bims-mikwok Biomed News
on Mitochondrial quality control
Issue of 2021‒04‒18
twenty-six papers selected by
Avinash N. Mukkala
University of Toronto

  1. J Cell Biol. 2021 Jun 07. pii: e202006043. [Epub ahead of print]220(6):
      Here, we report that acute reduction in mitochondrial translation fidelity (MTF) causes ubiquitination of the inner mitochondrial membrane (IMM) proteins, including TRAP1 and CPOX, which occurs selectively in mitochondria with a severed outer mitochondrial membrane (OMM). Ubiquitinated IMM recruits the autophagy machinery. Inhibiting autophagy leads to increased accumulation of mitochondria with severed OMM and ubiquitinated IMM. This process occurs downstream of the accumulation of cytochrome c/CPOX in a subset of mitochondria heterogeneously distributed throughout the cell ("mosaic distribution"). Formation of mosaic mitochondria, OMM severing, and IMM ubiquitination require active mitochondrial translation and mitochondrial fission, but not the proapoptotic proteins Bax and Bak. In contrast, in Parkin-overexpressing cells, MTF reduction does not lead to the severing of the OMM or IMM ubiquitination, but it does induce Drp1-independent ubiquitination of the OMM. Furthermore, high-cytochrome c/CPOX mitochondria are preferentially targeted by Parkin, indicating that in the context of reduced MTF, they are mitophagy intermediates regardless of Parkin expression. In sum, Parkin-deficient cells adapt to mitochondrial proteotoxicity through a Drp1-mediated mechanism that involves the severing of the OMM and autophagy targeting ubiquitinated IMM proteins.
  2. Cell Rep. 2021 Apr 13. pii: S2211-1247(21)00261-8. [Epub ahead of print]35(2): 108947
      During mitochondrial fission, key molecular and cellular factors assemble on the outer mitochondrial membrane, where they coordinate to generate constriction. Constriction sites can eventually divide or reverse upon disassembly of the machinery. However, a role for membrane tension in mitochondrial fission, although speculated, has remained undefined. We capture the dynamics of constricting mitochondria in mammalian cells using live-cell structured illumination microscopy (SIM). By analyzing the diameters of tubules that emerge from mitochondria and implementing a fluorescence lifetime-based mitochondrial membrane tension sensor, we discover that mitochondria are indeed under tension. Under perturbations that reduce mitochondrial tension, constrictions initiate at the same rate, but are less likely to divide. We propose a model based on our estimates of mitochondrial membrane tension and bending energy in living cells which accounts for the observed probability distribution for mitochondrial constrictions to divide.
    Keywords:  fluorescence lifetime; fluorescent tension sensor; membrane tension; microtubules; mitochondrial division; mitochondrial dynamics; super-resolution microscopy
  3. Cell Rep. 2021 Apr 13. pii: S2211-1247(21)00297-7. [Epub ahead of print]35(2): 108983
      Preclinical models of ischemia/reperfusion injury (RI) demonstrate the deleterious effects of permeability transition pore complex (PTPC) opening in the first minutes upon revascularization of the occluded vessel. The ATP synthase c subunit (Csub) influences PTPC activity in cells, thus impacting tissue injury. A conserved glycine-rich domain in Csub is classified as critical because, when mutated, it modifies ATP synthase properties, protein interaction with the mitochondrial calcium (Ca2+) uniporter complex, and the conductance of the PTPC. Here, we document the role of a naturally occurring mutation in the Csub-encoding ATP5G1 gene at the G87 position found in two ST-segment elevation myocardial infarction (STEMI) patients and how PTPC opening is related to RI in patients affected by the same disease. We report a link between the expression of ATP5G1G87E and the response to hypoxia/reoxygenation of human cardiomyocytes, which worsen when compared to those expressing the wild-type protein, and a positive correlation between PTPC and RI.
    Keywords:  ATP synthase; PTP; STEMI patients; cardiovascular diseases; glycine-rich domain; ischemia; mitochondria; reperfusion injury; subunit c
  4. Stem Cell Reports. 2021 Apr 03. pii: S2213-6711(21)00152-1. [Epub ahead of print]
      Mitochondrial state changes were shown to be critical for stem cell function. However, variation in the mitochondrial content in stem cells and the implication, if any, on differentiation is poorly understood. Here, using cellular and molecular studies, we show that the planarian pluripotent stem cells (PSCs) have low mitochondrial mass compared with their progenitors. Transplantation experiments provided functional validation that neoblasts with low mitochondrial mass are the true PSCs. Further, the mitochondrial mass correlated with OxPhos and inhibiting the transition to OxPhos dependent metabolism in cultured cells resulted in higher PSCs. In summary, we show that low mitochondrial mass is a hallmark of PSCs in planaria and provide a mechanism to isolate live, functionally active, PSCs from different cell cycle stages (G0/G1 and S, G2/M). Our study demonstrates that the change in mitochondrial metabolism, a feature of PSCs is conserved in planaria and highlights its role in organismal regeneration.
