bims-mitmed Biomed News
on Mitochondrial medicine
Issue of 2022‒06‒12
twenty-six papers selected by
Dario Brunetti
Fondazione IRCCS Istituto Neurologico


  1. Cell. 2022 May 30. pii: S0092-8674(22)00590-6. [Epub ahead of print]
      The mitochondrial genome encodes 13 components of the oxidative phosphorylation system, and altered mitochondrial transcription drives various human pathologies. A polyadenylated, non-coding RNA molecule known as 7S RNA is transcribed from a region immediately downstream of the light strand promoter in mammalian cells, and its levels change rapidly in response to physiological conditions. Here, we report that 7S RNA has a regulatory function, as it controls levels of mitochondrial transcription both in vitro and in cultured human cells. Using cryo-EM, we show that POLRMT dimerization is induced by interactions with 7S RNA. The resulting POLRMT dimer interface sequesters domains necessary for promoter recognition and unwinding, thereby preventing transcription initiation. We propose that the non-coding 7S RNA molecule is a component of a negative feedback loop that regulates mitochondrial transcription in mammalian cells.
    Keywords:  7S RNA; POLRMT; SUV3; cryo-EM; dimer; mitochondria; mtDNA; mtEXO; non-coding RNA; transcription
    DOI:  https://doi.org/10.1016/j.cell.2022.05.006
  2. Front Cell Dev Biol. 2022 ;10 788516
      In this study, we examine the cause and progression of mitochondrial diseases linked to the loss of mtRNase P, a three-protein complex responsible for processing and cleaving mitochondrial transfer RNAs (tRNA) from their nascent transcripts. When mtRNase P function is missing, mature mitochondrial tRNA levels are decreased, resulting in mitochondrial dysfunction. mtRNase P is composed of Mitochondrial RNase P Protein (MRPP) 1, 2, and 3. MRPP1 and 2 have their own enzymatic activity separate from MRPP3, which is the endonuclease responsible for cleaving tRNA. Human mutations in all subunits cause mitochondrial disease. The loss of mitochondrial function can cause devastating, often multisystemic failures. When mitochondria do not provide enough energy and metabolites, the result can be skeletal muscle weakness, cardiomyopathy, and heart arrhythmias. These symptoms are complex and often difficult to interpret, making disease models useful for diagnosing disease onset and progression. Previously, we identified Drosophila orthologs of each mtRNase P subunit (Roswell/MRPP1, Scully/MRPP2, Mulder/MRPP3) and found that the loss of each subunit causes lethality and decreased mitochondrial tRNA processing in vivo. Here, we use Drosophila to model mtRNase P mitochondrial diseases by reducing the level of each subunit in skeletal and heart muscle using tissue-specific RNAi knockdown. We find that mtRNase P reduction in skeletal muscle decreases adult eclosion and causes reduced muscle mass and function. Adult flies exhibit significant age-progressive locomotor defects. Cardiac-specific mtRNase P knockdowns reduce fly lifespan for Roswell and Scully, but not Mulder. Using intravital imaging, we find that adult hearts have impaired contractility and exhibit substantial arrhythmia. This occurs for roswell and mulder knockdowns, but with little effect for scully. The phenotypes shown here are similar to those exhibited by patients with mitochondrial disease, including disease caused by mutations in MRPP1 and 2. These findings also suggest that skeletal and cardiac deficiencies induced by mtRNase P loss are differentially affected by the three subunits. These differences could have implications for disease progression in skeletal and heart muscle and shed light on how the enzyme complex functions in different tissues.
    Keywords:  MRPP; arrythmia; cardiomyopathy; drosophila; intravital imaging; mitochondrial RNase P; mitochondrial disease; skeletal muscle
    DOI:  https://doi.org/10.3389/fcell.2022.788516
  3. Front Pediatr. 2022 ;10 812408
      Leigh syndrome is a neurodegenerative disorder that presents with fluctuation and stepwise deterioration, such as neurodevelopmental delay and regression, dysarthria, dysphagia, hypotonia, dystonia, tremor, spasticity, epilepsy, and respiratory problems. The syndrome characteristically presents symmetric necrotizing lesions in the basal ganglia, brainstem, cerebellum, thalamus, and spinal cord on cranial magnetic resonance imaging. To date, more than 85 genes are known to be associated with Leigh syndrome. Here, we present a rare case of a child who developed Leigh syndrome due to pathogenic variants of NDUFAF6, which encodes an assembly factor of complex I, a respiratory chain subunit. A targeted next-generation sequencing analysis related to mitochondrial disease revealed a missense variant (NM_152416.4:c.371T > C; p.Ile124Thr) and a frameshift variant (NM_152416.4:c.233_242del; p.Leu78GInfs*10) inherited biparentally. The proband underwent physical therapy and nutrient cocktail therapy, but his physical impairment gradually worsened.
