a myriad of human diseases as well as with the natural process of aging. Hydrogen sulfide (H2S), which is biosynthesized by organisms ranging from bacteria to man, influences a broad range of physiological functions. A highly touted molecular mechanism by which H2S exerts its cellular effects is via post-translational modification of the thiol redox proteome, converting cysteine thiols to persulfides, in a process referred to as protein persulfidation. The physiological relevance of this modification in the context of specific signal transmission pathways remains to be rigorously established, while a general protective role for protein persulfidation against hyper-oxidation of the cysteine proteome is better supported. A second mechanism by which H2S modulates redox homeostasis is via remodeling the redox metabolome, targeting the electron transfer chain and perturbing the major redox nodes i.e. CoQ/CoQH2, NAD+/NADH and FAD/FADH2. The metabolic changes that result from H2S-induced redox changes fan out from the mitochondrion to other compartments. In this review, we discuss recent developments in elucidating the roles of H2S and its oxidation products on redox homeostasis and its role in protecting the thiol proteome.
    Keywords:  Sulfide oxidation pathway; mitochondrial bioenergetics; persulfidation; reactive sulfur species; redox metabolome; redox proteome; sulfide quinone oxidoreductase
  30. J Autoimmun. 2021 Mar 10. pii: S0896-8411(21)00038-X. [Epub ahead of print]119 102630
      OBJECTIVES: Literature suggests that neutrophils of patients with rheumatoid arthritis (RA) are primed to respond to N-formyl methionine group (formylated peptides). Animal models indicate that formylated peptides contribute to joint damage via neutrophil recruitment and inflammation in joints. Non-steroidal anti-inflammatory drugs are also known to inhibit formyl peptide-induced neutrophil activation. The predominant source of formylated peptides in sterile inflammatory conditions like RA is mitochondria, organelles with prokaryotic molecular signatures. However, there is no direct evidence of mitochondrial formyl peptides (mtNFPs) in the circulation of patients with RA and their potential role in neutrophil-mediated inflammation in RA, including their clinical significance.METHODS: Levels of mtNFPs (total fMet, MT-ND6) were analyzed using ELISA in plasma and serum obtained from patients in 3 cross-sectional RA cohorts (n = 275), a longitudinal inception cohort (n = 192) followed for a median of 8 years, and age/gender-matched healthy controls (total n = 134). Neutrophil activation assays were done in the absence or presence of formyl peptide receptor 1 (FPR1) inhibitor cyclosporine H.
    RESULTS: Elevated levels of total fMet were observed in the circulation of patients with RA as compared to healthy controls (p < 0.0001) associating with disease activity and could distinguish patients with the active disease from patients with inactive disease or patients in remission. Baseline levels of total fMet correlated with current and future joint involvement, respectively and predicted the development of rheumatoid nodules (OR = 1.2, p = 0.04). Further, total fMet levels improved the prognostic ability of ACPA in predicting erosive disease (OR of 7.9, p = 0.001). Total fMet levels correlated with markers of inflammation and neutrophil activation. Circulating mtNFPs induced neutrophil activation in vitro through FPR1-dependent mechanisms.
    CONCLUSIONS: Circulating mtNFPs could be novel biomarkers of disease monitoring and prognosis for RA and in investigating neutrophil-mediated inflammation in RA. We propose, FPR1 as a novel therapeutic target for RA.
    Keywords:  Clinical biomarkers; Formyl peptide receptor 1; Mitochondria; Neutrophils; Rheumatoid arthritis
  31. Mol Genet Metab Rep. 2021 Jun;27 100738
      Background: In infancy multiple acyl-CoA dehydrogenase deficiency (MADD) is commonly a severe inherited metabolic disease caused by genetic defects in electron transfer flavoprotein (ETF) or ETF ubiquinone oxidoreductase. Both enzymes require flavin adenine dinucleotide (FAD) as a cofactor. Riboflavin (vitamin B2) is a precursor in the synthesis of FAD. MADD can be detected by newborn screening (NBS) based on elevation of multiple acylcarnitines.Methods: We present the results of two children whose NBS results and subsequent confirmatory testing resulted in a suspected diagnosis of MADD. In parallel in both children vitamin B12 deficiency was detected.
