bims-mikwok Biomed News
on Mitochondrial quality control
Issue of 2021‒10‒10
eleven papers selected by
Avinash N. Mukkala
University of Toronto

  1. J Biol Chem. 2021 Oct 05. pii: S0021-9258(21)01082-6. [Epub ahead of print] 101279
      Mitochondria are essential organelles that carry out a number of pivotal metabolic processes and maintain cellular homeostasis. Mitochondrial dysfunction caused by various stresses is associated with many diseases such as type 2 diabetes, obesity, cancer, heart failure, neurodegenerative disorders, and aging. Therefore, it is important to understand the stimuli that induce mitochondrial stress. However, broad analysis of mitochondrial stress has not been carried out to date. Here, we present a set of fluorescent tools, called mito-Pain (mitochondrial PINK1 accumulation index), which enables the labeling of stressed mitochondria. Mito-Pain utilizes PINK1 stabilization on mitochondria and quantifies mitochondrial stress levels by comparison with PINK1-GFP, which is stabilized under mitochondrial stress, and RFP-Omp25, which is constitutively localized on mitochondria. To identify compounds that induce mitochondrial stress, we screened a library of 3374 compounds using mito-Pain and identified 57 compounds as mitochondrial stress inducers. Furthermore, we classified each compound into several categories based on mitochondrial response: depolarization, mitochondrial morphology, or Parkin recruitment. Parkin recruitment to mitochondria was often associated with mitochondrial depolarization and aggregation, suggesting that Parkin is recruited to heavily damaged mitochondria. In addition, many of the compounds led to various mitochondrial morphological changes, including fragmentation, aggregation, elongation, and swelling, with or without Parkin recruitment or mitochondrial depolarization. We also found that several compounds induced an ectopic response of Parkin, leading to the formation of cytosolic puncta dependent on PINK1. Thus, mito-Pain enables the detection of stressed mitochondria under a wide variety of conditions and provide insights into mitochondrial quality control systems.
    Keywords:  PTEN‐induced putative kinase 1 (PINK1); Parkin; mitochondria; mitochondrial membrane potential; mitochondrial sensor; mitochondrial stress
  2. J Cell Sci. 2021 Oct 01. pii: jcs240465. [Epub ahead of print]134(19):
      Mitochondria, which resemble their α-proteobacteria ancestors, are a major cellular asset, producing energy 'on the cheap' through oxidative phosphorylation. They are also a liability. Increased oxidative phosphorylation means increased oxidative stress, and damaged mitochondria incite inflammation through release of their bacteria-like macromolecules. Mitophagy (the selective macroautophagy of mitochondria) controls mitochondria quality and number to manage these risky assets. Parkin, BNIP3 and NIX were identified as being part of the first mitophagy pathways identified in mammals over a decade ago, with additional pathways, including that mediated by FUNDC1 reported more recently. Loss of Parkin or PINK1 function causes Parkinson's disease, highlighting the importance of mitophagy as a quality control mechanism in the brain. Additionally, mitophagy is induced in idiopathic Parkinson's disease and Alzheimer's disease, protects the heart and other organs against energy stress and lipotoxicity, regulates metabolism by controlling mitochondrial number in brown and beige fat, and clears mitochondria during terminal differentiation of glycolytic cells, such as red blood cells and neurons. Despite its importance in disease, mitophagy is likely dispensable under physiological conditions. This Review explores the in vivo roles of mitophagy in mammalian systems, focusing on the best studied examples - mitophagy in neurodegeneration, cardiomyopathy, metabolism, and red blood cell development - to draw out common themes.
    Keywords:  Mitochondria quality control; Neurodegeneration; PRKN; Park2; Park6
  3. Elife. 2021 10 05. pii: e69207. [Epub ahead of print]10
      The Connexin43 gap junction gene GJA1 has one coding exon, but its mRNA undergoes internal translation to generate N-terminal truncated isoforms of Connexin43 with the predominant isoform being only 20 kDa in size (GJA1-20k). Endogenous GJA1-20k protein is not membrane bound and has been found to increase in response to ischemic stress, localize to mitochondria, and mimic ischemic preconditioning protection in the heart. However, it is not known how GJA1-20k benefits mitochondria to provide this protection. Here, using human cells and mice, we identify that GJA1-20k polymerizes actin around mitochondria which induces focal constriction sites. Mitochondrial fission events occur within about 45 s of GJA1-20k recruitment of actin. Interestingly, GJA1-20k mediated fission is independent of canonical Dynamin-Related Protein 1 (DRP1). We find that GJA1-20k-induced smaller mitochondria have decreased reactive oxygen species (ROS) generation and, in hearts, provide potent protection against ischemia-reperfusion injury. The results indicate that stress responsive internally translated GJA1-20k stabilizes polymerized actin filaments to stimulate non-canonical mitochondrial fission which limits ischemic-reperfusion induced myocardial infarction.