    Keywords:  Leigh syndrome; NDUFAF6; complex I deficiency; mitochondrial disease; neurodegenerative disorder
    DOI:  https://doi.org/10.3389/fped.2022.812408
  4. Front Cell Dev Biol. 2022 ;10 924272
      
    Keywords:  OXPHOS; mitochondria; mitochondrial dysfunction; mitochondrial proteome; mitochondrial quality control; mtDNA
    DOI:  https://doi.org/10.3389/fcell.2022.924272
  5. Int J Mol Sci. 2022 May 25. pii: 5933. [Epub ahead of print]23(11):
      Dysfunctional mitochondria are linked to several neurodegenerative diseases. Metabolic defects, a symptom which can result from dysfunctional mitochondria, are also present in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, the most frequent, dominantly inherited neurodegenerative ataxia worldwide. Mitochondrial dysfunction has been reported for several neurodegenerative disorders and ataxin-3 is known to deubiquitinylate parkin, a key protein required for canonical mitophagy. In this study, we analyzed mitochondrial function and mitophagy in a patient-derived SCA3 cell model. Human fibroblast lines isolated from SCA3 patients were immortalized and characterized. SCA3 patient fibroblasts revealed circular, ring-shaped mitochondria and featured reduced OXPHOS complexes, ATP production and cell viability. We show that wildtype ataxin-3 deubiquitinates VDAC1 (voltage-dependent anion channel 1), a member of the mitochondrial permeability transition pore and a parkin substrate. In SCA3 patients, VDAC1 deubiquitination and parkin recruitment to the depolarized mitochondria is inhibited. Increased p62-linked mitophagy, autophagosome formation and autophagy is observed under disease conditions, which is in line with mitochondrial fission. SCA3 fibroblast lines demonstrated a mitochondrial phenotype and dysregulation of parkin-VDAC1-mediated mitophagy, thereby promoting mitochondrial quality control via alternative pathways.
    Keywords:  Machado–Joseph disease; VDAC1 ubiquitination; ataxin-3; mitochondria dysfunction; spinocerebellar ataxia type 3
    DOI:  https://doi.org/10.3390/ijms23115933
  6. Molecules. 2022 May 29. pii: 3494. [Epub ahead of print]27(11):
      Mitochondrial diseases (MDs) may result from mutations affecting nuclear or mitochondrial genes, encoding mitochondrial proteins, or non-protein-coding mitochondrial RNA. Despite the great variability of affected genes, in the most severe cases, a neuromuscular and neurodegenerative phenotype is observed, and no specific therapy exists for a complete recovery from the disease. The most used treatments are symptomatic and based on the administration of antioxidant cocktails combined with antiepileptic/antipsychotic drugs and supportive therapy for multiorgan involvement. Nevertheless, the real utility of antioxidant cocktail treatments for patients affected by MDs still needs to be scientifically demonstrated. Unfortunately, clinical trials for antioxidant therapies using α-tocopherol, ascorbate, glutathione, riboflavin, niacin, acetyl-carnitine and coenzyme Q have met a limited success. Indeed, it would be expected that the employed antioxidants can only be effective if they are able to target the specific mechanism, i.e., involving the central and peripheral nervous system, responsible for the clinical manifestations of the disease. Noteworthily, very often the phenotypes characterizing MD patients are associated with mutations in proteins whose function does not depend on specific cofactors. Conversely, the administration of the antioxidant cocktails might determine the suppression of endogenous oxidants resulting in deleterious effects on cell viability and/or toxicity for patients. In order to avoid toxicity effects and before administering the antioxidant therapy, it might be useful to ascertain the blood serum levels of antioxidants and cofactors to be administered in MD patients. It would be also worthwhile to check the localization of mutations affecting proteins whose function should depend (less or more directly) on the cofactors to be administered, for estimating the real need and predicting the success of the proposed cofactor/antioxidant-based therapy.
