Dysfunctional mitochondrial translation and combined oxidative phosphorylation deficiency in a mouse model of hepatoencephalopathy due to Gfm1 mutations

2021 ◽  
Vol 36 (1) ◽  
Author(s):  
Miguel Molina‐Berenguer ◽  
Ferran Vila‐Julià ◽  
Sandra Pérez‐Ramos ◽  
Maria Teresa Salcedo‐Allende ◽  
Yolanda Cámara ◽  
...  
Keyword(s):  
Mouse Model ◽  
Oxidative Phosphorylation ◽  
Mitochondrial Translation

scholarly journals Bioenergetic defects in muscle fibers of RYR1 mutant knock-in mice associated with malignant hyperthermia

2020 ◽  
Vol 295 (45) ◽  
pp. 15226-15235 ◽  
Author(s):  
Leon Chang ◽  
Xiaochen Liu ◽  
Christine P. Diggle ◽  
John P. Boyle ◽  
Philip M. Hopkins ◽  
...  
Keyword(s):  
Mouse Model ◽  
Oxidative Phosphorylation ◽  
Malignant Hyperthermia ◽  
Receptor Gene ◽  
Muscle Fibers ◽  
Acid Oxidation ◽  
Mouse Muscle ◽  
Metabolic Defects ◽  
Expression Of Genes ◽  
Consumption Rates

Mutations in the skeletal muscle ryanodine receptor gene (RYR1) can cause susceptibility to malignant hyperthermia (MH), a potentially lethal genetic condition triggered by volatile anesthetics. MH is associated with hypermetabolism, which has directed research interest into oxidative phosphorylation and muscle bioenergetics. The most common cause of MH in the United Kingdom is the c.7300G>A RYR1 variant, which is present in ∼16% of MH families. Our study focuses on the MH susceptible G2435R-RYR1 knock-in mouse model, which is the murine equivalent of the human c.7300G>A genotype. Using a combination of transcriptomics, protein expression, and functional analysis, we investigated adult muscle fiber bioenergetics in this mouse model. RNA-Seq data showed reduced expression of genes associated with mitochondria and fatty acid oxidation in RYR1 mutants when compared with WT controls. Mitochondrial function was assessed by measuring oxygen consumption rates in permeabilized muscle fibers. Comparisons between WT and homozygous G2435R-RYR1 mitochondria showed a significant increase in complex I–facilitated oxidative phosphorylation in mutant muscle. Furthermore, we observed a gene-dose-specific increase in reactive oxygen species production in G2435R-RYR1 muscle fibers. Collectively, these findings provide evidence of metabolic defects in G2435R-RYR1 knock-in mouse muscle under basal conditions. Differences in metabolic profile could be the result of differential gene expression in metabolic pathways, in conjunction with mitochondrial damage accumulated from chronic exposure to increased oxidative stress.

scholarly journals Paradoxical neuronal hyperexcitability in a mouse model of mitochondrial pyruvate import deficiency

2020 ◽  
Author(s):  
Andres De la Rossa ◽  
Marine H. Laporte ◽  
Simone Astori ◽  
Thomas Marissal ◽  
Sylvie Montessuit ◽  
...  
Keyword(s):  
Mouse Model ◽  
Oxidative Phosphorylation ◽  
Calcium Homeostasis ◽  
Ketone Bodies ◽  
Synaptic Activity ◽  
Channel Activity ◽  
Energy Status ◽  
Neuronal Hyperexcitability ◽  
Neuronal Excitation ◽  
Mitochondrial Pyruvate Carrier

