bims-mikwok Biomed News
on Mitochondrial quality control
Issue of 2022‒10‒16
nineteen papers selected by
Avinash N. Mukkala
University of Toronto
  1. Rapid fractionation of mitochondria from mouse liver and heart reveals in vivo metabolite compartmentation.
  2. Does Disruption of Optic Atrophy-1 (OPA1) Contribute to Cell Death in HL-1 Cardiomyocytes Subjected to Lethal Ischemia-Reperfusion Injury?
  3. BNIP3L/NIX regulates both mitophagy and pexophagy.
  4. Neuronal induction of BNIP3-mediated mitophagy slows systemic aging in Drosophila.
  5. Mitochondrial network expansion and dynamic redistribution during islet morphogenesis in zebrafish larvae.
  6. Mitochondrial network configuration influences sarcomere and myosin filament structure in striated muscles.
  7. NSP4 and ORF9b of SARS-CoV-2 Induce Pro-Inflammatory Mitochondrial DNA Release in Inner Membrane-Derived Vesicles.
  8. Knockdown of TMEM160 leads to an increase in reactive oxygen species generation and the induction of the mitochondrial unfolded protein response.
  9. Cyclophilin D knockout mice do not accumulate succinate during cardiac ischemia.
  10. Lipid and protein content profiling of isolated native autophagic vesicles.
  11. Universal dynamics of mitochondrial networks: a finite-size scaling analysis.
  12. Evaluation of Mitochondrial Turnover Using Fluorescence Microscopy in Drosophila.
  13. Lipid droplet turnover at the lysosome inhibits growth of hepatocellular carcinoma in a BNIP3-dependent manner.
  14. Evaluation of 10°C as the optimal storage temperature for aspiration-injured donor lungs in a large animal transplant model.
  15. Impaired TFEB activation and mitophagy as a cause of PPP3/calcineurin inhibitor-induced pancreatic β-cell dysfunction.
  16. Two type I topoisomerases maintain DNA topology in human mitochondria.
  17. Mitochondrial Unfolded Protein Response in Ischemia-Reperfusion Injury.
  18. INF2-mediated actin filament reorganization confers intrinsic resilience to neuronal ischemic injury.
  19. Precise and Simultaneous Quantification of Mitochondrial DNA Heteroplasmy and Copy Number by Digital PCR.
  1. FEBS Lett. 2022 Oct 11.
    Rapid fractionation of mitochondria from mouse liver and heart reveals in vivo metabolite compartmentation.
    Fay M Allen, Ana S H Costa, Anja V Gruszczyk, Georgina R Bates, Hiran A Prag, Efterpi Nikitopoulou, Carlo Viscomi, Christian Frezza, Andrew M James, Michael P Murphy.
      The compartmentation and distribution of metabolites between mitochondria and the rest of the cell is a key parameter of cell signalling and pathology. Here, we have developed a rapid fractionation procedure that enables us to take mouse heart and liver from in vivo and within ~ 30 seconds stabilise the distribution of metabolites between mitochondria and the cytosol by rapid cooling, homogenisation and dilution. This is followed by centrifugation of mitochondria through an oil layer to separate mitochondrial and cytosolic fractions for subsequent metabolic analysis. Using this procedure revealed the in vivo compartmentation of mitochondrial metabolites and will enable assessment of the distribution of metabolites between the cytosol and mitochondria during a range of situations in vivo.
    Keywords:  compartmentation; in vivo; ischemia; metabolites; mitochondria; rapid fractionation
    DOI:  https://doi.org/10.1002/1873-3468.14511
  2. Cells. 2022 Sep 30. pii: 3083. [Epub ahead of print]11(19):
    Does Disruption of Optic Atrophy-1 (OPA1) Contribute to Cell Death in HL-1 Cardiomyocytes Subjected to Lethal Ischemia-Reperfusion Injury?
    Andrew R Kulek, Vishnu V R Undyala, Anthony R Anzell, Sarita Raghunayakula, Lee Ann MacMillan-Crow, Thomas H Sanderson, Karin Przyklenk.