    Keywords:  FACS; differentiation; mitochondria; neoblasts; planaria; pluripotency; stem cells
  5. Int J Biochem Cell Biol. 2021 Apr 09. pii: S1357-2725(21)00060-1. [Epub ahead of print] 105976
      The impairment of the CFTR channel activity, a cAMP-activated chloride (Cl-) channel responsible for cystic fibrosis (CF), has been associated with a variety of mitochondrial alterations such as modified gene expression, impairment in oxidative phosphorylation, increased reactive oxygen species (ROS), and a disbalance in calcium homeostasis. The mechanisms by which these processes occur in CF are not fully understood. Previously, we demonstrated a reduced MTND4 expression and a failure in the mitochondrial complex I (mCx-I) activity in CF cells. Here we hypothesized that the activity of CFTR might modulate the mitochondrial fission/fusion balance, explaining the decreased mCx-I. The mitochondrial morphology and the levels of mitochondrial dynamic proteins MFN1 and DRP1 were analysed in IB3-1 CF cells, and S9 (IB3-1 expressing wt-CFTR), and C38 (IB3-1 expressing a truncated functional CFTR) cells. The mitochondrial morphology of IB3-1 cells compared to S9 and C38 cells showed that the impaired CFTR activity induced a fragmented mitochondrial network with increased rounded mitochondria and shorter branches. Similar results were obtained by using the CFTR pharmacological inhibitors CFTR(inh)-172 and GlyH101 on C38 cells. These morphological changes were accompanied by modifications in the levels of the mitochondrial dynamic proteins MFN1, DRP1, and p(616)-DRP1. IB3-1 CF cells treated with Mdivi-1, an inhibitor of the mitochondrial fission, restored the mCx-I activity to values similar to those seen in S9 and C38 cells. These results suggest that the mitochondrial fission/fusion balance is regulated by the CFTR activity and might be a potential target to treat the impaired mCx-I activity in CF.
    Keywords:  CFTR; Cystic fibrosis; DRP1; MFN1; Mdivi-1; Mitochondrial dynamics
  6. Can J Cardiol. 2021 Apr 07. pii: S0828-282X(21)00174-4. [Epub ahead of print]
      BACKGROUND: Downregulation of claudin-5 in the heart is associated with the end-stage heart failure. However, the underlying mechanism of claudin-5 is unclear. Here we investigated the molecular actions of claudin-5 in perspective of mitochondria in cardiomyocytes to better understand the role of claudin-5 in cardioprotection during ischemia.METHODS AND RESULTS: Claudin-5 was detected in the murine heart tissue and the neonatal rat cardiomyocytes (NRCM). Its protein level was severely decreased after myocardial ischemia/reperfusion (I/R; 30 min/24 h) or hypoxia/reoxygenation (H/R; 24 h/4 h). Claudin-5 was present in the mitochondria of NRCM as determined by confocal microscopy. H/R-induced downregulation of claudin-5 was accompanied by mitochondrial fragmentation. The protein level of mitofusin 2 (Mfn2) was dramatically decreased while the expression of dynamin-related protein (Drp) 1 was significantly increased after H/R. H/R-induced mitochondrial swelling and fission were observed by transmission electron microscope (TEM). Overexpression of claudin-5 by adenoviral infection reversed these structural disintegration of mitochondria. The mitochondria-centered intrinsic pathway of apoptosis triggered by H/R and indicated by the expression of cytochrome c and cleaved caspase 3 in the cytoplasm of NRCMs was also reduced by overexpressing claudin-5. Overexpression of claudin-5 in mouse heart also significantly decreased cleaved caspase 3 expression and the infarct size in ischemic heart with improved systolic function.
    CONCLUSION: We demonstrated for the first time the presence of claudin-5 in the mitochondria in cardiomyocytes and provided the firm evidence for the cardioprotective role of claudin-5 in the preservation of mitochondrial dynamics and cell fate against hypoxia- or ischemia-induced stress.