    Keywords:  GJA1-20k; actin dynamics; cell biology; human; ischemia/reperfusion; mitochondria; mitochondria dynamics; mouse; organ protection
  4. Life Sci. 2021 Oct 01. pii: S0024-3205(21)00992-9. [Epub ahead of print] 120005
      Renal ischemia-reperfusion injury (IRI) is a leading cause of acute kidney injury (AKI) and may influence renal graft survival. In this study, we investigate the involvement of SIRT3 and DRP1 in mitochondrial autophagy and AKI in a mouse model of IRI. Autophagy was detected in the absence of SIRT3, and hypoxic reoxygenation (H/R) experiments using renal tubular epithelial cells NRK52E were performed in vitro to validate these results. We found that autophagosomes increased following IRI and that the expression of autophagy-related genes was up-regulated. The inhibition of autophagy with 3-methyladenine exacerbated IRI, whereas the DRP1 inhibitor Mdivi-1 reversed this inhibition. Mdivi-1 did not reverse the inhibition of autophagy in the absence of SIRT3. During IRI, Mdivi-1 reduced autophagy and DRP1 expression, whereas SIRT3 overexpression attenuated this condition. Rescue experiment showed that autophagy was increased when both SIRT3 or DRP1 were over- or under-expressed or just DRP1 was under-expressed but expression was reduced when just SIRT3 was under-expressed. However, the expression of DRP1-related molecules was reduced when SIRT3 was overexpressed and when DRP1 was under-expressed. Taken together, these findings indicate that SIRT3 protects against kidney damage from IRI by modulating the DRP1 pathway to induce mitochondrial autophagy.
    Keywords:  Acute kidney injury; Ischemia-reperfusion injury; Mitophagy; SIRT3, DRP1
  5. Mitochondrion. 2021 Oct 01. pii: S1567-7249(21)00140-9. [Epub ahead of print]
      Although alterations in cellular mitochondrial DNA (mtDNA) content have been linked to various pathological conditions, the mechanisms that govern mtDNA copy number (mtCN) control remain poorly understood. Moreover, techniques for mtDNA quantification do not allow for direct comparisons of absolute mtCNs between labs. Here we report the development of a direct droplet digital PCR technique for the determination of mtCNs in whole-cell lysates. Using this technique, we demonstrate that cellular mtDNA content can fluctuate in culture by as much as 50% and provide evidence for both cell proliferation-coupled and uncoupled mtDNA replication.
    Keywords:  Mitochondrial DNA; X-ray; cell cycle; cell proliferation; mtDNA copy number; mtDNA replication
  6. J Cell Mol Med. 2021 Oct 08.
      Transient ischaemia and reperfusion in liver tissue induce hepatic ischaemia/reperfusion (I/R) tissue injury and a profound inflammatory response in vivo. Hepatic I/R can be classified into warm I/R and cold I/R and is characterized by three main types of cell death, apoptosis, necrosis and autophagy, in rodents or patients following I/R. Warm I/R is observed in patients or animal models undergoing liver resection, haemorrhagic shock, trauma, cardiac arrest or hepatic sinusoidal obstruction syndrome when vascular occlusion inhibits normal blood perfusion in liver tissue. Cold I/R is a condition that affects only patients who have undergone liver transplantation (LT) and is caused by donated liver graft preservation in a hypothermic environment prior to entering a warm reperfusion phase. Under stress conditions, autophagy plays a critical role in promoting cell survival and maintaining liver homeostasis by generating new adenosine triphosphate (ATP) and organelle components after the degradation of macromolecules and organelles in liver tissue. This role of autophagy may contribute to the protection of hepatic I/R-induced liver injury; however, a considerable amount of evidence has shown that autophagy inhibition also protects against hepatic I/R injury by inhibiting autophagic cell death under specific circumstances. In this review, we comprehensively discuss current strategies and underlying mechanisms of autophagy regulation that alleviates I/R injury after liver resection and LT. Directed autophagy regulation can maintain liver homeostasis and improve liver function in individuals undergoing warm or cold I/R. In this way, autophagy regulation can contribute to improving the prognosis of patients undergoing liver resection or LT.