    Results: Biochemical profiles normalized rapidly in both children under supplementation with riboflavin. After extensive work-up of both cases including molecular genetic studies there was no indication of MADD. Vitamin B12 deficiency in both children was caused by maternal vitamin B12 deficiency and was rapidly corrected by oral supplementation with vitamin B12 or (partial) formula feeding. As both vitamin B12 and riboflavin have similar food sources we postulate that in these cases positive NBS for MADD was caused by combined maternal vitamin B deficiencies.
    Conclusion: The differential diagnosis of maternally caused vitamin B deficiencies should be considered in children with abnormal NBS results for MADD, especially in the presence of normal molecular genetic analysis or in case of associated findings of other maternal vitamin B deficiencies like vitamin B12 or folic acid deficiency.
    Keywords:  Maternal; Mother and child health; Multiple acyl-CoA dehydrogenase deficiency; Newborn screening; Vitamin B12 deficiency
  32. J Clin Invest. 2021 Mar 15. pii: 147734. [Epub ahead of print]131(6):
      Leber's hereditary optic neuropathy (LHON) is the most common mitochondrial disease and in most cases is caused by mutations in mitochondrial DNA-encoded (mtDNA-encoded) respiratory complex I subunit ND1, ND4, or ND6. In this issue of the JCI, Stenton et al. describe biallelic mutations in a nuclear DNA-encoded gene, DNAJC30, establishing recessively inherited LHON (arLHON). Functional studies suggest that DNAJC30 is a protein chaperone required for exchange of damaged complex I subunits. Hallmark mtDNA LHON features were also found in arLHON, including incomplete penetrance, male predominance, and positive response to idebenone therapy. These results extend complex I-deficient phenotypes to include recessively inherited optic neuropathy, with important clinical implications for genetic counseling and therapeutic considerations.
  33. Dev Cell. 2021 Mar 09. pii: S1534-5807(21)00162-3. [Epub ahead of print]
      Neuronal activity increases energy consumption and requires balanced production to maintain neuronal function. How activity is coupled to energy production remains incompletely understood. Here, we report that Rheb regulates mitochondrial tricarboxylic acid cycle flux of acetyl-CoA by activating pyruvate dehydrogenase (PDH) to increase ATP production. Rheb is induced by synaptic activity and lactate and dynamically trafficked to the mitochondrial matrix through its interaction with Tom20. Mitochondria-localized Rheb protein is required for activity-induced PDH activation and ATP production. Cell-type-specific gain- and loss-of-function genetic models for Rheb reveal reciprocal changes in PDH phosphorylation/activity, acetyl-CoA, and ATP that are not evident with genetic or pharmacological manipulations of mTORC1. Mechanistically, Rheb physically associates with PDH phosphatase (PDP), enhancing its activity and association with the catalytic E1α-subunit of PDH to reduce PDH phosphorylation and increase its activity. Findings identify Rheb as a nodal point that balances neuronal activity and neuroenergetics via Rheb-PDH axis.
    Keywords:  Rheb; mTORC1; mitochondria; neuroenergetics; neuronal activity; pyruvate dehydrogenase
  34. Mol Neurodegener. 2021 Mar 19. 16(1): 17
      The most common mutation in the Leucine-rich repeat kinase 2 gene (LRRK2), G2019S, causes familial Parkinson's Disease (PD) and renders the encoded protein kinase hyperactive. While targeting LRRK2 activity is currently being tested in clinical trials as a therapeutic avenue for PD, to date, the molecular effects of chronic LRRK2 inhibition have not yet been examined in vivo. We evaluated the utility of newly available phospho-antibodies for Rab substrates and LRRK2 autophosphorylation to examine the pharmacodynamic response to treatment with the potent and specific LRRK2 inhibitor, MLi-2, in brain and peripheral tissue in G2019S LRRK2 knock-in mice. We report higher sensitivity of LRRK2 autophosphorylation to MLi-2 treatment and slower recovery in washout conditions compared to Rab GTPases phosphorylation, and we identify pS106 Rab12 as a robust readout of downstream LRRK2 activity across tissues. The downstream effects of long-term chronic LRRK2 inhibition in vivo were evaluated in G2019S LRRK2 knock-in mice by phospho- and total proteomic analyses following an in-diet administration of MLi-2 for 10 weeks. We observed significant alterations in endolysosomal and trafficking pathways in the kidney that were sensitive to MLi-2 treatment and were validated biochemically. Furthermore, a subtle but distinct biochemical signature affecting mitochondrial proteins was observed in brain tissue in the same animals that, again, was reverted by kinase inhibition. Proteomic analysis in the lung did not detect any major pathway of dysregulation that would be indicative of pulmonary impairment. This is the first study to examine the molecular underpinnings of chronic LRRK2 inhibition in a preclinical in vivo PD model and highlights cellular processes that may be influenced by therapeutic strategies aimed at restoring LRRK2 physiological activity in PD patients.
