bims-mikwok Biomed News
on Mitochondrial quality control
Issue of 2020‒08‒23
seven papers selected by
Avinash N. Mukkala
University of Toronto
  1. Placental Mitochondria Therapy for Cerebral Ischemia-Reperfusion Injury in Mice.
  2. The very low number of calcium-induced permeability transition pores in the single mitochondrion.
  3. An optimized procedure for quantitative analysis of mitophagy with the mtKeima system using flow cytometry.
  4. NRF-1 and HIF-1α contribute to modulation of human VDAC1 gene promoter during starvation and hypoxia in HeLa cells.
  5. Phosphorylation of cyclophilin D at serine 191 regulates mitochondrial permeability transition pore opening and cell death after ischemia-reperfusion.
  6. Mitochondrial electron transport chain: Oxidative phosphorylation, oxidant production, and methods of measurement.
  7. Trauma-Hemorrhage Stimulates Immune Defense, Mitochondrial Dysfunction, Autophagy and Apoptosis in Pig Liver at 72 H.
  1. Stroke. 2020 Aug 21. STROKEAHA120030152
    Placental Mitochondria Therapy for Cerebral Ischemia-Reperfusion Injury in Mice.
    Yoshihiko Nakamura, Eng H Lo, Kazuhide Hayakawa.
      BACKGROUND AND PURPOSE: There is an urgent need to develop adjunct therapies that can be added onto reperfusion for acute ischemic stroke. Recently, mitochondrial transplantation has emerged as a promising therapeutic approach for boosting brain tissue protection. In this proof-of-concept study, we investigate the feasibility of using placenta as a source for mitochondrial transplantation in a mouse model of transient focal cerebral ischemia-reperfusion.METHODS: Mitochondria-enriched fractions were isolated from cryopreserved mouse placenta. Mitochondrial purity and JC1 membrane potentials were assessed by flow cytometry. Adenosine triphosphate and mitochondrial proteins were measured by luminescence intensity and western blot, respectively. Therapeutic efficacy of mitochondrial fractions was assessed in a mouse model of transient focal cerebral ischemia-reperfusion.
    RESULTS: Flow cytometry analysis demonstrated that about 87% of placental mitochondria were viable and maintained JC1 membrane potentials after isolation. Placental mitochondrial fractions contained adenosine triphosphate equivalent to mitochondrial fractions isolated from skeletal muscle and brown fat tissue. Normalized mitochondrial antioxidant enzymes (glutathione reductase, MnSOD [manganese superoxide dismutase]) and HSP70 (heat shock protein 70) were highly preserved in placental mitochondrial fractions. Treatment with placental mitochondrial fractions immediately after reperfusion significantly decreased infarction after focal cerebral ischemia in mice.
    CONCLUSIONS: Cryopreserved placenta can be a feasible source for viable mitochondrial isolation. Transplantation with placental mitochondria may amplify beneficial effects of reperfusion in stroke.
    Keywords:  membrane potentials; mitochondrial proteins; placenta; proof of concept study; stroke
    DOI:  https://doi.org/10.1161/STROKEAHA.120.030152
  2. J Gen Physiol. 2020 Oct 05. pii: e202012631. [Epub ahead of print]152(10):
    The very low number of calcium-induced permeability transition pores in the single mitochondrion.
    Maria A Neginskaya, Jasiel O Strubbe, Giuseppe F Amodeo, Benjamin A West, Shoshana Yakar, Jason N Bazil, Evgeny V Pavlov.
      Mitochondrial permeability transition (PT) is a phenomenon of stress-induced increase in nonspecific permeability of the mitochondrial inner membrane that leads to disruption of oxidative phosphorylation and cell death. Quantitative measurement of the membrane permeability increase during PT is critically important for understanding the PT's impact on mitochondrial function. The elementary unit of PT is a PT pore (PTP), a single channel presumably formed by either ATP synthase or adenine nucleotide translocator (ANT). It is not known how many channels are open in a single mitochondrion during PT, which makes it difficult to quantitatively estimate the overall degree of membrane permeability. Here, we used wide-field microscopy to record mitochondrial swelling and quantitatively measure rates of single-mitochondrion volume increase during PT-induced high-amplitude swelling. PT was quantified by calculating the rates of water flux responsible for measured volume changes. The total water flux through the mitochondrial membrane of a single mitochondrion during PT was in the range of (2.5 ± 0.4) × 10-17 kg/s for swelling in 2 mM Ca2+ and (1.1 ± 0.2) × 10-17 kg/s for swelling in 200 µM Ca2+. Under these experimental conditions, a single PTP channel with ionic conductance of 1.5 nS could allow passage of water at the rate of 0.65 × 10-17 kg/s. Thus, we estimate the integral ionic conductance of the whole mitochondrion during PT to be 5.9 ± 0.9 nS for 2 mM concentration of Ca2+ and 2.6 ± 0.4 nS for 200 µM of Ca2+. The number of PTPs per mitochondrion ranged from one to nine. Due to the uncertainties in PTP structure and model parameters, PTP count results may be slightly underestimated. However, taking into account that each mitochondrion has ∼15,000 copies of ATP synthases and ANTs, our data imply that PTP activation is a rare event that occurs only in a small subpopulation of these proteins.