    Keywords:  Claudin-5; Hypoxia/reoxygenation; Mirochondrial fragmentation; Mitochondrial dynamics; Myocardial ischemia/reperfusion
  7. Mol Immunol. 2021 Apr 07. pii: S0161-5890(21)00115-2. [Epub ahead of print]135 1-11
      Neutrophils play a key role in the innate immunity with their ability to generate and release inflammatory mediators that promote the inflammatory response and consequently restore the hemostasis. As active participants in several steps of the normal inflammatory response, neutrophils are also involved in chronic inflammatory diseases such as asthma, atherosclerosis, and arthritis. Given their dual role in the modulation of inflammation, regulating the inflammatory response of neutrophils has been suggested as an important therapeutic approach by numerous researchers. The neutrophils have a relatively short lifespan, which can be problematic for some in vitro experiments. To address this issue, researchers have used the human monomyelocyte cell line PLB-985 as an in vitro model for exploratory experiments addressing neutrophil-related physiological functions. PLB-985 cells can be differentiated into a neutrophil-like phenotype upon exposure to several agonists, including dimethyl sulfoxide (DMSO). Whether this differentiation of PLB-985 affects important features related to the neutrophil's normal functions (i.e., mitochondrial activity, eicosanoid production) remains elusive, and characterizing these changes will be the focal point of this study. Our results indicate that the differentiation affected the proliferation of PLB-985 cells, without inducing apoptosis. A significant decrease in mitochondrial respiration was observed in differentiated PLB-985 cells. However, the overall mitochondria content was not affected. Immunoblotting with mitochondrial antibodies revealed a strong modulation of the succinate dehydrogenase A, superoxide dismutase 2, ubiquinol-cytochrome c reductase core protein 2 and ATP synthase subunit α in differentiated PLB-985 cells. Finally, eicosanoids (leukotriene B4, 12-hydroxyheptadecatrienoic and 15-hydroxyeicosatetraenoic acids) production was significantly increased in differentiated cells. In summary, our data demonstrate that the differentiation process of PLB-985 cells does not impact their viability despite a reduced respiratory state of the cells. This process is also accompanied by modulation of the inflammatory state of the cell. Of importance, our data suggest that PLB-985 cells could be suitable in vitro candidates to study mitochondrial-related dysfunctions in inflammatory diseases.
    Keywords:  Cell respiration; Cell viability; Eicosanoids; Inflammation; Lipoxygenase; Mitochondria; Netosis
  8. Mitochondrion. 2021 Apr 08. pii: S1567-7249(21)00048-9. [Epub ahead of print]
      Cell-free mitochondrial DNA (cf-mtDNA) is a marker of inflammatory disease and a predictor of mortality, but little is known about cf-mtDNA in relation to psychobiology. A systematic review of the literature reveals that blood cf-mtDNA varies in relation to common real-world stressors including psychopathology, acute psychological stress, and exercise. Moreover, cf-mtDNA is inducible within minutes and exhibits high intra-individual day-to-day variation, highlighting the dynamic regulation of cf-mtDNA levels. We discuss current knowledge on the mechanisms of cf-mtDNA release, its forms of transport ("cell-free" does not mean "membrane-free"), potential physiological functions, putative cellular and neuroendocrine triggers, and factors that may contribute to cf-mtDNA removal from the circulation. A review of in vitro, pre-clinical, and clinical studies shows conflicting results around the dogma that physiological forms of cf-mtDNA are pro-inflammatory, opening the possibility of other physiological functions, including the cell-to-cell transfer of whole mitochondria. Finally, to enhance the reproducibility and biological interpretation of human cf-mtDNA research, we propose guidelines for blood collection, cf-mtDNA isolation, quantification, and reporting standards, which can promote concerted advances by the community. Defining the mechanistic basis for cf-mtDNA signaling is an opportunity to elucidate the role of mitochondria in brain-body interactions and psychopathology.
    Keywords:  cell-free DNA; mitochondria; mtDNA; non-inflammatory effects; psychosocial stress; standard protocol
  9. Mitochondrion. 2021 Apr 08. pii: S1567-7249(21)00050-7. [Epub ahead of print]
      Alzheimer's disease (AD) is the inoperable, incapacitating, neuropsychiatric, and degenerative manifestation that drastically affects human life quality. The current medications target extra-neuronal senile plaques, oxidative stress, neuroinflammation, intraneuronal neurofibrillary tangles, cholinergic deficits, and excitotoxicity. Among novel pathways and targets, bioenergetic and resultant mitochondrial dysfunction has been recognized as essential factors that decide the neuronal fate and consequent neurodegeneration in AD. The crucial attributes of mitochondria, including bioenergesis, signaling, sensing, integrating, and transmitting biological signals contribute to optimum networking of neuronal dynamics and make them indispensable for cell survival. In AD, mitochondrial dysfunction and mitophagy are a preliminary and critical event that aggravates the pathological cascade. Stress is known to promote and exaggerate the neuropathological alteration during neurodegeneration and metabolic impairments, especially in the cortico-limbic system, besides adversely affecting the normal physiology and mitochondrial dynamics. Stress involves the allocation of energy resources for neuronal survival. Chronic and aggravated stress response leads to excessive release of glucocorticoids by activation of the hypothalamic-pituitary-adrenal (HPA) axis. By acting through their receptors, glucocorticoids influence adverse mitochondrial changes and alter mtDNA transcription, mtRNA expression, hippocampal mitochondrial network, and ultimately mitochondrial physiology. Chronic stress also affects mitochondrial dynamics by changing metabolic and neuro-endocrinal signalling, aggravating oxidative stress, provoking inflammatory mediators, altering tropic factors, influencing gene expression, and modifying epigenetic pathways. Thus, exploring chronic stress-induced glucocorticoid dysregulation and resultant bio-behavioral and psychosomatic mitochondrial alterations may be a feasible narrative to investigate and unravel the mysterious pathobiology of AD.