      Disruption of mitochondrial structure/function is well-recognized to be a determinant of cell death in cardiomyocytes subjected to lethal episodes of ischemia-reperfusion (IR). However, the precise mitochondrial event(s) that precipitate lethal IR injury remain incompletely resolved. Using the in vitro HL-1 cardiomyocyte model, our aims were to establish whether: (1) proteolytic processing of optic atrophy protein-1 (OPA1), the inner mitochondrial membrane protein responsible for maintaining cristae junction integrity, plays a causal, mechanistic role in determining cardiomyocyte fate in cells subjected to lethal IR injury; and (2) preservation of OPA1 may contribute to the well-documented cardioprotection achieved with ischemic preconditioning (IPC) and remote ischemic conditioning. We report that HL-1 cells subjected to 2.5 h of simulated ischemia displayed increased activity of OMA1 (the metalloprotease responsible for proteolytic processing of OPA1) during the initial 45 min following reoxygenation. This was accompanied by processing of mitochondrial OPA1 (i.e., cleavage to yield short-OPA1 peptides) and release of short-OPA1 into the cytosol. However, siRNA-mediated knockdown of OPA1 content did not exacerbate lethal IR injury, and did not attenuate the cardioprotection seen with IPC and a remote preconditioning stimulus, achieved by transfer of 'reperfusate' medium (TRM-IPC) in this cell culture model. Taken together, our results do not support the concept that maintenance of OPA1 integrity plays a mechanistic role in determining cell fate in the HL-1 cardiomyocyte model of lethal IR injury, or that preservation of OPA1 underlies the cardioprotection seen with ischemic conditioning.
    Keywords:  OMA1 cristae remodeling; OPA1; apoptosis; cardiomyocyte; ischemia-reperfusion injury; ischemic preconditioning; mitochondrial morphosis; remote ischemic conditioning
    DOI:  https://doi.org/10.3390/cells11193083
  3. EMBO J. 2022 Oct 10. e111115
    BNIP3L/NIX regulates both mitophagy and pexophagy.
    Léa P Wilhelm, Juan Zapata-Muñoz, Beatriz Villarejo-Zori, Stephanie Pellegrin, Catarina Martins Freire, Ashley M Toye, Patricia Boya, Ian G Ganley.
      Mitochondria and peroxisomes are closely related metabolic organelles, both in terms of origin and in terms of function. Mitochondria and peroxisomes can also be turned over by autophagy, in processes termed mitophagy and pexophagy, respectively. However, despite their close relationship, it is not known if both organelles are turned over under similar conditions, and if so, how this might be coordinated molecularly. Here, we find that multiple selective autophagy pathways are activated upon iron chelation and show that mitophagy and pexophagy occur in a BNIP3L/NIX-dependent manner. We reveal that the outer mitochondrial membrane-anchored NIX protein, previously described as a mitophagy receptor, also independently localises to peroxisomes and drives pexophagy. We show this process happens in vivo, with mouse tissue that lacks NIX having a higher peroxisomal content. We further show that pexophagy is stimulated under the same physiological conditions that activate mitophagy, including cardiomyocyte and erythrocyte differentiation. Taken together, our work uncovers a dual role for NIX, not only in mitophagy but also in pexophagy, thus illustrating the interconnection between selective autophagy pathways.
    Keywords:  autophagy; mitochondria; mitophagy; peroxisomes; pexophagy
    DOI:  https://doi.org/10.15252/embj.2022111115
  4. Nat Aging. 2022 Jun;2(6): 494-507
    Neuronal induction of BNIP3-mediated mitophagy slows systemic aging in Drosophila.
    Edward T Schmid, Jung-Hoon Pyo, David W Walker.
      The effects of aging on the brain are widespread and can have dramatic implications on the overall health of an organism. Mitochondrial dysfunction is a hallmark of brain aging, but, the interplay between mitochondrial quality control, neuronal aging, and organismal health is not well understood. Here, we show that aging leads to a decline in mitochondrial autophagy (mitophagy) in the Drosophila brain with a concomitant increase in mitochondrial content. We find that induction of BCL2-interacting protein 3 (BNIP3), a mitochondrial outer membrane protein, in the adult nervous system induces mitophagy and prevents the accumulation of dysfunctional mitochondria in the aged brain. Importantly, neuronal induction of BNIP3-mediated mitophagy increases organismal longevity and healthspan. Furthermore, BNIP3-mediated mitophagy in the nervous system improves muscle and intestinal homeostasis in aged flies, indicating cell non-autonomous effects. Our findings identify BNIP3 as a therapeutic target to counteract brain aging and prolong overall organismal health with age.