AbstractA large number of neuropathologies, including cerebral ischemia and diverse mitochondriopathies, in which neurons experience a deficit in oxidative phosphorylation, and consequently in ATP, are frequently accompanied by severe seizures. This observation is paradoxical given that neuronal excitation imposes a high demand of ATP in neurons. The mechanisms underlying neuronal hyperexcitation in these pathologies remains unclear. Most of the ATP synthesized in neurons derives primarily from pyruvate-mediated oxidative phosphorylation, a process that relies on import of pyruvate into mitochondria occuring exclusively via the mitochondrial pyruvate carrier. To address the question of how neurons can be hyperexcitable with reduced levels of ATP, we generated mice in which the mitochondrial pyruvate carrier was genetically inactivated in adult glutamatergic neurons. We found that, despite decreased levels of oxidative phosphorylation in these excitatory neurons, mice were normal at rest. In response to mild inhibition of GABA mediated synaptic activity they rapidly developed severe seizures and died, whereas under similar conditions the behaviour of control mice remained unchanged. We show that neurons with a deficient mitochondrial pyruvate carrier are intrinsically hyperexcitable as a consequence of impaired calcium homeostasis, which reduces M-type potassium channel activity. Provision of ketone bodies restores energy status, calcium homeostasis and M-channel activity and attenuates seizures in animals fed a ketogenic diet.One Sentence SummaryDecreased OXPHOS and Ca2+-mediated neuronal hyperexcitability lead to seizure in a mouse model of mitochondrial pyruvate import deficiency.

scholarly journals Inhibition of Human Peptide Deformylase Disrupts Mitochondrial Function

2010 ◽  
Vol 30 (21) ◽  
pp. 5099-5109 ◽  
Author(s):  
Sindy Escobar-Alvarez ◽  
Jeffrey Gardner ◽  
Aneesh Sheth ◽  
Giovanni Manfredi ◽  
Guangli Yang ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Oxidative Phosphorylation ◽  
Respiratory Function ◽  
Aerobic Glycolysis ◽  
Peptide Deformylase ◽  
Protein Accumulation ◽  
Human Homolog ◽  
Mitochondrial Translation ◽  
And Control

ABSTRACT Deformylases are metalloproteases in bacteria, plants, and humans that remove the N-formyl-methionine off peptides in vitro. The human homolog of peptide deformylase (HsPDF) resides in the mitochondria, along with its putative formylated substrates; however, the cellular function of HsPDF remains elusive. Here we report on the function of HsPDF in mitochondrial translation and oxidative phosphorylation complex biogenesis. Functional HsPDF appears to be necessary for the accumulation of mitochondrial DNA-encoded proteins and assembly of new respiratory complexes containing these proteins. Consequently, inhibition of HsPDF reduces respiratory function and cellular ATP levels, causing dependence on aerobic glycolysis for cell survival. A series of structurally different HsPDF inhibitors and control peptidase inhibitors confirmed that inhibition of HsPDF decreases mtDNA-encoded protein accumulation. Therefore, HsPDF appears to have a role in maintenance of mitochondrial respiratory function, and this function is analogous to that of chloroplast PDF.

Chronic Inhibition of Mitochondrial Translation Alters Metabolism and Stemness in AML

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1364-1364
Author(s):  
Jhas Bozhena ◽  
Shrivani Sriskanthadevan ◽  
Marko Skrtic ◽  
Mahadeo A. Sukhai ◽  
Veronique Voisin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Oxygen Consumption ◽  
Membrane Potential ◽  
Oxidative Phosphorylation ◽  
Mitochondrial Membrane ◽  
Wild Type ◽  
Mitochondrial Translation ◽  
Mitochondrial Mass ◽  
Cox 2 ◽  
Cox 1