    Keywords:  Alzheimer’s Disorder; Bioenergetics; Glucocorticoids; Mitochondria; Stress; mtDNA
  10. Neurosurgery. 2021 Apr 16. pii: nyab105. [Epub ahead of print]
      BACKGROUND: Peripheral nerve injuries result in muscle denervation and apoptosis of the involved muscle, which subsequently reduces mitochondrial content and causes muscle atrophy. The local injection of mitochondria has been suggested as a useful tool for restoring the function of injured nerves or the brain.OBJECTIVE: To determine outcomes following the administration of isolated mitochondria into denervated muscle after nerve injury that have not been investigated.
    METHODS: Muscle denervation was conducted in a sciatic nerve crushed by a vessel clamp and the denervated gastrocnemius muscle was subjected to 195 μg hamster green fluorescent protein (GFP)-mitochondria intramuscular infusion for 10 min.
    RESULTS: The mitochondria were homogeneously distributed throughout the denervated muscle after intramuscular infusion. The increases in caspase 3, 8-oxo-dG, Bad, Bax, and ratio of Bax/Bcl-2 levels in the denervated muscle were attenuated by mitochondrial infusion, and the downregulation of Bcl-2 expression was prevented by mitochondrial infusion. In addition, the decrease in the expression of desmin and the acetylcholine receptor was counteracted by mitochondrial infusion; this effect paralleled the amount of distributed mitochondria. The restoration of the morphology of injured muscles and nerves was augmented by the local infusion of mitochondria. Mitochondrial infusion also led to improvements in sciatic functional indexes, compound muscle action potential amplitudes, and conduction latencies as well as the parameters of CatWalk (Noldus) gait analysis.
    CONCLUSION: The local infusion of mitochondria can successfully prevent denervated muscle atrophy and augment nerve regeneration by reducing oxidative stress in denervated muscle.
    Keywords:  Denervated muscle; Mitochondria transplantation; Nerve crush injury
  11. Ann Transl Med. 2021 Mar;9(6): 479
      Background: Studies have shown that the ability of the myocardium to tolerate ischemia becomes significantly compromised with age. During ischemia, several endogenous protective signals are activated to protect the heart from injury, among which extracellular-signal regulated kinase (ERK) 1/2 signaling has been established as playing a pivotal role. However, in aging hearts, the activation of ERK1/2 is compromised. Mitogen-activated protein kinase/ERK kinase (MEK) is a major regulator of ERK1/2 signaling. In the present study, we investigated whether transduction of CaMEK, a constitutively activated MEK, using adeno-associated virus serotype 9 (AAV9) could protect the aging heart against ischemia.Methods: Myocardial ischemia models were established in aging mice and senescent cardiomyocytes, and AAV9-mediated delivery of CaMEK was applied. Echocardiography, fluorescent staining, transmission electron microscopy, flow cytometry, and immunoblotting were used to explore the effects of CaMEK and their underlying mechanism.
    Results: AAV9-CaMEK activated ERK1/2 signaling and exerted cardioprotective effects against ischemia in aging hearts. Specifically, CaMEK transduction decreased dynamin-related protein-1 (Drp1) expression and phosphorylation at serine 616, resulting in improved mitochondrial morphology and function in aging ischemic hearts. Furthermore, CaMEK transduction exerted similar protective effects in senescent cardiomyocytes under hypoxia. Meanwhile, with the inhibition of ERK1/2 signaling in senescent cardiomyocytes under hypoxia, the opposite effects were observed, including an increase in mitochondrial fragmentation and aggravation of mitochondrial dysfunction and cell apoptosis.
    Conclusions: Our results suggested that AAV9-CaMEK alleviated ischemia-induced myocardium injury in the aging heart, at least in part, through inhibition of mitochondrial fragmentation. Therefore, AAV9-CaMEK is a potential intervention for prevention of ischemia-induced injury of the aging myocardium.
    Keywords:  Aging; ERK1/2 signaling; apoptosis; mitochondria; myocardial ischemia
  12. Methods Mol Biol. 2021 ;2281 265-272
      The mitochondrial single-stranded DNA-binding protein (mtSSB) regulates the function of the mitochondrial DNA (mtDNA) replisome. In vitro, mtSSB stimulates the activity of enzymatic components of the replisome, namely mtDNA helicase and DNA polymerase gamma (Pol γ). We have demonstrated that the stimulatory properties of mtSSB result from its ability to organize the single-stranded DNA template in a specific manner. Here we present methods employing electron microscopy and enzymatic assays to characterize and classify the mtSSB-DNA complexes and their effects on the activity of Pol γ.