    DOI:  https://doi.org/10.1085/jgp.202012631
  3. Biotechniques. 2020 Aug 18.
    An optimized procedure for quantitative analysis of mitophagy with the mtKeima system using flow cytometry.
    Nathaniel J Winsor, Samuel A Killackey, Dana J Philpott, Stephen E Girardin.
      Mitophagy is the process by which mitochondria are selectively targeted and removed via autophagic machinery to maintain mitochondrial homeostasis in the cell. Recently, flow cytometry-based assays that utilize the fluorescent mtKeima reporter system have allowed for quantitative assessment of mitophagy at a single-cell level. However, clear guidelines for appropriate flow cytometry workflow and downstream analysis are lacking and studies using flow cytometry in mtKeima-expressing cells often display incorrect and arbitrary binary mitophagic or nonmitophagic cutoffs that prevent proper quantitative analyses. In this paper we propose a novel method of mtKeima data analysis that preserves subtle differences present within flow cytometry data in a manner that ensures reproducibility.
    Keywords:  flow cytometry assays; gating strategies; mitophagy; mtKeima; ratio gating
    DOI:  https://doi.org/10.2144/btn-2020-0071
  4. Biochim Biophys Acta Bioenerg. 2020 Aug 15. pii: S0005-2728(20)30139-0. [Epub ahead of print] 148289
    NRF-1 and HIF-1α contribute to modulation of human VDAC1 gene promoter during starvation and hypoxia in HeLa cells.
    Francesca Guarino, Federica Zinghirino, Lia Mela, Xena Giada Pappalardo, François Ichas, Vito De, Angela Messina.
      VDAC (Voltage Dependent Anion Channel) is a family of pore forming protein located in the outer mitochondrial membrane. Its channel property ensures metabolites exchange between mitochondria and the rest of the cell resulting in metabolism and bioenergetics regulation, and in cell death and life switch. VDAC1 is the best characterized and most abundant isoform, and is involved in many pathologies, as cancer or neurodegenerative diseases. However, little information is available about its gene expression regulation in normal and/or pathological conditions. In this work, we explored VDAC1 gene expression regulation in normal conditions and in the contest of some metabolic and energetic mitochondrial dysfunction and cell stress as example. The core of the putative promoter region was characterized in terms of transcription factors responsive elements both by bioinformatic studies and promoter activity experiments. In particular, we found an abundant presence of NRF-1 sites, together with other transcription factors binding sites involved in cell growth, proliferation, development, and we studied their prevalence in gene activity. Furthermore, upon depletion of nutrients or controlled hypoxia, as detected in various pathologies, we found that VDAC1 transcripts levels were significantly increased in a time related manner. VDAC1 promoter activity was also validated by gene reporter assays. According to PCR real-time experiments, it was confirmed that VDAC1 promoter activity is further stimulated when cells are exposed to stress. A bioinformatic survey suggested HIF-1α, besides NRF-1, as a most active TFBS. Their validation was obtained by TFBS mutagenesis and TF overexpression experiments. In conclusion, we experimentally demonstrated the involvement of both NRF-1 and HIF-1α in the regulation of VDAC1 promoter activation at basal level and in some peculiar cell stress conditions.
    Keywords:  VDAC1; expression; hypoxia; mitochondria; regulation; stress
    DOI:  https://doi.org/10.1016/j.bbabio.2020.148289
  5. Cell Death Dis. 2020 Aug 19. 11(8): 661
    Phosphorylation of cyclophilin D at serine 191 regulates mitochondrial permeability transition pore opening and cell death after ischemia-reperfusion.
    Stephen Hurst, Fabrice Gonnot, Maya Dia, Claire Crola Da Silva, Ludovic Gomez, Shey-Shing Sheu.