    Keywords:  Autophagy; Intestinal barrier dysfunction; Intestinal stem cell; Mito-QC; Mitophagy; Muscle aging; Neuronal aging
    DOI:  https://doi.org/10.1038/s43587-022-00214-y
  5. FEBS Lett. 2022 Oct 10.
    Mitochondrial network expansion and dynamic redistribution during islet morphogenesis in zebrafish larvae.
    Julia Freudenblum, Dirk Meyer, Robin A Kimmel.
      Mitochondria, organelles critical for energy production, modify their shape and location in response to developmental state and metabolic demands. Mitochondria are altered in diabetes, but the mechanistic basis is poorly defined, due to difficulties in assessing mitochondria within an intact organism. Here, we use in vivo imaging in transparent zebrafish larvae to demonstrate filamentous, interconnected mitochondrial networks within islet cells. Mitochondrial movements highly resemble what has been reported for human islet cells in vitro, showing conservation in behavior across species and cellular context. During islet development, mitochondrial content increases with emergence of cell motility, and mitochondria disperse within fine protrusions. Overall, this work presents quantitative analysis of mitochondria within their native environment and provides insights into mitochondrial behavior during organogenesis.
    Keywords:  Mitochondria; image analysis; in vivo imaging; islet; pancreas; zebrafish
    DOI:  https://doi.org/10.1002/1873-3468.14508
  6. Nat Commun. 2022 Oct 13. 13(1): 6058
    Mitochondrial network configuration influences sarcomere and myosin filament structure in striated muscles.
    Prasanna Katti, Alexander S Hall, Hailey A Parry, Peter T Ajayi, Yuho Kim, T Bradley Willingham, Christopher K E Bleck, Han Wen, Brian Glancy.
      Sustained muscle contraction occurs through interactions between actin and myosin filaments within sarcomeres and requires a constant supply of adenosine triphosphate (ATP) from nearby mitochondria. However, it remains unclear how different physical configurations between sarcomeres and mitochondria alter the energetic support for contractile function. Here, we show that sarcomere cross-sectional area (CSA) varies along its length in a cell type-dependent manner where the reduction in Z-disk CSA relative to the sarcomere center is closely coordinated with mitochondrial network configuration in flies, mice, and humans. Further, we find myosin filaments near the sarcomere periphery are curved relative to interior filaments with greater curvature for filaments near mitochondria compared to sarcoplasmic reticulum. Finally, we demonstrate variable myosin filament lattice spacing between filament ends and filament centers in a cell type-dependent manner. These data suggest both sarcomere structure and myofilament interactions are influenced by the location and orientation of mitochondria within muscle cells.
    DOI:  https://doi.org/10.1038/s41467-022-33678-y
  7. Cells. 2022 Sep 23. pii: 2969. [Epub ahead of print]11(19):
    NSP4 and ORF9b of SARS-CoV-2 Induce Pro-Inflammatory Mitochondrial DNA Release in Inner Membrane-Derived Vesicles.
    Md Imam Faizan, Rituparna Chaudhuri, Shakti Sagar, Sarah Albogami, Nisha Chaudhary, Iqbal Azmi, Areej Akhtar, Syed Mansoor Ali, Rohit Kumar, Jawed Iqbal, Mohan C Joshi, Gaurav Kharya, Pankaj Seth, Soumya Sinha Roy, Tanveer Ahmad.