    Keywords:  CRAT deficiency; Leigh syndrome; Leigh-like syndromes; MEGDEL; MELAS; MERRF; SLC25A10 and DIC deficiency; aminoacyl tRNA synthetase; antioxidant cocktails; cofactors; complex I; dietary supplement; encephalomyopathies; mitochondrial carriers; mitochondrial diseases; mitochondrial dysfunction; mitochondrial impairment; peptide-based treatments; phospholipids; type I NADH dehydrogenase; vitamins
    DOI:  https://doi.org/10.3390/molecules27113494
  7. Neurochem Res. 2022 Jun 04.
      Type 2 diabetes (T2DM) is a well known risk factor for Alzheimer's disease. Mitochondria are the center of intracellular energy metabolism and the main source of reactive oxygen species. Mitochondrial dysfunction has been identified as a key factor in diabetes-associated brain alterations contributing to neurodegenerative events. Defective insulin signaling may act in concert with neurodegenerative mechanisms leading to abnormalities in mitochondrial structure and function. Mitochondrial dysfunction triggers neuronal energy exhaustion and oxidative stress, leading to brain neuronal damage and cognitive impairment. The normality of mitochondrial function is basically maintained by mitochondrial quality control mechanisms. In T2DM, defects in the mitochondrial quality control pathway in the brain have been found to lead to mitochondrial dysfunction and cognitive impairment. Here, we discuss the association of mitochondrial dysfunction with T2DM and cognitive impairment. We also review the molecular mechanisms of mitochondrial quality control and impacts of mitochondrial quality control on the progression of cognitive impairment in T2DM.
    Keywords:  Cognitive dysfunction; Diabetes; Mitochondria; Mitochondrial quality control
    DOI:  https://doi.org/10.1007/s11064-022-03631-y
  8. Front Cell Dev Biol. 2022 ;10 892069
      The redox activity of cytochrome c oxidase (COX), the terminal oxidase of the mitochondrial respiratory chain (MRC), depends on the incorporation of iron and copper into its catalytic centers. Many mitochondrial proteins have specific roles for the synthesis and delivery of metal-containing cofactors during COX biogenesis. In addition, a large set of different factors possess other molecular functions as chaperones or translocators that are also necessary for the correct maturation of these complexes. Pathological variants in genes encoding structural MRC subunits and these different assembly factors produce respiratory chain deficiency and lead to mitochondrial disease. COX deficiency in Drosophila melanogaster, induced by downregulated expression of three different assembly factors and one structural subunit, resulted in decreased copper content in the mitochondria accompanied by different degrees of increase in the cytosol. The disturbances in metal homeostasis were not limited only to copper, as some changes in the levels of cytosolic and/or mitochondrial iron, manganase and, especially, zinc were observed in several of the COX-deficient groups. The altered copper and zinc handling in the COX defective models resulted in a transcriptional response decreasing the expression of copper transporters and increasing the expression of metallothioneins. We conclude that COX deficiency is generally responsible for an altered mitochondrial and cellular homeostasis of transition metals, with variations depending on the origin of COX assembly defect.
    Keywords:  copper; cytochrome c oxidase; iron; manganese; metal homeostasis; mitochondrial respiratory chain; zinc
    DOI:  https://doi.org/10.3389/fcell.2022.892069
  9. Neural Regen Res. 2022 Dec;17(12): 2563-2575
      Neuronal disorders are associated with a profound loss of mitochondrial functions caused by various stress conditions, such as oxidative and metabolic stress, protein folding or import defects, and mitochondrial DNA alteration. Cells engage in different coordinated responses to safeguard mitochondrial homeostasis. In this review, we will explore the contribution of mitochondrial stress responses that are activated by the organelle to perceive these dangerous conditions, keep them under control and rescue the physiological condition of nervous cells. In the sections to come, particular attention will be dedicated to analyzing how compensatory mitochondrial hyperfusion, mitophagy, mitochondrial unfolding protein response, and apoptosis impact human neuronal diseases. Finally, we will discuss the relevance of the new concept: the "mito-inflammation", a mitochondria-mediated inflammatory response that is recently found to cover a relevant role in the pathogenesis of diverse inflammatory-related diseases, including neuronal disorders.