Abstract Abstract 1364 Recently, we demonstrated that the anti-bacterial agent tigecycline preferentially induces death in AML and AML stem cells over normal hematopoietic cells through the inhibition of mitochondrial translation. This heightened sensitivity was due to increased mitochondrial mass and reliance on oxidative metabolism in the AML cells compared to normal hematopoietic cells. Here, we sought to better understand the mechanisms of sensitivity and resistance to inhibitors of mitochondrial translation. To establish cells resistant to tigecycline, we exposed TEX leukemia cells to increasing concentrations of tigecycline over 4 months and selected a population of TEX cells resistant to tigecycline (RTEX+TIG) with an IC50 > 24 μM (versus an IC50 of 2.8 + 0.4 μM in wild type cells). We then profiled oxidative metabolism in the resistant cells. In RTEX+TIG cells, levels of Cox-1 and Cox-2, subunits of respiratory complex IV in the electron transport chain that are translated by mitochondrial ribosomes, were undetectable. In contrast, Cox-4 that is part of the same respiratory chain, but translated in the cytoplasm, was only slightly reduced. RTEX+TIG cells also had undetectable levels of oxygen consumption and increased rates of glycolysis compared to wild type cells. Moreover, RTEX+TIG cells were more sensitive to inhibitors of glycolysis and more resistant to hypoxia, thus demonstrating the functional importance to the change in their metabolic status. RTEX+TIG cells also had reduced mitochondrial membrane potential by 44.4 + 7.2% and reduced mitochondrial mass compared to wild type cells. Morphologically, RTEX+TIG cells had abnormally swollen mitochondria with irregular cristae structures. To understand the molecular basis for the metabolic changes in the RTEX+TIG cells, we performed RNA sequencing of the RTEX+TIG cells and wild type TEX cells. Unbiased analysis, by two independent approaches, of the promoter sequences of transcripts upregulated 1.5-fold or greater in RTEX+TIG cells demonstrated a significant over-representation of binding sites for the hypoxia-inducible factor 1 alpha HIF1α :HIF1β transcription factor complex. Specifically, a subset of HIF1α target genes involved in energy balance and cellular metabolism were coordinately upregulated in RTEX+TIG cells, corresponding with our phenotypic observations of the metabolic state of these cells. We validated the upregulation of HIF1α mRNA and protein by Q-RTPCR and immunoblotting. Strikingly, upon removal of tigecycline from RTEX+TIG cells, the cells re-established aerobic metabolism and oxidative phosphorylation. Levels of Cox-1 and Cox-2, oxygen consumption, glycolysis, mitochondrial mass and mitochondrial membrane potential returned to wild type levels. However, HIF1α remained elevated. Upon re-treatment with tigecycline, the cells remained resistant and the glycolytic phenotype was re-established. TEX cells display features of leukemia stem cells, including differentiation, self-renewal and hierarchical organization. Interestingly, RTEX+TIG cells were more differentiated and had reduced stemness compared to the wild type TEX cells. By immunohistochemistry, RTEX+TIG had increased non-specific esterase activity (NSE). In addition, RTEX+TIG cells had reduced clonogenic growth and ability to engraft immune deficient mice compared to wild type cells. Moreover, RNA sequencing data showed reduced expression of stem cell maintenance genes in RTEX+TIG cells. Depletion of mitochondrial DNA via prolonged exposure of parental cell lines to cationic lipophilic agents such as ethidium bromide produces rho-zero cells that have irreversibly lost mitochondrially translated proteins. These cells lack a functional respiratory chain and cannot derive energy from oxidative phosphorylation. Instead, these cells rely on glycolysis for their energy supply. Here, we have produced a reversible rho-zero like metabolic phenotype by sustained inhibition of mitochondrial translation. This work, therefore, highlights mechanisms of metabolic adaption to inhibition of oxidative phosphorylation. Finally, these data suggest a unique role for metabolism in differentiation and stemness in AML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Recurrent erosion of COA1/MITRAC15 demonstrates gene dispensability in oxidative phosphorylation

2021 ◽  
Author(s):  
Sagar Sharad Shinde ◽  
Sandhya Sharma ◽  
Lokdeep Teekas ◽  
Ashutosh Sharma ◽  
Nagarjun Vijay
Keyword(s):  
Skeletal Muscle ◽  
Oxidative Phosphorylation ◽  
Gene Loss ◽  
Chromosomal Rearrangements ◽  
Genomic Region ◽  
Glycolytic Pathway ◽  
Mitochondrial Translation ◽  
Closely Related Species ◽  
Genome Assemblies

Skeletal muscle fibers rely upon either oxidative phosphorylation or glycolytic pathway to achieve muscular contractions that power mechanical movements. Species with energy-intensive adaptive traits that require sudden bursts of energy have a greater dependency on fibers that use the glycolytic pathway. Glycolytic fibers have decreased reliance on OXPHOS and lower mitochondrial content compared to oxidative fibers. Hence, we hypothesized that adaptive gene loss might have occurred within the OXPHOS pathway in lineages that largely depend on glycolytic fibers. The protein encoded by the COA1/MITRAC15 gene with conserved orthologs found in budding yeast to humans promotes mitochondrial translation. We show that gene disrupting mutations have accumulated within the COA1/MITRAC15 gene in the cheetah, several species of galliforms, and rodents. The genomic region containing COA1/MITRAC15 is a well-established evolutionary breakpoint region in mammals. Careful inspection of genome assemblies of closely related species of rodents and marsupials suggests two independent COA1/MITRAC15 gene loss events co-occurring with chromosomal rearrangements. Besides recurrent gene loss events, we document changes in COA1/MITRAC15 exon structure in primates and felids. The detailed evolutionary history presented in this study reveals the intricate link between skeletal muscle fiber composition and dispensability of the chaperone-like role of the COA1/MITRAC15 gene.

