bims-mikwok Biomed News
on Mitochondrial quality control
Issue of 2022‒01‒02
eight papers selected by
Avinash N. Mukkala
University of Toronto

  1. Methods Mol Biol. 2022 ;2445 227-239
      Mitophagy, a process of selective elimination of mitochondria by autophagy, is a mechanism of mitochondrial quality control that maintains mitochondrial network functionality. The elimination of damaged mitochondria through autophagy requires two steps: induction of general autophagy and priming of damaged mitochondria for selective autophagic recognition. Mitophagy impairment is linked to various pathologies; thus, removal of malfunctioning or even harmful mitochondria is vital to cellular physiology. Here, we describe methods that can be applied to the investigation of mitophagy.
    Keywords:  Autophagy; Confocal microscopy; Flow cytometry; Mitochondria; Mitophagy; Respiration
  2. Am J Physiol Cell Physiol. 2021 12 29.
      Selective autophagy of the mitochondria, known as mitophagy, is a major mitochondrial quality control pathway in the heart that is involved in removing unwanted or dysfunctional mitochondria from the cell. Baseline mitophagy is critical for maintaining the fitness of the mitochondrial population by continuous turnover of aged and less functional mitochondria. Mitophagy is also critical in adapting to stress associated with mitochondrial damage or dysfunction. The removal of damaged mitochondria prevents ROS-mediated damaged to proteins and DNA and suppresses activation of inflammation and cell death. Impairments in mitophagy are associated with the pathogenesis of many diseases, including cancers, inflammatory diseases, neurodegeneration, and cardiovascular disease. Mitophagy is a highly regulated and complex process that requires the coordination of labeling dysfunctional mitochondria for degradation while simultaneously promoting de novo autophagosome biogenesis adjacent to the cargo. In this review, we provide an update on our current understanding of these steps in mitophagy induction and discuss the physiological and pathophysiological consequences of altered mitophagy in the heart.
    Keywords:  Parkin; autophagy; heart; mitochondria; mitophagy
  3. Methods Mol Biol. 2022 ;2445 207-226
      Damaged, dysfunctional, or excess mitochondria are removed from cells via a selective form of macroautophagy termed mitophagy. The clearance of mitochondria during mitophagy is mediated by double-membrane vesicles called autophagosomes, which encapsulate mitochondria that have been tagged for mitophagic removal before delivering them to lysosomes for degradation. A variety of different mitophagy pathways exist that differ in their mechanisms of initiation but share a common pathway of autophagosome formation. Autophagosome biogenesis is regulated by a number of autophagy factors which translocate from the cytosol to spatially defined focal points (foci) on the mitochondrial surface after mitophagy has been initiated. The functional analysis of autophagosome biogenesis requires the use of microscopy-based techniques which assess the recruitment of autophagy factors to mitophagic foci representing autophagosome formation sites. Here, we describe a routine method for the quantitative 3D analysis of mitophagic foci in PINK1/Parkin mitophagy immunofluorescence samples through the application of object-based image analysis (OBIA) to 3D confocal imaging datasets. The approach enables unbiased high-throughput characterisation of autophagosome biogenesis during mitophagy.
    Keywords:  ImageJ/FIJI; Object-based image analysis (OBIA); PINK1/Parkin mitophagy; Phagophore biogenesis; Regions of interest (ROI)
  4. J Biol Chem. 2021 Dec 24. pii: S0021-9258(21)01350-8. [Epub ahead of print] 101540
      Persistent inactivity promotes skeletal muscle atrophy, marked by mitochondrial aberrations that affect strength, mobility, and metabolic health leading to the advancement of disease. Mitochondrial quality control (MQC) pathways include biogenesis (synthesis), mitophagy/lysosomal turnover, and the mitochondrial unfolded protein response (UPRmt) which serve to maintain an optimal organelle network. Tumor suppressor p53 has been implicated in regulating muscle mitochondria in response to cellular stress; however, its role in the context of muscle disuse has yet to be explored, and whether p53 is necessary for MQC remains unclear. To address this, we subjected p53 muscle-specific knockout (mKO) and wild-type (WT) mice to unilateral denervation. Transcriptomic and pathway analyses revealed dysregulation of pathways pertaining to mitochondrial function, and especially turnover, in mKO muscle following denervation. Protein and mRNA data of the MQC pathways indicated activation of the UPRmt and mitophagy-lysosome systems along with reductions in mitochondrial biogenesis and content in WT and mKO tissue following chronic denervation. However, p53 ablation also attenuated the expression of autophagy/mitophagy machinery, reduced autophagic flux, and enhanced lysosomal dysfunction. While similar reductions in mitochondrial biogenesis and content were observed between genotypes, MQC dysregulation exacerbated mitochondrial dysfunction in mKO fibers, evidenced by elevated reactive oxygen species (ROS). Moreover, acute experiments indicate that p53 mediates the expression of transcriptional regulators of MQC pathways as early as 1 day following denervation. Together, our data illustrate exacerbated mitochondrial dysregulation with denervation stress in p53 mKO tissue, thus indicating that p53 contributes to organellar maintenance via regulation of MQC pathways during muscle atrophy.