    Keywords:  DNA polymerase γ; Electron microscopy; Mitochondrial DNA replication; Mitochondrial single-stranded DNA-binding protein; Nucleoprotein complexes
  13. Nat Commun. 2021 Apr 16. 12(1): 2304
      Mitochondria play a pivotal role in the generation of signals coupling metabolism with neurotransmitter release, but a role for mitochondrial-produced ROS in regulating neurosecretion has not been described. Here we show that endogenously produced hydrogen peroxide originating from axonal mitochondria (mtH2O2) functions as a signaling cue to selectively regulate the secretion of a FMRFamide-related neuropeptide (FLP-1) from a pair of interneurons (AIY) in C. elegans. We show that pharmacological or genetic manipulations that increase mtH2O2 levels lead to increased FLP-1 secretion that is dependent upon ROS dismutation, mitochondrial calcium influx, and cysteine sulfenylation of the calcium-independent PKC family member PKC-1. mtH2O2-induced FLP-1 secretion activates the oxidative stress response transcription factor SKN-1/Nrf2 in distal tissues and protects animals from ROS-mediated toxicity. mtH2O2 levels in AIY neurons, FLP-1 secretion and SKN-1 activity are rapidly and reversibly regulated by exposing animals to different bacterial food sources. These results reveal a previously unreported role for mtH2O2 in linking diet-induced changes in mitochondrial homeostasis with neuropeptide secretion.
  14. Cell Rep. 2021 Apr 13. pii: S2211-1247(21)00266-7. [Epub ahead of print]35(2): 108952
      The mechanisms controlling the post-natal maturation of astrocytes play a crucial role in ensuring correct synaptogenesis. We show that mitochondrial biogenesis in developing astrocytes is necessary for coordinating post-natal astrocyte maturation and synaptogenesis. The astrocytic mitochondrial biogenesis depends on the transient upregulation of metabolic regulator peroxisome proliferator-activated receptor gamma (PPARγ) co-activator 1α (PGC-1α), which is controlled by metabotropic glutamate receptor 5 (mGluR5). At tissue level, the loss or downregulation of astrocytic PGC-1α sustains astrocyte proliferation, dampens astrocyte morphogenesis, and impairs the formation and function of neighboring synapses, whereas its genetic re-expression is sufficient to restore the mitochondria compartment and correct astroglial and synaptic defects. Our findings show that the developmental enhancement of mitochondrial biogenesis in astrocytes is a critical mechanism controlling astrocyte maturation and supporting synaptogenesis, thus suggesting that astrocytic mitochondria may be a therapeutic target in the case of neurodevelopmental and psychiatric disorders characterized by impaired synaptogenesis.
    Keywords:  PGC-1α; astrocyte; mGluR5; mitochondria
  15. Biochim Biophys Acta Bioenerg. 2021 Apr 13. pii: S0005-2728(21)00064-5. [Epub ahead of print] 148431
      High altitude pulmonary edema (HAPE) is experienced by non-acclimatized sea level individuals on exposure to high altitude hypoxic conditions. Available evidence suggests that genetic factors and perturbed mitochondrial redox status may play an important role in HAPE pathophysiology. However, the precise mechanism has not been fully understood. In the present study, sequencing of mitochondrial DNA (mtDNA) from HAPE subjects and acclimatized controls was performed to identify pathogenic mutations and to determine their role in HAPE. Hypobaric hypoxia induced oxidative stress and metabolic alterations were also assessed in HAPE subjects. mtDNA copy number, mitochondrial oxidative phosphorylation (mtOXPHOS) activity, mitochondrial biogenesis were measured to determine mitochondrial functions. The data revealed that the mutations in Complex I genes affects the secondary structure of protein in HAPE subjects. Further, increased oxidative stress during hypobaric hypoxia, reduced mitochondrial biogenesis and mtOXPHOS activity induced metabolic reprogramming that might contribute to mitochondrial dysfunctions in HAPE individuals. Haplogroup analysis suggests that mtDNA haplogroup H2a2a1 has potential contribution in the pathobiology of HAPE in lowlanders. This study suggests contribution of altered mitochondrial functions in HAPE susceptibility.
    Keywords:  HAPE; Hypobaric hypoxia; Mitochondria; Oxidative phosphorylation; Oxidative stress; mtDNA
  16. Free Radic Biol Med. 2021 Apr 01. pii: S0891-5849(21)00198-2. [Epub ahead of print]168 247-257
      Methicillin-resistant Staphylococcus aureus (MRSA) is the leading cause of bacterial pneumonia, featured with exuberant inflammatory cytokine production, extensive oxidative stress and tissue injury. The Keap1/Nrf2 system is the major apparatus essential for host defense against oxidative and electrophilic stresses of both exogenous and endogenous origins, representing a logical target for host-directed strategy to treat severe inflammatory diseases including MRSA-induced pneumonia. In an effort to search therapeutics for bacterial pneumonia, we identify rosmarinic acid (RA) as a covalent modifier of Keap1 and hence an activator of Nrf2. Specifically, RA forms a covalent bond with the cysteine 151 of Keap1 in BTB domain, and blocks its association with Nrf2 for proteasome-mediated degradation. Consequently, RA treatment caused the increased Nrf2 nuclear translocation to initiate antioxidant and mitochondrial biogenic programs, as well as macrophage bactericidal activity through inducing autophagic pathway, which eventually led to expedited bacterial eradication, inflammation resolution, and disease recovery. Collectively, our findings establish RA as a specific inducer of Nrf2 and show its potential to prevent MRSA pneumonia.