scholarly journals Recurrent erosion of COA1/MITRAC15 exemplifies conditional gene dispensability in oxidative phosphorylation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sagar Sharad Shinde ◽  
Sandhya Sharma ◽  
Lokdeep Teekas ◽  
Ashutosh Sharma ◽  
Nagarjun Vijay
Keyword(s):  
Skeletal Muscle ◽  
Oxidative Phosphorylation ◽  
Gene Loss ◽  
Chromosomal Rearrangements ◽  
Genomic Region ◽  
Mitochondrial Translation ◽  
Closely Related Species ◽  
Genome Assemblies ◽  
Exon Structure

AbstractSkeletal muscle fibers rely upon either oxidative phosphorylation or the glycolytic pathway with much less reliance on oxidative phosphorylation to achieve muscular contractions that power mechanical movements. Species with energy-intensive adaptive traits that require sudden bursts of energy have a greater dependency on glycolytic fibers. Glycolytic fibers have decreased reliance on OXPHOS and lower mitochondrial content compared to oxidative fibers. Hence, we hypothesized that gene loss might have occurred within the OXPHOS pathway in lineages that largely depend on glycolytic fibers. The protein encoded by the COA1/MITRAC15 gene with conserved orthologs found in budding yeast to humans promotes mitochondrial translation. We show that gene disrupting mutations have accumulated within the COA1 gene in the cheetah, several species of galliform birds, and rodents. The genomic region containing COA1 is a well-established evolutionary breakpoint region in mammals. Careful inspection of genome assemblies of closely related species of rodents and marsupials suggests two independent COA1 gene loss events co-occurring with chromosomal rearrangements. Besides recurrent gene loss events, we document changes in COA1 exon structure in primates and felids. The detailed evolutionary history presented in this study reveals the intricate link between skeletal muscle fiber composition and the occasional dispensability of the chaperone-like role of the COA1 gene.

scholarly journals Inhibition of Oxidative Phosphorylation Reverses Bone Marrow Hypoxia Visualized in Imageable Syngeneic B-ALL Mouse Model

2020 ◽  
Vol 10 ◽  
Author(s):  
Mateusz Rytelewski ◽  
Karine Harutyunyan ◽  
Natalia Baran ◽  
Saradhi Mallampati ◽  
M. Anna Zal ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Oxidative Phosphorylation

scholarly journals An RMND1 Mutation Causes Encephalopathy Associated with Multiple Oxidative Phosphorylation Complex Deficiencies and a Mitochondrial Translation Defect

2012 ◽  
Vol 91 (4) ◽  
pp. 737-743 ◽  
Author(s):  
Alexandre Janer ◽  
Hana Antonicka ◽  
Emilie Lalonde ◽  
Tamiko Nishimura ◽  
Florin Sasarman ◽  
...  
Keyword(s):  
Oxidative Phosphorylation ◽  
Mitochondrial Translation

scholarly journals Inhibition of Mitochondrial Translation By the Marine Natural Product Elatol Shows Potent Antileukemia Activity

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4342-4342
Author(s):  
Priyanka Maiti ◽  
Tyler A. Cunningham ◽  
Antonio Barrientos ◽  
Jonathan H. Schatz
Keyword(s):  
Stem Cells ◽  
Protein Synthesis ◽  
Oxidative Phosphorylation ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Hematologic Malignancies ◽  
Myelogenous Leukemia ◽  
Mitochondrial Translation ◽  
Signaling Inhibitors