    Keywords:  lysosome; mitochondria; mitochondrial biogenesis; mitochondrial quality control; mitophagy; muscle atrophy; p53; skeletal muscle; transcriptomics; unfolded protein response (UPR)
  5. Redox Biol. 2021 Dec 24. pii: S2213-2317(21)00385-2. [Epub ahead of print]49 102225
      BACKGROUND: Neutrophils play a role in innate immunity and are critical for clearance of Staphylococcus aureus. Current understanding of neutrophil bactericidal effects is that NADPH oxidase produces reactive oxygen species (ROS), mediating bacterial killing. Neutrophils also contain numerous mitochondria; since these organelles lack oxidative metabolism, their function is unclear. We hypothesize that mitochondria in human neutrophils contribute to the bactericidal capacity of S. aureus.METHODS: and Findings: Using human neutrophils isolated from healthy volunteers (n = 13; 7 females, 6 males), we show that mitochondria are critical in the immune response to S. aureus. Using live-cell and fixed confocal, and transmission electron microscopy, we show mitochondrial tagging of bacteria prior to ingestion and surrounding of phagocytosed bacteria immediately upon engulfment. Further, we demonstrate that mitochondria are ejected from intact neutrophils and engage bacteria during vital NETosis. Inhibition of the mitochondrial electron transport chain at Complex III, but not Complex I, attenuates S. aureus killing by 50 ± 7%, comparable to the NADPH oxidase inhibitor apocynin. Similarly, mitochondrial ROS scavenging using MitoTEMPO attenuates bacterial killing 112 ± 60% versus vehicle control. Antimycin A treatment also reduces mitochondrial ROS production by 50 ± 12% and NETosis by 53 ± 5%.
    CONCLUSIONS: We identify a previously unrecognized role for mitochondria in human neutrophils in the killing of S. aureus. Inhibition of electron transport chain Complex III significantly impairs antimicrobial activity. This is the first demonstration that vital NETosis, an early event in the antimicrobial response, occurring within 5 min of bacterial exposure, depends on the function of mitochondrial Complex III. Mitochondria join NADPH oxidase as bactericidal ROS generators that mediate the bactericidal activities of human neutrophils.
    Keywords:  Electron transport chain complex III; Immunity; Neutrophil extracellular trap (NET); Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases; Phagocytosis; Staphylococcus aureus
  6. J Biol Chem. 2021 Dec 22. pii: S0021-9258(21)01342-9. [Epub ahead of print] 101532
      Hepatic ischemia/reperfusion (I/R) injury is an inflammation-mediated process arising from ischemia/reperfusion-elicited stress in multiple cell types, causing liver damage during surgical procedures and often resulting in liver failure. Endoplasmic reticulum (ER) stress triggers the activation of the unfolded protein response (UPR) and is implicated in tissue injuries, including hepatic I/R injury. However, the cellular mechanism that links the UPR signaling to local inflammatory responses during hepatic I/R injury remains largely obscure. Here, we report that IRE1α, a critical ER-resident transmembrane signal transducer of the UPR, plays an important role in promoting Kupffer cell-mediated liver inflammation and hepatic I/R injury. Utilizing a mouse model in which IRE1α is specifically ablated in myeloid cells, we found that abrogation of IRE1α markedly attenuated necrosis and cell death in the liver, accompanied by reduced neutrophil infiltration and liver inflammation following hepatic I/R injury. Mechanistic investigations in mice as well as in primary Kupffer cells revealed that loss of IRE1α in Kupffer cells not only blunted the activation of the NLRP3 inflammasome and IL-1β production, but also suppressed the expression of the inducible nitric oxide synthase (iNos) and pro-inflammatory cytokines. Moreover, pharmacological inhibition of IRE1α's RNase activity was able to attenuate inflammasome activation and iNos expression in Kupffer cells, leading to alleviation of hepatic I/R injury in mice. Collectively, these results demonstrate that Kupffer cell IRE1α mediates local inflammatory damage during hepatic I/R injury. Our findings suggest that IRE1α RNase activity may serve as a promising target for therapeutic treatment of ischemia/reperfusion-associated liver inflammation and dysfunction.