    Keywords:  Autophagy; Keap1; Methicillin-resistant Staphylococcus aureus; Mitochondrial homeostasis; Nrf2; Rosmarinic acid
  17. Front Cell Dev Biol. 2021 ;9 626117
      Mammalian mitochondria are emerging as a critical stress-responsive contributor to cellular life/death and developmental outcomes. Maintained as an organellar network distributed throughout the cell, mitochondria respond to cellular stimuli and stresses through highly sensitive structural dynamics, particularly in energetically demanding cell settings such as cardiac and muscle tissues. Fusion allows individual mitochondria to form an interconnected reticular network, while fission divides the network into a collection of vesicular organelles. Crucially, optic atrophy-1 (OPA1) directly links mitochondrial structure and bioenergetic function: when the transmembrane potential across the inner membrane (ΔΨm) is intact, long L-OPA1 isoforms carry out fusion of the mitochondrial inner membrane. When ΔΨm is lost, L-OPA1 is cleaved to short, fusion-inactive S-OPA1 isoforms by the stress-sensitive OMA1 metalloprotease, causing the mitochondrial network to collapse to a fragmented population of organelles. This proteolytic mechanism provides sensitive regulation of organellar structure/function but also engages directly with apoptotic factors as a major mechanism of mitochondrial participation in cellular stress response. Furthermore, emerging evidence suggests that this proteolytic mechanism may have critical importance for cell developmental programs, particularly in cardiac, neuronal, and stem cell settings. OMA1's role as a key mitochondrial stress-sensitive protease motivates exciting new questions regarding its mechanistic regulation and interactions, as well as its broader importance through involvement in apoptotic, stress response, and developmental pathways.
    Keywords:  OMA1; OPA1; apoptosis; development; mitochondria
  18. ACS Chem Neurosci. 2021 Apr 12.
      Mitochondrial dysfunction and elevated ROS generation are predominant contributors of neuronal death that is responsible for the diabetes-related cognitive impairments. Emerging evidence has demonstrated that long noncoding RNA-MEG3 can serve as an important regulator in the pathogenesis of diabetes. However, the underlying mechanisms remain to be further clarified. Here, it was observed that MEG3 was significantly down-regulated in STZ (streptozotocin)-induced diabetic rats. MEG3 overexpression noticeably improved diabetes-induced cognitive dysfunctions, accompanied by the abatement of Rac1 activation and ROS production, as well as the inhibition of mitochondria-associated apoptosis. Furthermore, either MEG3 overexpression or Rac1 inhibition promoted FUNDC1 dephosphorylation and suppressed oxidative stress and neuro-inflammation. Similarly, in vitro studies confirmed that hyperglycemia also down-regulated MEG3 expression in PC12 cells. MEG3 reintroduction protected PC12 cells against hyperglycemia-triggered neurotoxicity by improving mitochondrial fitness and repressing mitochondria-mediated apoptosis. Moreover, these neuroprotective effects of MEG3 relied on FUNDC1-related mitophagy, since silencing of FUNDC1 abolished these beneficial outcomes. Additionally, MEG3 rescued HG-induced neurotoxicity was involved in inhibiting Rac1 expression via interaction with Rac1 3'UTR. Conversely, knockdown of MEG3 showed opposite effects. NSC23766, a specific inhibitor of Rac1, fully abolished harmful effects of MEG3 depletion. Consistently, knockdown of Rac1 potentiated FUNDC1-associated mitophagy. Meanwhile, colocalization of Rac1 and FUNDC1 was found in mitochondria under hyperglycemia, which was interrupted by MEG3 overexpression. Furthermore, silencing of Rac1 promoted PGAM5 expression, and FUNDC1 strongly interacted with LC3 in Rac1-deleted cells. Altogether, our findings suggested that the Rac1/ROS axis may be a downstream signaling pathway for MEG3-induced neuroprotection, which was involved in FUNDC1-associated mitophagy.