    Keywords:  Alzheimer’s disease; Parkinson’s disease; UPR mt; apoptosis; mito-inflammation; mitochondrial dynamics; mitophagy; multiple sclerosis; neurodegeneration
    DOI:  https://doi.org/10.4103/1673-5374.339473
  10. Neurosci Res. 2022 Jun 07. pii: S0168-0102(22)00172-9. [Epub ahead of print]
      BAX is a Bcl-2 family protein acting on apoptosis. It also promotes mitochondrial fusion by interacting with the mitochondrial fusion protein Mitofusin (Mfn1 and Mfn2). Neuronal mitochondria are important for the development and modification of dendritic spines, which are subcellular compartments accommodating excitatory synapses in postsynaptic neurons. The abundance of dendritic mitochondria influences dendritic spine development. Mitochondrial fusion is essential for mitochondrial homeostasis. Here, we show that in the hippocampal neuron of BAX knockout mice, mitochondrial fusion is impaired, leading to decreases in mitochondrial length and total mitochondrial mass in dendrites. Notably, BAX knockout mice also have fewer dendritic spines and less cellular Adenosine 5'triphosphate (ATP) in dendrites. The spine and ATP changes are abolished by restoring mitochondria fusion via overexpressing Mfn1 and Mfn2. These findings indicate that BAX-mediated mitochondrial fusion in neurons is crucial for the development of dendritic spines and the maintenance of cellular ATP levels.
    Keywords:  ATP; Mfn; mitochondria; spine
    DOI:  https://doi.org/10.1016/j.neures.2022.06.002
  11. EMBO Mol Med. 2022 Jun 07. e15851
      Aberrant localization of proteins to mitochondria disturbs mitochondrial function and contributes to the pathogenesis of Huntington's disease (HD). However, the crucial factors and the molecular mechanisms remain elusive. Here, we found that heat shock transcription factor 1 (HSF1) accumulates in the mitochondria of HD cell models, a YAC128 mouse model, and human striatal organoids derived from HD induced pluripotent stem cells (iPSCs). Overexpression of mitochondria-targeting HSF1 (mtHSF1) in the striatum causes neurodegeneration and HD-like behavior in mice. Mechanistically, mtHSF1 facilitates mitochondrial fission by activating dynamin-related protein 1 (Drp1) phosphorylation at S616. Moreover, mtHSF1 suppresses single-stranded DNA-binding protein 1 (SSBP1) oligomer formation, which results in mitochondrial DNA (mtDNA) deletion. The suppression of HSF1 mitochondrial localization by DH1, a unique peptide inhibitor, abolishes HSF1-induced mitochondrial abnormalities and ameliorates deficits in an HD animal model and human striatal organoids. Altogether, our findings describe an unsuspected role of HSF1 in contributing to mitochondrial dysfunction, which may provide a promising therapeutic target for HD.
    Keywords:  Huntington's disease; heat shock transcription factor 1; human striatal organoids; mitochondrial DNA; single-stranded DNA-binding protein 1
    DOI:  https://doi.org/10.15252/emmm.202215851
  12. Neurobiol Dis. 2022 Jun 03. pii: S0969-9961(22)00173-5. [Epub ahead of print] 105781
      INTRODUCTION: Mitochondrial dysfunction is observed in Alzheimer's disease (AD). However, the relationship between functional mitochondrial deficits and AD pathologies is not well established in human subjects.METHODS: Post-mortem human brain tissue from 11 non-demented (ND) and 12 AD subjects was used to examine mitochondrial electron transport chain (ETC) function. Data were analyzed by neuropathology diagnosis and Apolipoprotein E (APOE) genotype. Relationships between AD pathology and mitochondrial function were determined.
    RESULTS: AD subjects had reductions in brain cytochrome oxidase (COX) function and complex II Vmax. APOE ε4 carriers had COX, complex II and III deficits. AD subjects had reduced expression of Complex I-III ETC proteins, no changes were observed in APOE ε4 carriers. No correlation between p-Tau Thr 181 and mitochondrial outcomes was observed, although brains from non-demented subjects demonstrated positive correlations between Aβ concentration and COX Vmax.
    DISCUSSION: These data support a dysregulated relationship between brain mitochondrial function and Aβ pathology in AD.