      Circulating cell-free mitochondrial DNA (cf-mtDNA) has been found in the plasma of severely ill COVID-19 patients and is now known as a strong predictor of mortality. However, the underlying mechanism of mtDNA release is unexplored. Here, we show a novel mechanism of SARS-CoV-2-mediated pro-inflammatory/pro-apoptotic mtDNA release and a rational therapeutic stem cell-based approach to mitigate these effects. We systematically screened the effects of 29 SARS-CoV-2 proteins on mitochondrial damage and cell death and found that NSP4 and ORF9b caused extensive mitochondrial structural changes, outer membrane macropore formation, and the release of inner membrane vesicles loaded with mtDNA. The macropore-forming ability of NSP4 was mediated through its interaction with BCL2 antagonist/killer (BAK), whereas ORF9b was found to inhibit the anti-apoptotic member of the BCL2 family protein myeloid cell leukemia-1 (MCL1) and induce inner membrane vesicle formation containing mtDNA. Knockdown of BAK and/or overexpression of MCL1 significantly reversed SARS-CoV-2-mediated mitochondrial damage. Therapeutically, we engineered human mesenchymal stem cells (MSCs) with a simultaneous knockdown of BAK and overexpression of MCL1 (MSCshBAK+MCL1) and named these cells IMAT-MSCs (intercellular mitochondrial transfer-assisted therapeutic MSCs). Upon co-culture with SARS-CoV-2-infected or NSP4/ORF9b-transduced airway epithelial cells, IMAT-MSCs displayed functional intercellular mitochondrial transfer (IMT) via tunneling nanotubes (TNTs). The mitochondrial donation by IMAT-MSCs attenuated the pro-inflammatory and pro-apoptotic mtDNA release from co-cultured epithelial cells. Our findings thus provide a new mechanistic basis for SARS-CoV-2-induced cell death and a novel therapeutic approach to engineering MSCs for the treatment of COVID-19.
    Keywords:  BCL2 antagonist/killer; NSP4; ORF9b; SARS-CoV-2; intercellular mitochondrial transfer; mitochondrial DNA; myeloid cell leukemia-1; tunneling nanotubes
    DOI:  https://doi.org/10.3390/cells11192969
  8. FEBS Open Bio. 2022 Oct 10.
    Knockdown of TMEM160 leads to an increase in reactive oxygen species generation and the induction of the mitochondrial unfolded protein response.
    Kosei Yamashita, Misa Haraguchi, Masato Yano.
      Transmembrane protein 160 (TMEM160) was recently reported to be localized to the mitochondrial inner membrane, but mitochondrial function was noted to be unaffected by loss of TMEM160. In contrast to these previously published findings, we report here that the absence of TMEM160 influences intracellular responses. After confirming that TMEM160 is localized in the inner mitochondrial membrane, we knocked down TMEM160 in human cultured cells and analyzed the changes in cellular responses. TMEM160 depletion led to an upregulation of the mitochondrial chaperone HSPD1, suggesting that depletion induced the mitochondrial unfolded protein response (UPRmt ). Indeed, the expression of key transcription factors that induce the UPRmt (ATF4, ATF5, and DDIT3) was increased following TMEM160 depletion. Expression of the mitochondrial protein import-receptors TOMM22 and TOMM20 was also enhanced. In addition, we observed a significant increase in reactive oxygen species (ROS) generation following TMEM160 depletion. Glutathione S-transferases, which detoxify the products of oxidative stress, were also upregulated in TMEM160-depleted cells. Immunoblot analysis was performed to detect proteins modified by 4-hydroxynonenal (which is released after the peroxidation of lipids by ROS): the expression patterns of 4-hydroxynonenal-modified proteins were altered after TMEM160 depletion, suggesting that depletion enhanced degadation of these proteins. HSPD1, TOMM22, ATF4, ATF5, and DDIT3 remained upregulated after ROS was scavenged by N-acetylcysteine, suggesting that once the UPRmt is induced by TMEM160 depletion, it is not suppresed by the subsequent detoxification of ROS. These findings suggest that TMEM160 may suppress ROS generation and stabilize mitochondrial protein(s).
    Keywords:  TMEM160; mitochondria; mitochondrial unfolded protein response; oxidative stress; reactive oxygen species
    DOI:  https://doi.org/10.1002/2211-5463.13496
  9. J Mol Cell Cardiol. 2022 Oct 06. pii: S0022-2828(22)00531-4. [Epub ahead of print]173 73-74
    Cyclophilin D knockout mice do not accumulate succinate during cardiac ischemia.
    Hiran A Prag, Duvaraka Kula-Alwar, Paolo Bernardi, Fabio Di Lisa, Michael P Murphy, Thomas Krieg.