    Keywords:  ER stress; Hepatic ischemia/reperfusion injury; IRE1α; Inflammation; Kupffer cells
  7. Free Radic Biol Med. 2021 Dec 22. pii: S0891-5849(21)01156-4. [Epub ahead of print]179 119-132
      Mitochondria are key organelles involved in cellular survival, differentiation, and death induction. In this regard, mitochondrial morphology and/or function alterations are involved in stress-induced adaptive pathways, priming mitochondria for mitophagy or apoptosis induction. We have previously shown that the mitochondriotropic antioxidant AntiOxCIN4 (100 μM; 48 h) presented significant cytoprotective effect without affecting the viability of human hepatoma-derived (HepG2) cells. Moreover, AntiOxCIN4 (12.5 μM; 72 h) caused a mild increase of reactive oxygen species (ROS) levels without toxicity to primary human skin fibroblasts (PHSF). As Nrf2 is a master regulator of the oxidative stress response inducing antioxidant-encoding gene expression, we hypothesized that AntiOxCIN4 could increase the resistance of human hepatoma-derived HepG2 to oxidative stress by Nrf2-dependent mechanisms, in a process mediated by mitochondrial ROS (mtROS). Here we showed that after an initial decrease in oxygen consumption paralleled by a moderate increase in superoxide anion levels, AntiOxCIN4 led to a time-dependent Nrf2 translocation to the nucleus. This was followed later by a 1.5-fold increase in basal respiration and a 1.2-fold increase in extracellular acidification. AntiOxCIN4 treatment enhanced mitochondrial quality by triggering the clearance of defective organelles by autophagy and/or mitophagy, coupled with increased mitochondrial biogenesis. AntiOxCIN4 also up-regulated the cellular antioxidant defense system. AntiOxCIN4 seems to have the ability to maintain hepatocyte redox homeostasis, regulating the electrophilic/nucleophilic tone, and preserve cellular physiological functions. The obtained data open a new avenue to explore the effects of AntiOxCIN4 in the context of preserving hepatic mitochondrial function in disorders, such as NASH/NAFLD and type II diabetes.
    Keywords:  Antioxidant defenses; Autophagy; Caffeic acid; Dietary antioxidants; Mitochondria; Mitochondria-targeted antioxidants; Nrf2
  8. Eur Surg Res. 2021 Dec 24.
      Hemorrhage control often poses a great challenge for clinicians due to trauma-induced coagulopathy (TIC). The pathogenesis of TIC is not completely revealed; however, growing evidence attributes a central role to altered platelet biology. The activation of thrombocytes and subsequent clot formation are highly energetic processes being tied to mitochondrial activity, and the inhibition of the electron transport chain (ETC) impedes on thrombogenesis, suggesting the potential role of mitochondria in TIC. Our present study protocol provides a guide to quantitatively characterize the derangements of mitochondrial functions in TIC. One hundred eleven severely injured (Injury Severity Score ≥16), bleeding trauma patients with an age of 18 or greater will be included in this prospective observational study. Patients receiving oral antiplatelet agents including cyclooxygenase-1 or adenosine diphosphate receptor inhibitors (aspirin, clopidogrel, prasugrel, and ticagrelor) will be excluded from the final analysis. Hemorrhage will be confirmed and assessed with computer tomography. Conventional laboratory markers of hemostasis such as prothrombin time and international normalized ratio (INR) will be measured and rotational thromboelastometry (ROTEM) will be performed directly upon patient arrival. Platelets will be isolated from venous blood samples and subjected to high-resolution fluororespirometry (Oxygraph-2k, Oroboros Instruments, Innsbruck, Austria) to evaluate the efficacy of mitochondrial respiration. Oxidative phosphorylation (OxPhos), coupling of the ETC, mitochondrial superoxide formation, mitochondrial membrane potential changes and extramitochondrial Ca2+-movement will be recorded. The association between OxPhos capacity of platelet mitochondria and numerical parameters of ROTEM aggregometry will constitute our primary outcome. The relation between OxPhos capacity and results of viscoelastic assays and conventional markers of hemostasis will serve as secondary outcomes. The association of the OxPhos capacity of platelet mitochondria upon patient arrival to the need for massive blood transfusion (MBT) and 24-hour mortality will constitute our tertiary outcomes. Mitochondrial dysfunction and its importance in TIC in are yet to be assessed for the deeper understanding of this common, life-threatening condition. Disclosure of mitochondria-mediated processes in thrombocytes may reveal new therapeutic targets in the management of hemorrhaging trauma patients, thereby leading to a reduction of potentially preventable mortality. The present protocol was registered to on 12 August 2021, under the reference number NCT05004844.