    Keywords:  Alzheimer's disease; Aβ; Brain; Cytochrome oxidase; Mitochondria
    DOI:  https://doi.org/10.1016/j.nbd.2022.105781
  13. J Cell Sci. 2022 Jun 09. pii: jcs.259924. [Epub ahead of print]
      Peroxisome membrane dynamics and division are essential to adapt the peroxisomal compartment to cellular needs. The peroxisomal membrane protein PEX11β, and the tail-anchored adaptor proteins FIS1 (mitochondrial fission protein 1) and MFF (mitochondrial fission factor), which recruit the fission GTPase DRP1 (dynamin-related protein 1) to both peroxisomes and mitochondria, are key factors of peroxisomal division. The current model suggests MFF is essential for peroxisome division, whereas the role of FIS1 is unclear. Here, we reveal that PEX11β can promote peroxisome division in the absence of MFF in a DRP1- and FIS1-dependent manner. We also demonstrate that MFF permits peroxisome division independent of PEX11β and restores peroxisome morphology in PEX11β-deficient patient cells. Moreover, targeting of PEX11β to mitochondria induces mitochondrial division indicating the potential for PEX11β to modulate mitochondrial dynamics. Our findings suggest the existence of an alternative, MFF-independent pathway in peroxisome division and report a function for FIS1 in peroxisome division.
    Keywords:  FIS1; MFF; Mitochondria; Organelle division; PEX11; Peroxisomes
    DOI:  https://doi.org/10.1242/jcs.259924
  14. Int J Mol Sci. 2022 Jun 04. pii: 6297. [Epub ahead of print]23(11):
      Friedreich's ataxia (FRDA) is a rare genetic disorder caused by mutations in the gene frataxin, encoding for a mitochondrial protein involved in iron handling and in the biogenesis of iron-sulphur clusters, and leading to progressive nervous system damage. Although the overt manifestations of FRDA in the nervous system are mainly observed in the neurons, alterations in non-neuronal cells may also contribute to the pathogenesis of the disease, as recently suggested for other neurodegenerative disorders. In FRDA, the involvement of glial cells can be ascribed to direct effects caused by frataxin loss, eliciting different aberrant mechanisms. Iron accumulation, mitochondria dysfunction, and reactive species overproduction, mechanisms identified as etiopathogenic in neurons in FRDA, can similarly affect glial cells, leading them to assume phenotypes that can concur to and exacerbate neuron loss. Recent findings obtained in FRDA patients and cellular and animal models of the disease have suggested that neuroinflammation can accompany and contribute to the neuropathology. In this review article, we discuss evidence about the involvement of neuroinflammatory-related mechanisms in models of FRDA and provide clues for the modulation of glial-related mechanisms as a possible strategy to improve disease features.
    Keywords:  astrocytes; frataxin; iron; microglia; neurons
    DOI:  https://doi.org/10.3390/ijms23116297
  15. Front Mol Biosci. 2022 ;9 830650
      Friedreich's ataxia (FA) is an inherited multisystemic neuro- and cardio-degenerative disorder. Seventy-four clinical trials are listed for FA (including past and present), but none are considered FDA/EMA-approved therapy. To date, FA therapeutic strategies have focused along two main lines using a single-drug approach: a) increasing frataxin and b) enhancing downstream pathways, including antioxidant levels and mitochondrial function. Our novel strategy employed a combinatorial approach to screen approved compounds to determine if a combination of molecules provided an additive or synergistic benefit to FA cells and/or animal models. Eight single drug molecules were administered to FA fibroblast patient cells: nicotinamide riboside, hemin, betamethasone, resveratrol, epicatechin, histone deacetylase inhibitor 109, methylene blue, and dimethyl fumarate. We measured their individual ability to induce FXN transcription and mitochondrial biogenesis in patient cells. Single-drug testing highlighted that dimethyl fumarate and resveratrol increased these two parameters. In addition, the simultaneous administration of these two drugs was the most effective in terms of FXN mRNA and mitobiogenesis increase. Interestingly, this combination also improved mitochondrial functions and reduced reactive oxygen species in neurons and cardiomyocytes. Behavioral tests in an FA mouse model treated with dimethyl fumarate and resveratrol demonstrated improved rotarod performance. Our data suggest that dimethyl fumarate is effective as a single agent, and the addition of resveratrol provides further benefit in some assays without showing toxicity. Therefore, they could be a valuable combination to counteract FA pathophysiology. Further studies will help fully understand the potential of a combined therapeutic strategy in FA pathophysiology.