    Keywords:  FUNDC1; MEG3; Rac1; cognitive dysfunctions; diabetes; mitophagy
  19. Biochem J. 2021 Apr 12. pii: BCJ20200975. [Epub ahead of print]
      Inorganic polyphosphate (polyP) is a linear polymer composed of up to a few hundred orthophosphates linked together by high-energy phosphoanhydride bonds, identical to those found in ATP. In mammalian mitochondria, polyP has been implicated in multiple processes, including energy metabolism, ion channels function, and the regulation of calcium signaling. However, the specific mechanisms of all these effects of polyP within the organelle remain poorly understood. The central goal of this study was to investigate how mitochondrial polyP participates in the regulation of the mammalian cellular energy metabolism. To accomplish this, we created HEK293 cells depleted of mitochondrial polyP, through the stable expression of the polyP hydrolyzing enzyme (scPPX). We found that these cells have significantly reduced rates of oxidative phosphorylation (OXPHOS), while their rates of glycolysis were elevated. Consistent with this, metabolomics assays confirmed increased levels of metabolites involved in glycolysis in these cells, compared with the wild-type samples. At the same time, key respiratory parameters of the isolated mitochondria were unchanged, suggesting that respiratory chain activity is not affected by the lack of mitochondrial polyP. However, we detected that mitochondria from cells that lack mitochondrial polyP are more fragmented when compared with those from wild-type cells. Based on these results, we propose that mitochondrial polyP plays an important role as a regulator of the metabolic switch between OXPHOS and glycolysis.
    Keywords:  glycolysis; inorganic polyphosphates; mitochondrial bioenergetics; oxidative phosphorylation; polyP
  20. Mitochondrion. 2021 Apr 07. pii: S1567-7249(21)00042-8. [Epub ahead of print]
      Calcium (Ca2+) plays fundamental and diverse roles in brain cells as a second messenger of many signaling pathways. Given the high energy demand in the brain and the generally non-regenerative state of neurons, the role of brain mitochondrial calcium [Ca2+]m in particular, in regulating ATP generation and determination of cell fate by initiation or inhibition of programmed cell death (PCD) becomes critical. Since [Ca2+]m signaling has a central role in brain physiology, it represents an ideal target for viruses to hijack the Ca2+ machinery to favor their own persistence, replication and/or dissemination by modulating cell death. This review discusses the ways by which neurotropic viruses are known to exploit the [Ca2+]m signaling of their host cells to regulate cell death in the brain, particularly in neurons. We hope our review will highlight the importance of [Ca2+]m handling in the virus-infected brain and stimulate further studies towards exploring novel [Ca2+]m related therapeutic strategies for viral effects on the brain.
    Keywords:  Mitochondrial calcium; apoptosis; astrocytes; neurons; neurotropic viruses; survival
  21. Sci Signal. 2021 Apr 13. pii: eabc7931. [Epub ahead of print]14(678):
      The RIG-I-like receptor (RLR) signaling pathway is pivotal for innate immunity against invading viruses, and dysregulation of this molecular cascade has been linked to various diseases. Here, we identified dimethylarginine dimethylaminohydrolase 2 (DDAH2) as a potent regulator of the RLR-mediated antiviral response in human and mouse. Overexpression of DDAH2 attenuated RLR signaling, whereas loss of DDAH2 function enhanced RLR signaling and suppressed viral replication ex vivo and in mice. Upon viral infection, DDAH2 relocated to mitochondria, where it induced the production of nitric oxide (NO) and the activation of dynamin-related protein 1 (Drp1), which promoted mitochondrial fission and blocked the activation of innate immune responses mediated by mitochondrial antiviral signaling (MAVS). TANK-binding kinase 1 (TBK1), a kinase downstream of MAVS, inhibited DDAH2 by phosphorylating DDAH2 at multiple sites. Our study thus identifies a reciprocal inhibitory loop between the DDAH2-NO cascade and the RLR signaling pathway that fine-tunes the antiviral immune response.
  22. J Immunol Res. 2021 ;2021 6670495
      At present, liver ischemia-reperfusion (IR) injury is still a great challenge for clinical liver partial resection and liver transplantation. The innate immunity regulated by liver macrophages orchestrates the cascade of IR inflammation and acts as a bridge. As a specific macrophage subunit of vacuolar ATPase, ATP6V0D2 (V-ATPase D2 subunit) has been shown to promote the formation of autophagolysosome in vitro. Our research fills a gap which has existed in the study of inflammatory stress about the V-ATPase subunit ATP6V0D2 in liver macrophages. We first found that the expression of specific ATP6V0D2 in liver macrophages was upregulated with the induction of inflammatory cascade after liver IR surgery, and knockdown of ATP6V0D2 resulted in increased secretion of proinflammatory factors and chemokines, which enhanced activation of NLRP3 and aggravation of liver injury. Further studies found that the exacerbated activation of NLRP3 was related to the autophagic flux regulated by ATP6V0D2. Knocking down ATP6V0D2 impaired the formation of autophagolysosome and aggravated liver IR injury through nonspecific V-ATPase activation independent of V-ATPase-Notchl-Hesl signal axis. In general, we illustrated that the expression of ATP6V0D2 in liver macrophages was upregulated after liver IR, and by gradually promoting the formation of autophagolysosomes to increase autophagy flux to limit the activation of liver inflammation, this regulation is independent of the Notch1-Hes1 signal axis.