      
    Keywords:  Cyclophilin D; Cyclosporin A; Ischemia/reperfusion injury; Mitochondrial permeability transition pore; Reactive oxygen species; Succinate
    DOI:  https://doi.org/10.1016/j.yjmcc.2022.09.006
  10. EMBO Rep. 2022 Oct 10. e53065
    Lipid and protein content profiling of isolated native autophagic vesicles.
    Daniel Schmitt, Süleyman Bozkurt, Pascale Henning-Domres, Heike Huesmann, Stefan Eimer, Laura Bindila, Christian Behrends, Emily Boyle, Florian Wilfling, Georg Tascher, Christian Münch, Christian Behl, Andreas Kern.
      Autophagy is responsible for clearance of an extensive portfolio of cargoes, which are sequestered into vesicles, called autophagosomes, and are delivered to lysosomes for degradation. The pathway is highly dynamic and responsive to several stress conditions. However, the phospholipid composition and protein contents of human autophagosomes under changing autophagy rates are elusive so far. Here, we introduce an antibody-based FACS-mediated approach for the isolation of native autophagic vesicles and ensured the quality of the preparations. Employing quantitative lipidomics, we analyze phospholipids present within human autophagic vesicles purified upon basal autophagy, starvation, and proteasome inhibition. Importantly, besides phosphoglycerides, we identify sphingomyelin within autophagic vesicles and show that the phospholipid composition is unaffected by the different conditions. Employing quantitative proteomics, we obtain cargo profiles of autophagic vesicles isolated upon the different treatment paradigms. Interestingly, starvation shows only subtle effects, while proteasome inhibition results in the enhanced presence of ubiquitin-proteasome pathway factors within autophagic vesicles. Thus, here we present a powerful method for the isolation of native autophagic vesicles, which enabled profound phospholipid and cargo analyses.
    Keywords:  autophagic vesicles; autophagy; cargo profiling; lipid profiling; vesicle isolation
    DOI:  https://doi.org/10.15252/embr.202153065
  11. Sci Rep. 2022 Oct 12. 12(1): 17074
    Universal dynamics of mitochondrial networks: a finite-size scaling analysis.
    Nahuel Zamponi, Emiliano Zamponi, Sergio A Cannas, Dante R Chialvo.
      Evidence from models and experiments suggests that the networked structure observed in mitochondria emerges at the critical point of a phase transition controlled by fission and fusion rates. If mitochondria are poised at criticality, the relevant network quantities should scale with the system's size. However, whether or not the expected finite-size effects take place has not been demonstrated yet. Here, we first provide a theoretical framework to interpret the scaling behavior of mitochondrial network quantities by analyzing two conceptually different models of mitochondrial dynamics. Then, we perform a finite-size scaling analysis of real mitochondrial networks extracted from microscopy images and obtain scaling exponents comparable with critical exponents from models and theory. Overall, we provide a universal description of the structural phase transition in mammalian mitochondria.
    DOI:  https://doi.org/10.1038/s41598-022-14946-9
  12. Bio Protoc. 2022 Sep 05. pii: e4498. [Epub ahead of print]12(17):
    Evaluation of Mitochondrial Turnover Using Fluorescence Microscopy in Drosophila.
    Felipe Martelli.
      Mitochondrial dysfunction is associated with perturbations in the cellular oxidative status, changes in energy production and metabolic rate, and the onset of pathological processes. Classic methods of assessing mitochondrial dysfunction rely on indirect measures, such as evaluating mitochondrial DNA copy numbers, or direct but more costly and skilled techniques, such as electron microscopy. The protocol presented here was recently implemented to evaluate mitochondrial dysfunction in response to insecticide exposure in Drosophila melanogaster larvae, and it relies on the use of a previously established MitoTimer mutant strain. MitoTimer is a genetically engineered mitochondrial protein that shows green fluorescence when newly synthetized, irreversibly turning into red as mitochondria age. The protocol described here allows for the easy and direct assessment of shifts in mitochondrial turnover, with tissue-specific accuracy. This protocol can be adapted to assess changes in mitochondrial turnover in response to drugs, rearing conditions, and/or mutations in larva, pupa, or adult fruit flies.