  35. Aging (Albany NY). 2021 Mar 18. 13
      Declines in mitochondrial mass are thought to be a hallmark of mammalian aging, and a ketogenic diet (KD) may prevent the age-related decreases in mitochondrial content. The objective of this study was to investigate the impact of a KD on markers of mitochondrial mass. Mice were fed an isocaloric control diet (CD) or KD from 12 months of age. Tissues were collected after 1 month and 14 months of intervention, and a panel of commonly used markers of mitochondrial mass (mitochondrial enzyme activities and levels, mitochondrial to nuclear DNA ratio, and cardiolipin content) were measured. Our results showed that a KD stimulated activities of marker mitochondrial enzymes including citrate synthase, Complex I, and Complex IV in hindlimb muscle in aged mice. KD also increased the activity of citrate synthase and prevented an age-related decrease in Complex IV activity in aged brain. No other markers were increased in these tissues. Furthermore, the impacts of a KD on liver and kidney were mixed with no pattern indicative of a change in mitochondrial mass. In conclusion, results of the present study suggest that a KD induces tissue-specific changes in mitochondrial enzyme activities, or structure, rather than global changes in mitochondrial mass across tissues.
    Keywords:  brain; diet; kidney; liver; skeletal muscle
  36. Genome Biol Evol. 2021 Mar 19. pii: evab055. [Epub ahead of print]
      The last eukaryote common ancestor (LECA) possessed mitochondria and all key traits that make eukaryotic cells more complex than their prokaryotic ancestors, yet the timing of mitochondrial acquisition and the role of mitochondria in the origin of eukaryote complexity remain debated. Here we report evidence from gene duplications in LECA indicating an early origin of mitochondria. Among 163,545 duplications in 24,571 gene trees spanning 150 sequenced eukaryotic genomes, we identify 713 gene duplication events that occurred in LECA. LECA's bacterial derived genes include numerous mitochondrial functions and were duplicated significantly more often than archaeal derived and eukaryote specific genes. The surplus of bacterial derived duplications in LECA most likely reflects the serial copying of genes from the mitochondrial endosymbiont to the archaeal host's chromosomes. Clustering, phylogenies and likelihood ratio tests for 22.4 million genes from 5,655 prokaryotic and 150 eukaryotic genomes reveal no evidence for lineage specific gene acquisitions in eukaryotes, except from the plastid in the plant lineage. That finding, and the functions of bacterial genes duplicated in LECA, suggest that the bacterial genes in eukaryotes are acquisitions from the mitochondrion, followed by vertical gene evolution and differential loss across eukaryotic lineages, flanked by concomitant lateral gene transfer among prokaryotes. Overall, the data indicate that recurrent gene transfer via the copying of genes from a resident mitochondrial endosymbiont to archaeal host chromosomes preceded the onset of eukaryotic cellular complexity, favoring mitochondria-early over mitochondria-late hypotheses for eukaryote origin.
    Keywords:  endosymbiosis; eukaryote origin; evolution; gene duplication; gene transfer; paralogy
  37. Epigenetics Chromatin. 2021 Mar 19. 14(1): 14
      Epigenetic marks do not change the sequence of DNA but affect gene expression in a cell-type specific manner by altering the activities of regulatory elements. Development of new molecular biology assays, sequencing technologies, and computational approaches enables us to profile the human epigenome in three-dimensional structure genome-wide. Here we describe various molecular biology techniques and bioinformatic tools that have been developed to measure the activities of regulatory elements and their chromatin interactions. Moreover, we list currently available three-dimensional epigenomic data sets that are generated in various human cell types and tissues to assist in the design and analysis of research projects.