      The mitochondrial permeability transition pore (mPTP) plays a critical role in the pathogenesis of cardiovascular diseases, including ischemia/reperfusion injury. Although the pore structure is still unresolved, the mechanism through which cyclophilin D (CypD) regulates mPTP opening is the subject of intensive studies. While post-translational modifications of CypD have been shown to modulate pore opening, specific phosphorylation sites of CypD have not yet been identified. We hypothesized here that phosphorylation of CypD on a serine residue controls mPTP opening and subsequent cell death at reperfusion. We combined in silico analysis with in vitro and genetic manipulations to determine potential CypD phosphorylation sites and their effect on mitochondrial function and cell death. Importantly, we developed an in vivo intramyocardial adenoviral strategy to assess the effect of the CypD phosphorylation event on infarct size. Our results show that although CypD can potentially be phosphorylated at multiple serine residues, only the phosphorylation status at S191 directly impacts the ability of CypD to regulate the mPTP. Protein-protein interaction strategies showed that the interaction between CypD and oligomycin sensitivity-conferring protein (OSCP) was reduced by 45% in the phosphoresistant S191A mutant, whereas it was increased by 48% in the phosphomimetic S191E mutant cells. As a result, the phosphoresistant CypD S191A mutant was protected against 18 h starvation whereas cell death was significantly increased in phosphomimetic S191E group, associated with mitochondrial respiration alteration and ROS production. As in vivo proof of concept, in S191A phosphoresistant rescued CypD-KO mice developed significantly smaller infarct as compared to WT whereas infarct size was drastically increased in S191E phosphomimetic rescued mice. We conclude that CypD phosphorylation at S191 residue leads to its binding to OSCP and thus sensitizes mPTP opening for the subsequent cell death.
    DOI:  https://doi.org/10.1038/s41419-020-02864-5
  6. Redox Biol. 2020 Aug 06. pii: S2213-2317(20)30879-X. [Epub ahead of print] 101674
    Mitochondrial electron transport chain: Oxidative phosphorylation, oxidant production, and methods of measurement.
    Deirdre Nolfi-Donegan, Andrea Braganza, Sruti Shiva.
      The mitochondrial electron transport chain utilizes a series of electron transfer reactions to generate cellular ATP through oxidative phosphorylation. A consequence of electron transfer is the generation of reactive oxygen species (ROS), which contributes to both homeostatic signaling as well as oxidative stress during pathology. In this graphical review we provide an overview of oxidative phosphorylation and its inter-relationship with ROS production by the electron transport chain. We also outline traditional and novel translational methodology for assessing mitochondrial energetics in health and disease.
    Keywords:  Electron transport chain; Mitochondria; Mitochondrial reactive oxygen species; Oxidative phosphorylation
    DOI:  https://doi.org/10.1016/j.redox.2020.101674
  7. Shock. 2020 Aug 20.
    Trauma-Hemorrhage Stimulates Immune Defense, Mitochondrial Dysfunction, Autophagy and Apoptosis in Pig Liver at 72 H.
    Yulong Shi, Johannes Greven, Weijun Guo, Peng Luo, Ding Xu, Weikang Wang, Borna Relja, Jan T Vollrath, Eva Miriam Buhl, Klemens Horst, Eftychios Bolierakis, Felix Bläsius, Frank Hildebrand.
      Hepatic dysfunction frequently occurs after trauma-hemorrhage, resulting in severe pathophysiological responses that include leukocyte shifting and self-mediated mechanisms of cells, such as autophagy and apoptosis. This in vivo study aimed to characterize mitochondrial morphology, leukocyte reaction, and the processes of autophagy and apoptosis after TH in a long-term, large animal model.Liver tissue was taken from a porcine polytrauma hemorrhage (TH) model (hemorrhagic shock, blunt chest trauma, tibia fracture, and liver laceration) with an intensive care unit follow-up of 72 h. The ultrastructural changes of the liver tissue after TH were evaluated by transmission electron microscopy (TEM). The leukocyte phenotypes and autophagy and apoptosis pathways were elucidated by immunohistofluorescence (IHF), Western blot, and terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL).In addition to post-traumatic changes in the mitochondrial morphology, the biomarkers of anti-inflammatory macrophages (CD163) and reparative monocytes (CD11R3 and CD16) were upregulated, while the inducible nitric oxide synthase (iNOS) was downregulated after TH. Furthermore, the autophagy-related protein expressions of LC3 and Beclin-1 were upregulated, whereas the protein expression of P62 was downregulated after TH. Costaining showed that the macrophages were LC3 (or Beclin-1) positive and that CD163 was copositive and upregulated. Apoptosis biomarkers (cleaved-caspase-3/caspase-3 and Bcl-2) increased after TH, which is in line with TUNEL results.In conclusion, the observed findings indicate that mitochondrial dysfunction might be one trigger of hepatic autophagy and apoptosis after TH. These processes occur together with the activation of anti-inflammatory leukocytes in liver tissue. Further studies are needed to elucidate the potential therapeutic effects of inhibiting mitochondrial swelling during autophagy or apoptosis.
    DOI:  https://doi.org/10.1097/SHK.0000000000001556
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