    Keywords:  Dimethyl fumarate (DMF); Frataxin (FXN); Friedreich’s Ataxia (FA); Mitochondrial membrane potential (ΔΨm); Reactive Oxygen species (ROS); Resveratrol (Resv)
    DOI:  https://doi.org/10.3389/fmolb.2022.830650
  16. Autophagy. 2022 Jun 05. 1-2
      The protein TRIM5 is under intensive investigation related to its roles in antiviral defense, yet its underlying mechanisms of action remain elusive. In our study, we performed an unbiased identification of TRIM5-interacting partners and found proteins participating in a wide variety of cellular functions. We utilized this proteomics data set to uncover a role for TRIM5 in mitophagy, a mitochondrial quality control system that is impaired in multiple human diseases. Mitochondrial damage triggers the recruitment of TRIM5 to ER-mitochondria contact sites where TRIM5 colocalizes with markers of autophagosome biogenesis. Cells lacking TRIM5 are unable to carry out PRKN-dependent and PRKN-independent mitophagy pathways. TRIM5 knockout cells show reduced mitochondrial function and uncontrolled immune activation in response to mitochondrial damage; phenotypes consistent with a requirement for TRIM5 in mitophagy. Mechanistically, we found that TRIM5 is required for the recruitment of the autophagy initiation machinery to damaged mitochondria, where TRIM5 acts as a scaffold promoting interactions between protein markers of mitochondrial damage and the autophagy initiation machinery.
    Keywords:  APEX2; HIV-1; TRIM5α; autophagy; inflammation; mitochondria; mitophagy; restriction factor; tripartite-motif
    DOI:  https://doi.org/10.1080/15548627.2022.2084863
  17. Front Genome Ed. 2022 ;4 903139
      Friedreich's ataxia (FRDA) is an inherited, multisystemic disorder predominantly caused by GAA hyper expansion in intron 1 of frataxin (FXN) gene. This expansion mutation transcriptionally represses FXN, a mitochondrial protein that is required for iron metabolism and mitochondrial homeostasis, leading to neurodegerative and cardiac dysfunction. Current therapeutic options for FRDA are focused on improving mitochondrial function and increasing frataxin expression through pharmacological interventions but are not effective in delaying or preventing the neurodegeneration in clinical trials. Recent research on in vivo and ex vivo gene therapy methods in FRDA animal and cell models showcase its promise as a one-time therapy for FRDA. In this review, we provide an overview on the current and emerging prospects of gene therapy for FRDA, with specific focus on advantages of CRISPR/Cas9-mediated gene editing of FXN as a viable option to restore endogenous frataxin expression. We also assess the potential of ex vivo gene editing in hematopoietic stem and progenitor cells as a potential autologous transplantation therapeutic option and discuss its advantages in tackling FRDA-specific safety aspects for clinical translation.
    Keywords:  AAV; CRISPR/Cas9 gene editing; Friedreich’s ataxia; gene editing; gene therapy; hematopoietic stem and progenitor cells
    DOI:  https://doi.org/10.3389/fgeed.2022.903139
  18. Mol Psychiatry. 2022 Jun 03.
      Mitochondrial dysfunctions are central players in Alzheimer's disease (AD). In addition, impairments in mitophagy, the process of selective mitochondrial degradation by autophagy leading to a gradual accumulation of defective mitochondria, have also been reported to occur in AD. We provide an updated overview of the recent discoveries and advancements on mitophagic molecular dysfunctions in AD-derived fluids and cells as well as in AD brains. We discuss studies using AD cellular and animal models that have unraveled the contribution of relevant AD-related proteins (Tau, Aβ, APP-derived fragments and APOE) in mitophagy failure. In accordance with the important role of impaired mitophagy in AD, we report on various therapeutic strategies aiming at stimulating mitophagy in AD and we summarize the benefits of these potential therapeutic strategies in human clinical trials.