    Keywords:   Dissection ; Drosophila ; Fluorescence microscopy ; Insecticide ; Mitochondria ; Mitochondrial turnover ; Oxidative stress ; Tissue imaging
    DOI:  https://doi.org/10.21769/BioProtoc.4498
  13. Sci Adv. 2022 Oct 14. 8(41): eabo2510
    Lipid droplet turnover at the lysosome inhibits growth of hepatocellular carcinoma in a BNIP3-dependent manner.
    Damian E Berardi, Althea Bock-Hughes, Alexander R Terry, Lauren E Drake, Grazyna Bozek, Kay F Macleod.
      Hepatic steatosis is a major etiological factor in hepatocellular carcinoma (HCC), but factors causing lipid accumulation leading to HCC are not understood. We identify BNIP3 (a mitochondrial cargo receptor) as an HCC suppressor that mitigates against lipid accumulation to attenuate tumor cell growth. Targeted deletion of Bnip3 decreased tumor latency and increased tumor burden in a mouse model of HCC. This was associated with increased lipid in bnip3-/- HCC at early stages of disease, while lipid did not accumulate until later in tumorigenesis in wild-type mice, as Bnip3 expression was attenuated. Low BNIP3 expression in human HCC similarly correlated with increased lipid content and worse prognosis than HCC expressing high BNIP3. BNIP3 suppressed HCC cell growth by promoting lipid droplet turnover at the lysosome in a manner dependent on BNIP3 binding LC3. We have termed this process "mitolipophagy" because it involves the coordinated autophagic degradation of lipid droplets with mitochondria.
    DOI:  https://doi.org/10.1126/sciadv.abo2510
  14. J Heart Lung Transplant. 2022 Sep 08. pii: S1053-2498(22)02112-X. [Epub ahead of print]
    Evaluation of 10°C as the optimal storage temperature for aspiration-injured donor lungs in a large animal transplant model.
    Etienne Abdelnour-Berchtold, Aadil Ali, Cristina Baciu, Erika L Beroncal, Aizhou Wang, Olivia Hough, Mitsuaki Kawashima, Manyin Chen, Yu Zhang, Mingyao Liu, Tom Waddell, Ana C Andreazza, Shaf Keshavjee, Marcelo Cypel.
      BACKGROUND: Our recent work has challenged 4°C as an optimal lung preservation temperature by showing storage at 10°C to allow for the extension of preservation periods. Despite these findings, the impact of 10°C storage has not been evaluated in the setting of injured donor lungs.METHODS: Aspiration injury was created through bronchoscopic delivery of gastric juice (pH: 1.8). Injured donor lungs (n = 5/group) were then procured and blindly randomized to storage at 4°C (on ice) or at 10°C (in a thermoelectric cooler) for 12 hours. A third group included immediate transplantation. A left lung transplant was performed thereafter followed by 4 hours of graft evaluation.
    RESULTS: After transplantation, lungs stored at 10°C showed significantly better oxygenation when compared to 4°C group (343 ± 43 mm Hg vs 128 ± 76 mm Hg, p = 0.03). Active metabolism occurred during the 12 hours storage period at 10°C, producing cytoprotective metabolites within the graft. When compared to lungs undergoing immediate transplant, lungs preserved at 10°C tended to have lower peak airway pressures (p = 0.15) and higher dynamic lung compliances (p = 0.09). Circulating cell-free mitochondrial DNA within the recipient plasma was significantly lower for lungs stored at 10°C in comparison to those underwent immediate transplant (p = 0.048), alongside a tendency of lower levels of tissue apoptotic cell death (p = 0.075).
    CONCLUSIONS: We demonstrate 10°C as a potentially superior storage temperature for injured donor lungs in a pig model when compared to the current clinical standard (4°C) and immediate transplantation. Continuing protective metabolism at 10°C for donor lungs may result in better transplant outcomes.
    Keywords:  inflammasome; ischemia reperfusion injury (IRI); lung transplantation; mitochondrial health; necroptosis; organ preservation
    DOI:  https://doi.org/10.1016/j.healun.2022.08.025
  15. Autophagy. 2022 Oct 10. 1-15
    Impaired TFEB activation and mitophagy as a cause of PPP3/calcineurin inhibitor-induced pancreatic β-cell dysfunction.