Abstract Targeted signaling inhibitors for hematologic malignancies may lead to limited clinical efficacy due to the outgrowth of subpopulations with alternative pathways independent of the drug target. Relapse/refractory disease that results from treatment with targeted signaling inhibitors is a major hurdle in obtaining curative responses. Interestingly, work over the past decade or more has shown that chronic myelogenous leukemia (CML) stem cells (CD34+CD38-) are resistant to targeted signaling inhibitors, such as the BCR-ABL kinase class of inhibitors, often a problematic source of resistance leading to residual disease that may precipitate later progression (Hamilton et al., 2012). Recent studies have shown that some forms of lymphoma and leukemia cell have an energy metabolism highly dependent on mitochondrial oxidative phosphorylation (Ashton et al., 2018). Tigecycline, a US FDA approved antibiotic, has been shown to inhibit synthesis of mitochondrion-encoded proteins due to the similarity of bacterial and mitochondrial ribosomes, leading to selective lethality in hematologic malignancies reliant on enhanced oxidative phosphorylation (Norberg et al., 2017). Indeed, it was established that CML stem cells are reliant on upregulated oxidative phosphorylation, and combination treatment with the tyrosine-kinase inhibitor (TKI) imatinib and tigecycline eradicated therapy-resistant CML, both in vitro and in animal models (Kuntz et al., 2017). We have previously reported that elatol, the major compound from the red alga Laurencia microcladia, is effective against several non-Hodgkin lymphomas and primary chronic myelogenous leukemia cells (Peters et al., 2018). In vitro studies showed that elatol inhibits eIF4A1 helicase activity, suppressing cytoplasmic cap-dependent translation initiation. Further assessments using 35-S-methionine incorporation in HEK293T cells with or without single-digit micromolar concentrations of elatol for short time periods revealed strong downregulation of mitochondrion-encoded proteins as in Figure 1, (with no effect on mitochondrial transcription). This was confirmed in CML and acute lymphoblastic leukemia (ALL) cell lines whose 24-hour elatol LD50 ranged from high nanomolar to low micromolar concentrations. This potency was 10-40x higher than for tigecycline in side-by-side comparisons across several leukemia cell lines when compared at 72h. Additionally, we established that elatol does not affect integrity of small and large mitochondrial ribosomal units through sedimentation property analysis using sucrose gradients. Although the specific target on the mitochondrial translation apparatus remains elusive, we have uncovered that its mechanism of action differs from that of chloramphenicol, which inhibits translation elongation. In summary, we have performed proof-of-concept studies using HEK293T and HeLa cell lines, isolated mitochondria from HEK293T, and CML and ALL cell lines to reveal that elatol is a potent inhibitor of mitochondrial protein synthesis at concentrations that do not affect cytoplasmic protein synthesis and that this mechanism differs from chloramphenicol. Tigecycline's compelling preclinical data in combination with TKI informed design of a pending clinical trial (NCT02883036). Elatol's greatly improved potency provide a potential starting point for further optimization of this paradigm. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals The FASTK family proteins fine-tune mitochondrial RNA processing

PLoS Genetics ◽  
2021 ◽  
Vol 17 (11) ◽  
pp. e1009873
Author(s):  
Akira Ohkubo ◽  
Lindsey Van Haute ◽  
Danielle L. Rudler ◽  
Maike Stentenbach ◽  
Florian A. Steiner ◽  
...  
Keyword(s):  
Oxidative Phosphorylation ◽  
Cell Lines ◽  
Rna Processing ◽  
Family Members ◽  
Mitochondrial Rna ◽  
Mitochondrial Translation ◽  
Double Knockout ◽  
Chain Complexes ◽  
Transport Chain ◽  
Polycistronic Transcripts

Transcription of the human mitochondrial genome and correct processing of the two long polycistronic transcripts are crucial for oxidative phosphorylation. According to the tRNA punctuation model, nucleolytic processing of these large precursor transcripts occurs mainly through the excision of the tRNAs that flank most rRNAs and mRNAs. However, some mRNAs are not punctuated by tRNAs, and it remains largely unknown how these non-canonical junctions are resolved. The FASTK family proteins are emerging as key players in non-canonical RNA processing. Here, we have generated human cell lines carrying single or combined knockouts of several FASTK family members to investigate their roles in non-canonical RNA processing. The most striking phenotypes were obtained with loss of FASTKD4 and FASTKD5 and with their combined double knockout. Comprehensive mitochondrial transcriptome analyses of these cell lines revealed a defect in processing at several canonical and non-canonical RNA junctions, accompanied by an increase in specific antisense transcripts. Loss of FASTKD5 led to the most severe phenotype with marked defects in mitochondrial translation of key components of the electron transport chain complexes and in oxidative phosphorylation. We reveal that the FASTK protein family members are crucial regulators of non-canonical junction and non-coding mitochondrial RNA processing.

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