    DOI:  https://doi.org/10.1038/s41380-022-01631-6
  19. FASEB J. 2022 Jul;36(7): e22391
      Aerobic energy demands have led to the evolution of complex mitochondrial reticula in highly oxidative muscles, but the extent to which metabolic challenges can be met with adaptive changes in physiology of specific mitochondrial fractions remains unresolved. We examined mitochondrial mechanisms supporting adaptive increases in aerobic performance in deer mice (Peromyscus maniculatus) adapted to the hypoxic environment at high altitude. High-altitude and low-altitude mice were born and raised in captivity, and exposed as adults to normoxia or hypobaric hypoxia (12 kPa O2 for 6-8 weeks). Subsarcolemmal and intermyofibrillar mitochondria were isolated from the gastrocnemius, and a comprehensive substrate titration protocol was used to examine mitochondrial physiology and O2  kinetics by high-resolution respirometry and fluorometry. High-altitude mice had greater yield, respiratory capacity for oxidative phosphorylation, and O2 affinity (lower P50 ) of subsarcolemmal mitochondria compared to low-altitude mice across environments, but there were no species difference in these traits in intermyofibrillar mitochondria. High-altitude mice also had greater capacities of complex II relative to complexes I + II and higher succinate dehydrogenase activities in both mitochondrial fractions. Exposure to chronic hypoxia reduced reactive oxygen species (ROS) emission in high-altitude mice but not in low-altitude mice. Our findings suggest that functional changes in subsarcolemmal mitochondria contribute to improving aerobic performance in hypoxia in high-altitude deer mice. Therefore, physiological variation in specific mitochondrial fractions can help overcome the metabolic challenges of life at high altitude.
    Keywords:  bioenergetics; biological evolution; high-altitude hypoxia; muscle mitochondria; reactive oxygen species
    DOI:  https://doi.org/10.1096/fj.202200219R
  20. Exp Cell Res. 2022 Jun 01. pii: S0014-4827(22)00226-9. [Epub ahead of print] 113233
      Mitochondrial cloning is a promising approach to achieve homoplasmic mitochondrial DNA (mtDNA) mutations. We previously developed a microfluidic device that performs single mitochondrion transfer from a mtDNA-intact cell to a mtDNA-less (ρ0) cell by promoting cytoplasmic connection through a microtunnel between them. In the present study, we described a method for generating transmitochondrial cybrids using the microfluidic device. After achieving mitochondrial transfer between HeLa cells and thymidine kinase-deficient ρ0143B cells using the microfluidic device, selective culture was carried out using a pyruvate and uridine (PU)-absent and 5-bromo-2'-deoxyuridine-supplemented culture medium. The resulting cells contained HeLa mtDNA and 143B nuclei, but both 143B mtDNA and HeLa nuclei were absent in these cells. Additionally, these cells showed lower lactate production than parent ρ0143B cells and disappearance of PU auxotrophy for cell growth. These results suggest successful generation of transmitochondrial cybrids using the microfluidic device. Furthermore, we succeeded in selective harvest of generated transmitochondrial cybrids under a PU-supplemented condition by removing unfused ρ0 cells with puromycin-based selection in the microfluidic device.
    Keywords:  Cell fusion; Cybrid; Microfluidic device; Microtunnel; Mitochondrial transfer
    DOI:  https://doi.org/10.1016/j.yexcr.2022.113233
  21. Cell Rep. 2022 Jun 07. pii: S2211-1247(22)00689-1. [Epub ahead of print]39(10): 110912
      To elucidate the function of oxidative phosphorylation (OxPhos) during B cell differentiation, we employ CD23Cre-driven expression of the dominant-negative K320E mutant of the mitochondrial helicase Twinkle (DNT). DNT-expression depletes mitochondrial DNA during B cell maturation, reduces the abundance of respiratory chain protein subunits encoded by mitochondrial DNA, and, consequently, respiratory chain super-complexes in activated B cells. Whereas B cell development in DNT mice is normal, B cell proliferation, germinal centers, class switch to IgG, plasma cell maturation, and T cell-dependent as well as T cell-independent humoral immunity are diminished. DNT expression dampens OxPhos but increases glycolysis in lipopolysaccharide and B cell receptor-activated cells. Lipopolysaccharide-activated DNT-B cells exhibit altered metabolites of glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle and a lower amount of phosphatidic acid. Consequently, mTORC1 activity and BLIMP1 induction are curtailed, whereas HIF1α is stabilized. Hence, mitochondrial DNA controls the metabolism of activated B cells via OxPhos to foster humoral immunity.