    Keywords:  Analysis tools; Chromatin interactions; Databases; Epigenomics; Regulatory elements
  38. Mol Genet Metab. 2021 Mar 10. pii: S1096-7192(21)00057-3. [Epub ahead of print]
      Propionic Acidemia (PA) and Methylmalonic Acidemia (MMA) are inborn errors of metabolism affecting the catabolism of valine, isoleucine, methionine, threonine and odd-chain fatty acids. These are multi-organ disorders caused by the enzymatic deficiency of propionyl-CoA carboxylase (PCC) or methylmalonyl-CoA mutase (MUT), resulting in the accumulation of propionyl-coenzyme A (P-CoA) and methylmalonyl-CoA (M-CoA in MMA only). Primary metabolites of these CoA esters include 2-methylcitric acid (MCA), propionyl-carnitine (C3), and 3-hydroxypropionic acid, which are detectable in both PA and MMA, and methylmalonic acid, which is detectable in MMA patients only (Chapman et al., 2012). We deployed liver cell-based models that utilized PA and MMA patient-derived primary hepatocytes to validate a small molecule therapy for PA and MMA patients. The small molecule, HST5040, resulted in a dose-dependent reduction in the levels of P-CoA, M-CoA (in MMA) and the disease-relevant biomarkers C3, MCA, and methylmalonic acid (in MMA). A putative working model of how HST5040 reduces the P-CoA and its derived metabolites involves the conversion of HST5040 to HST5040-CoA driving the redistribution of free and conjugated CoA pools, resulting in the differential reduction of the aberrantly high P-CoA and M-CoA. The reduction of P-CoA and M-CoA, either by slowing production (due to increased demands on the free CoA (CoASH) pool) or enhancing clearance (to replenish the CoASH pool), results in a net decrease in the CoA-derived metabolites (C3, MCA and MMA (MMA only)). A Phase 2 study in PA and MMA patients will be initiated in the United States.
    Keywords:  Methylmalonic acidemia; Propionic acidemia
  39. Sci Adv. 2021 Mar;pii: eabe7520. [Epub ahead of print]7(12):
      The role of the nuclear genome in maintaining the stability of the mitochondrial genome (mtDNA) is incompletely known. mtDNA sequence variants can exist in a state of heteroplasmy, which denotes the coexistence of organellar genomes with different sequences. Heteroplasmic variants that impair mitochondrial capacity cause disease, and the state of heteroplasmy itself is deleterious. However, mitochondrial heteroplasmy may provide an intermediate state in the emergence of novel mitochondrial haplogroups. We used genome-wide genotyping data from 982,072 European ancestry individuals to evaluate variation in mitochondrial heteroplasmy and to identify the regions of the nuclear genome that affect it. Age, sex, and mitochondrial haplogroup were associated with the extent of heteroplasmy. GWAS identified 20 loci for heteroplasmy that exceeded genome-wide significance. This included a region overlapping mitochondrial transcription factor A (TFAM), which has multiple roles in mtDNA packaging, replication, and transcription. These results show that mitochondrial heteroplasmy has a heritable nuclear component.
  40. Redox Rep. 2021 Dec;26(1): 53-61
      Objectives: Although glutamate is an essential factor in the neuronal system, excess glutamate can produce excitotoxicity. We previously reported that Peroxiredoxin 5 (Prx5) protects neuronal cells from glutamate toxicity via its antioxidant effects. However, it is unclear whether cytosolic or mitochondrial Prx5 provides greater neuroprotection. Here, we investigated differences in the neuroprotective effects of cytosolic and mitochondrial Prx5.Methods: We analyzed patterns of cytosolic and mitochondrial H2O2 generation in glutamate toxicity using HyPer protein. And then, we confirmed the change of intracellular ROS level and apoptosis with respective methods. The mitochondrial dynamics was assessed with confocal microscope imaging and western blotting.Results: We found that the level of mitochondrial H2O2 greatly increased compared to cytosolic H2O2 and it affected cytosolic H2O2 generation after glutamate treatment. In addition, we confirmed that mitochondrial Prx5 provides more effective neuroprotection than cytosolic Prx5.Discussion: Overall, our study reveals the mechanisms of cytosolic and mitochondrial ROS in glutamate toxicity. Our findings suggest that mitochondrial ROS and Prx5 are attractive therapeutic targets and that controlling these factors be useful for the prevention of neurodegenerative diseases.