  23. Oxid Med Cell Longev. 2021 ;2021 6635955
      Receptor-interacting protein 3- (RIPK3-) modulated necroptosis plays a critical role in cardiac remodelling after myocardial infarction (MI). However, the precise regulatory mechanism is not fully elucidated yet. In the present study, we showed that RIPK3 expression was upregulated in myocardial tissue after MI in a mouse model by coronary artery ligation, as well as in the cardiomyocytes following hypoxic injury in vitro. The increase of RIPK3 expression was found to be accompanied by severe cardiac remodelling, cardiac dysfunction, and higher mortality. Elevated RIPK3 expression subsequently abrogated the AMPK pathway that was accompanied by inhibition of Parkin-mediated mitophagy. Loss of mitophagy increased the opening of mitochondrial permeability transition pore (mPTP), which ultimately induced the cardiomyocyte necroptosis. In contrast, genetic ablation of Ripk3 induced the AMPK/Parkin-mitophagy pathway, favouring a prosurvival state that eventually inhibited mPTP opening and induced the necroptosis of cardiomyocytes in the post-MI cardiac remodelling. In conclusion, our results revealed a key mechanism by which necroptosis could be mediated by RIPK3 via the AMPK/Parkin-mitophagy/mPTP opening axis, which provides a potential therapeutic target in the management of heart failure after MI.
  24. Methods Mol Biol. 2021 ;2281 313-322
      Defects in mitochondrial DNA (mtDNA) maintenance may lead to disturbances in mitochondrial homeostasis and energy production in eukaryotic cells, causing diseases. During mtDNA replication, the mitochondrial single-stranded DNA-binding protein (mtSSB) stabilizes and protects the exposed single-stranded mtDNA from nucleolysis; perhaps more importantly, it appears to coordinate the actions of both the replicative mtDNA helicase Twinkle and DNA polymerase gamma at the replication fork. Here, we describe a helicase stimulation protocol to test in vitro the functional interaction between mtSSB and variant forms of Twinkle. We show for the first time that the C-terminal tail of Twinkle is important for such an interaction, and that it negatively regulates helicase unwinding activity in a salt-dependent manner.
    Keywords:  Mitochondrial DNA replication; P66; Twinkle; dsDNA unwinding assay; mtSSB
  25. Geroscience. 2021 Apr 17.
      Mitochondria are organelles that provide energy to cells through ATP production. Mitochondrial dysfunction has long been postulated to mediate cellular declines that drive biological aging. Many well-characterized hallmarks of aging may involve underlying energetic defects that stem from loss of mitochondrial function with age. Why and how mitochondrial function declines with age is an open question and one that has been difficult to answer. Mitochondria are powered by an electrochemical gradient across the inner mitochondrial membrane known as the protonmotive force (PMF). This gradient decreases with age in several experimental models. However, it is unclear if a diminished PMF is a cause or a consequence of aging. Herein, we briefly review and define mitochondrial function, we summarize how PMF changes with age in several models, and we highlight recent studies that implicate PMF in aging biology. We also identify barriers that must be addressed for the field to progress. Emerging technology permits more precise in vivo study of mitochondria that will allow better understanding of cause and effect in metabolic models of aging. Once cause and effect can be discerned more precisely, energetics approaches to combat aging may be developed to prevent or reverse functional decline.
    Keywords:  AMPK; Autophagy; Membrane potential; Metabolism; mTOR
  26. Phys Biol. 2021 Apr 14.
      Recent experiments and thermodynamic arguments suggest that mitochondrial temperatures are higher than those of the cytoplasm. A "hot mitochondrion" calls for a closer examination of the energy balance that endows it with these claimed elevated temperatures. As a first step in this effort, we present here a semi-quantitative bookkeeping whereby, in one stroke, a formula is proposed that yields the rate of heat production in a typical mitochondrion and a formula for estimating the number of active ATP synthase molecules per mitochondrion. Scaling laws are shown to determine the number of active ATP synthase molecules in a mitochondrion and mitochondrial rate of heat production. Mitochondrial population of active ATP synthases and mitochondrial rate of heat production appear, both, to scale with cell volume. Four heterotrophic protozoa cell types are considered in this study. The studied cells, selected to cover a wide range of sizes (volumes) from ca. 100 μm3 to 1 million μm3, are estimated to exhibit a power per mitochondrion ranging from ca. 1 pW to 0.03 pW. The corresponding number of active ATP synthases per mitochondrion in these cells ranges from 5,000 to just about a hundred.
    Keywords:  Allometric power laws; Cellular volumes; Mitochondrial heat production; Mitochondrial thermodynamics; Number of ATP synthase molecules; Scaling laws