    Kihyoun Park, Seong Keun Sonn, Seungwoon Seo, Jinyoung Kim, Kyu Yeon Hur, Goo Taeg Oh, Myung-Shik Lee.
      Macroautophagy/autophagy or mitophagy plays crucial roles in the maintenance of pancreatic β-cell function. PPP3/calcineurin can modulate the activity of TFEB, a master regulator of lysosomal biogenesis and autophagy gene expression, through dephosphorylation. We studied whether PPP3/calcineurin inhibitors can affect the mitophagy of pancreatic β-cells and pancreatic β-cell function employing FK506, an immunosuppressive drug against graft rejection. FK506 suppressed rotenone- or oligomycin+antimycin-A-induced mitophagy measured by Mito-Keima localization in acidic lysosomes or RFP-LC3 puncta colocalized with TOMM20 in INS-1 insulinoma cells. FK506 diminished nuclear translocation of TFEB after treatment with rotenone or oligomycin+antimycin A. Forced TFEB nuclear translocation by a constitutively active TFEB mutant transfection restored impaired mitophagy by FK506, suggesting the role of decreased TFEB nuclear translocation in FK506-mediated mitophagy impairment. Probably due to reduced mitophagy, recovery of mitochondrial potential or quenching of mitochondrial ROS after removal of rotenone or oligomycin+antimycin A was delayed by FK506. Mitochondrial oxygen consumption was reduced by FK506, indicating reduced mitochondrial function by FK506. Likely due to mitochondrial dysfunction, insulin release from INS-1 cells was reduced by FK506 in vitro. FK506 treatment also reduced insulin release and impaired glucose tolerance in vivo, which was associated with decreased mitophagy and mitochondrial COX activity in pancreatic islets. FK506-induced mitochondrial dysfunction and glucose intolerance were ameliorated by an autophagy enhancer activating TFEB. These results suggest that diminished mitophagy and consequent mitochondrial dysfunction of pancreatic β-cells contribute to FK506-induced β-cell dysfunction or glucose intolerance, and autophagy enhancement could be a therapeutic modality against post-transplantation diabetes mellitus caused by PPP3/calcineurin inhibitors.
    Keywords:  Calcineurin; TFEB; mitophagy; pancreatic β-cell; post-transplantation diabetes mellitus
    DOI:  https://doi.org/10.1080/15548627.2022.2132686
  16. Nucleic Acids Res. 2022 Oct 10. pii: gkac857. [Epub ahead of print]
    Two type I topoisomerases maintain DNA topology in human mitochondria.
    Katja E Menger, James Chapman, Héctor Díaz-Maldonado, Mushtaq M Khazeem, Dasha Deen, Direnis Erdinc, John W Casement, Valeria Di Leo, Angela Pyle, Alejandro Rodríguez-Luis, Ian G Cowell, Maria Falkenberg, Caroline A Austin, Thomas J Nicholls.
      Genetic processes require the activity of multiple topoisomerases, essential enzymes that remove topological tension and intermolecular linkages in DNA. We have investigated the subcellular localisation and activity of the six human topoisomerases with a view to understanding the topological maintenance of human mitochondrial DNA. Our results indicate that mitochondria contain two topoisomerases, TOP1MT and TOP3A. Using molecular, genomic and biochemical methods we find that both proteins contribute to mtDNA replication, in addition to the decatenation role of TOP3A, and that TOP1MT is stimulated by mtSSB. Loss of TOP3A or TOP1MT also dysregulates mitochondrial gene expression, and both proteins promote transcription elongation in vitro. We find no evidence for TOP2 localisation to mitochondria, and TOP2B knockout does not affect mtDNA maintenance or expression. Our results suggest a division of labour between TOP3A and TOP1MT in mtDNA topology control that is required for the proper maintenance and expression of human mtDNA.
    DOI:  https://doi.org/10.1093/nar/gkac857
  17. Brain Res. 2022 Oct 06. pii: S0006-8993(22)00340-7. [Epub ahead of print] 148116
    Mitochondrial Unfolded Protein Response in Ischemia-Reperfusion Injury.