    Keywords:  Cytosolic ROS; HT22; Hydrogen peroxide; Hyper; Peroxiredoxin 5; apoptosis; glutamate; mitochondrial ROS
  41. Oxid Med Cell Longev. 2021 ;2021 6685468
      General anesthesia is a powerful and indispensable tool to ensure the accomplishment of surgical procedures or clinical examinations. Sevoflurane as an inhalational anesthetic without unpleasant odor is commonly used in clinical practice, especially for pediatric surgery. However, the toxicity caused by sevoflurane has gained growing attention. Mitochondria play a key role in maintaining cellular metabolism and survival. To maintain the stability of mitochondrial homeostasis, they are constantly going through fusion and fission. Also, damaged mitochondria need to be degraded by autophagy, termed as mitophagy. Accumulating evidence proves that sevoflurane exposure in young age could lead to cell toxicity by triggering the mitochondrial pathway of apoptosis, inducing the abnormalities of mitochondrial dynamics and mitophagy. In the present review, we focus on the current understanding of mitochondrial apoptosis, dynamics and mitophagy in cell function, the implications for cell toxicity in response to sevoflurane, and their underlying potential mechanisms.
  42. J Immunol. 2021 Mar 17. pii: ji2001016. [Epub ahead of print]
      Caseinolytic mitochondrial matrix peptidase proteolytic subunit (CLPP) is a serine protease that degrades damaged or misfolded mitochondrial proteins. CLPP-null mice exhibit growth retardation, deafness, and sterility, resembling human Perrault syndrome, but also display immune system alterations. However, the molecular mechanisms and signaling pathways underlying immunological changes in CLPP-null mice remain unclear. In this study, we report the steady-state activation of type I IFN signaling and antiviral gene expression in CLPP-deficient cells and tissues, resulting in marked resistance to RNA and DNA virus infection. Depletion of the cyclic GMP-AMP (cGAS)-stimulator of IFN genes (STING) DNA sensing pathway reduces steady-state IFN-I signaling and abrogates the broad antiviral phenotype of CLPP-null cells. Moreover, we report that CLPP deficiency leads to mitochondrial DNA (mtDNA) instability and packaging alterations. Pharmacological and genetic approaches to deplete mtDNA or inhibit cytosolic release markedly reduce antiviral gene expression, implicating mtDNA stress as the driver of IFN-I signaling in CLPP-null mice. Our work places the cGAS-STING-IFN-I innate immune pathway downstream of CLPP and may have implications for understanding Perrault syndrome and other human diseases involving CLPP dysregulation.
  43. Am J Physiol Gastrointest Liver Physiol. 2021 Mar 17.
      Nonalcoholic fatty liver disease (NAFLD) is typified by accumulating excess liver triacylglycerol, inflammation, and liver dysfunction. This study was aimed to investigate the role of mitochondrial DNA synthesis-induced activation of Absent in melanoma 2 (AIM2) inflammasome and pyroptosis in NAFLD. Mice were raised on a high-fat diet for 24 weeks to establish NAFLD models. F4/80 immunofluorescence was performed to reflect the inflammatory response in the liver of mice. Western blot, ELISA, and immunofluorescence were adopted to determine the expression of AIM2 inflammasome-related proteins and factors. EdU immunofluorescence was applied for the examination of mitochondrial DNA expression and flow cytometry for cell pyroptosis. Agarose gel electrophoresis was used to detect the integrity of extracted mouse mitochondrial DNA (mtDNA). The levels of AIM2 inflammasome-related proteins in the liver and the levels of IL-1β and IL-18 in serum were elevated in high-fat diet-induced NAFLD mice. AIM2 inflammasome activation and pyroptosis were triggered, and suppressed activation of AIM2 inflammasome alleviated the inflammation and pyroptosis in the liver of NAFLD mice. Mitochondria were severely damaged and mtDNA was synthesized after NAFLD modeling. Further, mtDNA treatment could promote palmitate (PA)-induced activation of AIM2 inflammasome and pyroptosis. Moreover, inhibition of IRF1 gene alleviated PA-induced AIM2 inflammasome activation and pyroptosis. In conclusion, mitochondrial DNA synthesis could enable AIM2 inflammasome activation and induce the hepatocyte pyroptosis, thereby exacerbating NAFLD.
    Keywords:  AIM2; Inflammasome; Mitochondrial DNA; Nonalcoholic fatty liver disease; Oxidative stress
  44. Front Pediatr. 2021 ;9 632293
      Hypertrophic cardiomyopathy (HCM) is a myocardial disease characterized by left ventricular hypertrophy not solely explained by abnormal loading conditions. Despite its rare prevalence in pediatric age, HCM carries a relevant risk of mortality and morbidity in both infants and children. Pediatric HCM is a large heterogeneous group of disorders. Other than mutations in sarcomeric genes, which represent the most important cause of HCM in adults, childhood HCM includes a high prevalence of non-sarcomeric causes, including inherited errors of metabolism (i.e., glycogen storage diseases, lysosomal storage diseases, and fatty acid oxidation disorders), malformation syndromes, neuromuscular diseases, and mitochondrial disease, which globally represent up to 35% of children with HCM. The age of presentation and the underlying etiology significantly impact the prognosis of children with HCM. Moreover, in recent years, different targeted approaches for non-sarcomeric etiologies of HCM have emerged. Therefore, the etiological diagnosis is a fundamental step in designing specific management and therapy in these subjects. The present review aims to provide an overview of the non-sarcomeric causes of HCM in children, focusing on the pathophysiology, clinical features, diagnosis, and treatment of these rare disorders.