    Keywords:  B lymphocyte; CP: Immunology; HIF1; TCA cycle; class switch recombination; germinal center; hypoxia inducible factor 1; mTOR; mammalian target of Rapamycin; mitochondrial DNA; mitochondrial respiration; oxidative phosphorylation; phosphatidic acid; plasma cell
    DOI:  https://doi.org/10.1016/j.celrep.2022.110912
  22. Diabetes Metab Syndr Obes. 2022 ;15 1687-1701
      Background: Mutations in mitochondrial DNA (mtDNA) are associated with type 2 diabetes mellitus (T2DM). In particular, m.A3243G is the most common T2DM-related mtDNA mutation in many families worldwide. However, the clinical features and pathophysiology of m.A3243G-induced T2DM are largely undefined.Methods: Two pedigrees with maternally inherited T2DM were underwent clinical, molecular and biochemical assessments. The mtDNA genes were PCR amplified and sequenced. Mitochondrial adenosine triphosphate (ATP) and reactive oxygen species (ROS) were measured in polymononuclear leukocytes derived from three patients with both the m.A3243G and m.T14502C mutations, three patients with only the m.A3243G mutation and three controls without these mutations. Moreover, GJB2, GJB3 and GJB6 mutations were screened by PCR-Sanger sequencing.
    Results: Members of the two pedigrees manifestated variable clinical phenotypes including diabetes and hearing and vision impairments. The age at onset of T2DM varied from 31 to 66 years, with an average of 41 years. Mutational analysis of mitochondrial genomes indicated the presence of the m.A3243G mutation in both pedigrees. Matrilineal relatives in one of the pedigrees harbored the coexisting of m.A3243G and m.T14502C mutations. Remarkably, the m.T14502C mutation, which causes the substitution of a conserved isoleucine for valine at position 58 in ND6 mRNA, may affect the mitochondrial respiratory chain functions. Biochemical analysis revealed that cell lines bearing both the m.A3243G and m.T14502C mutations exhibited greater reductions in ATP levels and increased ROS production compared with those carrying only the m.A3243G mutation. However, we did not find any mutations in the GJB2, GJB3 and GJB6 genes.
    Conclusion: Our study indicated that mitochondrial diabetes is associated with the tRNALeu(UUR) A3243G and ND6 T14502C mutations.
    Keywords:  ND6; T2DM; m.A3243G; m.T14502C; mt-tRNA; mutations
    DOI:  https://doi.org/10.2147/DMSO.S363978
  23. Am J Physiol Regul Integr Comp Physiol. 2022 Jun 07.
      The peroxisome proliferator activated receptor gamma co-activator 1 alpha (PGC-1α) is central to the regulation of cellular and mitochondrial energy homeostasis in mammals, but its role in other vertebrates remains unclear. Indeed, previous work suggests extensive structural and functional divergence of PGC-1α in teleosts but this remains to be directly tested. Here, we describe the initial characterization of heterozygous PGC-1α mutant zebrafish lines created by CRISPR-Cas9 disruptions of an evolutionarily conserved regulatory region of the PGC-1α proximal promoter. Using qPCR, we confirmed the disruption of PGC-1α gene expression in striated muscle, leading to a simultaneous 4-fold increase in mixed skeletal muscle PGC-1α mRNA levels and an opposite 4-fold downregulation in cardiac muscle. In mixed skeletal muscle, most downstream effector genes were largely unaffected yet two mitochondrial lipid transporters, carnitine palmitoyltransferase 1 and 2, were strongly induced. Conversely, PGC-1α depression in cardiac muscle reduced the expression of several transcriptional regulators (estrogen related receptor alpha, nuclear respiratory factor 1 and PGC-1β) without altering metabolic gene expression. Using high resolution respirometry, we determined that white muscle exhibited increased lipid oxidative capacity with little difference in markers of mitochondrial abundance. Finally, using whole animal intermittent respirometry, we show that mutant fish exhibit a 2-fold higher basal metabolism than their wildtype counterparts. Altogether, this new model confirms a central but complex role for PGC-1α in mediating energy utilization in zebrafish and we propose its use as a valuable tool to explore the intricate regulatory pathways of energy homeostasis in a popular biomedical model.
    Keywords:  CRISPR; Metabolism; co-activator; mitochondria; respirometry
    DOI:  https://doi.org/10.1152/ajpregu.00188.2021