    Ming-Xi Zhu, Xiao-Fei Ma, Xing Niu, Gui-Bo Fan, Yan Li.
      Mitochondrial unfolded protein response (UPRmt) is a mitochondrial stress response that activates the transcriptional program of mitochondrial chaperone proteins and proteases to keep protein homeostasis in mitochondria. Ischemia-reperfusion injury results in multiple severe clinical issues linked to high morbidity and mortality in various disorders. The pathophysiology and pathogenesis of ischemia-reperfusion injury are complex and multifactorial. Emerging evidence showed the roles of UPRmt signaling in ischemia-reperfusion injury. Herein, we discuss the regulatory mechanisms underlying UPRmt signaling in C. elegans and mammals. Furthermore, we review the recent studies into the roles and mechanisms of UPRmt signaling in ischemia-reperfusion injury of the heart, brain, kidney, and liver. Further research of UPRmt signaling will potentially develop novel therapeutic strategies against ischemia-reperfusion injury.
    Keywords:  ischemia-reperfusion injury; mitochondrial dysfunction; mitochondrial homeostasis; mitochondrial unfolded protein response
    DOI:  https://doi.org/10.1016/j.brainres.2022.148116
  18. Nat Commun. 2022 Oct 13. 13(1): 6037
    INF2-mediated actin filament reorganization confers intrinsic resilience to neuronal ischemic injury.
    Barbara Calabrese, Steven L Jones, Yoko Shiraishi-Yamaguchi, Michael Lingelbach, Uri Manor, Tatyana M Svitkina, Henry N Higgs, Andy Y Shih, Shelley Halpain.
      During early ischemic brain injury, glutamate receptor hyperactivation mediates neuronal death via osmotic cell swelling. Here we show that ischemia and excess NMDA receptor activation cause actin to rapidly and extensively reorganize within the somatodendritic compartment. Normally, F-actin is concentrated within dendritic spines. However, <5 min after bath-applied NMDA, F-actin depolymerizes within spines and polymerizes into stable filaments within the dendrite shaft and soma. A similar actinification occurs after experimental ischemia in culture, and photothrombotic stroke in mouse. Following transient NMDA incubation, actinification spontaneously reverses. Na+, Cl-, water, and Ca2+ influx, and spine F-actin depolymerization are all necessary, but not individually sufficient, for actinification, but combined they induce activation of the F-actin polymerization factor inverted formin-2 (INF2). Silencing of INF2 renders neurons vulnerable to cell death and INF2 overexpression is protective. Ischemia-induced dendritic actin reorganization is therefore an intrinsic pro-survival response that protects neurons from death induced by cell edema.
    DOI:  https://doi.org/10.1038/s41467-022-33268-y
  19. J Biol Chem. 2022 Oct 06. pii: S0021-9258(22)01018-3. [Epub ahead of print] 102574
    Precise and Simultaneous Quantification of Mitochondrial DNA Heteroplasmy and Copy Number by Digital PCR.
    Wendy K Shoop, Cassandra L Gorsuch, Sandra R Bacman, Carlos T Moraes.
      Mitochondrial DNA (mtDNA) is present in multiple copies and phenotypic consequences of mtDNA mutations depend on the mutant load surpassing a specific threshold. Additionally, changes in mtDNA copy number can impact mitochondrial ATP production, resulting in disease. Therefore, the precise determination of mtDNA heteroplasmy and copy number is crucial to the study of mitochondrial diseases. However, current methods can be imprecise, and quantifying small changes in either heteroplasmy or copy number is challenging. We developed a new approach to measure mtDNA heteroplasmy using a single digital PCR (dPCR) probe. This method is based on the observation that fluorescent-labeled probes in dPCR exhibit different intensities depending on the presence of a single nucleotide change in the sequence bound by the probe. This finding allowed us to precisely and simultaneously determine mtDNA copy number and heteroplasmy levels using duplex dPCR. We tested this approach in two different models (human and mouse), which proved faster and more internally controlled when compared to other published methods routinely used in the mitochondrial genetics field. We believe this approach could be broadly applicable to the detection and quantification of other mixed genetic variations.
    DOI:  https://doi.org/10.1016/j.jbc.2022.102574
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