    Keywords:  children; diagnosis; etiology; hypertrophic cardiomyopathy; treatment
  45. Aging Med (Milton). 2021 Mar;4(1): 47-52
      Objective: Age-associated decreases in immune functions are precipitated by a variety of mechanisms and affect nearly every immune cell subset. In myeloid cells, aging reduces numbers of phagocytes and impairs their functional abilities, including antigen presentation, phagocytosis, and bacterial clearance. Recently, we described an aging effect on several functions in monocytes, including impaired mitochondrial function and reduced inflammatory cytokine gene expression during stimulation with lipopolysaccharide. We hypothesized that circulating factors altered by the aging process underly these changes. Growth differentiation factor-15 (GDF-15) is a distant member of the transforming growth factor-β superfamily that has known anti-inflammatory effects in macrophages and has been shown to be highly differentially expressed during aging.Methods: We used biobanked plasma samples to assay circulating GDF-15 levels in subjects from our previous studies and examined correlations between GDF-15 and monocyte function.
    Results: Monocyte interleukin-6 production due to lipopolysaccharide stimulation was negatively correlated to plasma GDF-15. Additionally, GDF-15 was positively correlated to circulating CD16 + monocyte proportions and negatively correlated to monocyte mitochondrial respiratory capacity.
    Conclusions: These results suggest that GDF-15 is a potential circulating factor affecting a variety of monocyte functions and promoting monocyte immunosenescence and thus may be an attractive candidate for therapeutic intervention to ameliorate this.
    Keywords:  immune function; inflammaging; senescence; senescence‐associated secretory phenotype
  46. Methods Mol Biol. 2021 ;2212 155-168
      Epistasis is the interaction between genes or genetic variants (such as Single Nucleotide Polymorphisms or SNPs) that influences a phenotype or a disease outcome. Statistically and biologically, significant evidence of epistatic loci for several traits and diseases is well known in human, animals, and plants. However, there is no straightforward way to compute a large number of pairwise epistasis among millions of variants along the whole genome, relate them to phenotypes or diseases, and visualize them. The WISH-R package (WISH-R) was developed to address this technology gap to calculate epistatic interactions using a linear or generalized linear model on a genome-wide level using genomic data and phenotype/disease data in a fully parallelized environment, and visualize genome-wide epistasis in many ways. This method protocol chapter provides an easy-to-follow systematic guide to install this R software in computers on Win OS, Mac OS, and Linux platforms and can be downloaded from with a user guide. The WISH-R package has several inbuilt functions to reduce genotype data dimensionality and hence computational demand. WISH-R software can be used to build scale-free weighted SNP interaction networks and relate them to quantitative traits or phenotypes and case-control diseases outcomes. The software leads to integrating biological knowledge to identify disease- or trait-relevant SNP or gene modules, hub genes, potential biomarkers, and pathways related to complex traits and diseases.
    Keywords:  Biomarkers; Computing; Diseases; Epistasis; Phenotype; SNP modules; WISH-R
  47. J Am Soc Nephrol. 2021 Mar 15. pii: ASN.2020121742. [Epub ahead of print]
      Over the last 5 years, single cell methods have enabled the monitoring of gene and protein expression, genetic, and epigenetic changes in thousands of individual cells in a single experiment. With the improved measurement and the decreasing cost of the reactions and sequencing, the size of these datasets is increasing rapidly. The critical bottleneck remains the analysis of the wealth of information generated by single cell experiments. In this review, we give a simplified overview of the analysis pipelines, as they are typically used in the field today. We aim to enable researchers starting out in single cell analysis to gain an overview of challenges and the most commonly used analytical tools. In addition, we hope to empower others to gain an understanding of how typical readouts from single cell datasets are presented in the published literature.
    Keywords:  analysis; kidney; single cell RNA-sequencing; transcriptomics