    Keywords:  autophagy; cell death; ischaemia/reperfusion; liver resection; liver transplantation
  7. Lab Invest. 2021 Oct 04.
      Mitochondrial homeostasis is crucial for the function of pancreatic β-cells. ATP synthase inhibitory factor subunit 1 (IF1) is a mitochondrial protein interacting with ATP synthase to inhibit its enzyme activity. IF1 may also play a role in maintaining ATP synthase oligomerization and mitochondrial inner membrane formation. A recent study confirmed IF1 expresses in β-cells. IF1 knockdown in cultured INS-1E β-cells enhances glucose-induced insulin release. However, the role of IF1 in islet β-cells remains little known. The present study investigates islets freshly isolated from mouse lines with global IF1 knockout (IF1-/-) and overexpression (OE). The glucose-stimulated insulin secretion was increased in islets from IF1-/- mice but decreased in islets from IF1 OE mice. Transmitted Electronic Microscopic assessment of isolated islets revealed that the number of matured insulin granules (with dense core) was relatively higher in IF1-/-, but fewer in IF1 OE islets than those of controlled islets. The mitochondrial ultrastructure within β-cells of IF1 overexpressed islets was comparable with those of wild-type mice, whereas those in IF1-/- β-cells showed increased mitochondrial mass. Mitochondrial network analysis in cultured INS-1 β-cells showed a similar pattern with an increased mitochondrial network in IF1 knockdown cells. IF1 overexpressed INS-1 β-cells showed a compromised rate of mitochondrial oxidative phosphorylation with attenuated cellular ATP content. In contrast, INS-1 cells with IF1 knockdown showed markedly increased cellular respiration with improved ATP production. These results support that IF1 is a negative regulator of insulin production and secretion via inhibiting mitochondrial mass and respiration in β-cells. Therefore, inhibiting IF1 to improve β-cell function in patients can be a novel therapeutic strategy to treat diabetes.
  8. J Cell Biol. 2021 Dec 06. pii: e202006049. [Epub ahead of print]220(12):
      The cystine-glutamate antiporter, xCT, supports a glutathione synthesis program enabling cancer cells to cope with metabolically stressful microenvironments. Up-regulated xCT, in combination with glutaminolysis, leads to increased extracellular glutamate, which promotes invasive behavior by activating metabotropic glutamate receptor 3 (mGluR3). Here we show that activation of mGluR3 in breast cancer cells activates Rab27-dependent release of extracellular vesicles (EVs), which can transfer invasive characteristics to "recipient" tumor cells. These EVs contain mitochondrial DNA (mtDNA), which is packaged via a PINK1-dependent mechanism. We highlight mtDNA as a key EV cargo necessary and sufficient for intercellular transfer of invasive behavior by activating Toll-like receptor 9 in recipient cells, and this involves increased endosomal trafficking of pro-invasive receptors. We propose that an EV-mediated mechanism, through which altered cellular metabolism in one cell influences endosomal trafficking in other cells, is key to generation and dissemination of pro-invasive microenvironments during mammary carcinoma progression.
  9. Exp Cell Res. 2021 Oct 05. pii: S0014-4827(21)00415-8. [Epub ahead of print] 112861
      Ischemia reperfusion-induced acute kidney injury (IR-induced AKI) is a life-threatening disease with many complications. Mitofusin 2 (Mfn2) ubiquitination is related to AKI. But the underlying molecular mechanisms remain unknown. This study aimed to probe the mechanism of Mfn2 ubiquitination in IR-induced AKI development. In IR-induced AKI mouse models, orbital blood and urine were collected for assessing kidney function. The kidney injury, ultrastructure of mitochondria and histopathology in mice were evaluated after injection of G5, an ubiquitination inhibitor. Oxygen glucose deprivation/reoxygenation (OGD/R) models were established in HK-2 cells, and the mitochondria were extracted. Cell viability, apoptosis, oxidative stress, inflammatory reaction, mitochondrial membrane potential and ATP production were measured. Mfn2 ubiquitination in mouse and cell models was evaluated. si-SIRT3 and pcDNA3.1-SIRT3 were transfected into cell models. Consequently, kidney function in mice was impaired by IR-induced AKI. Mfn2 ubiquitination and degradation promoted IR-induced AKI. OGD/R induced renal tubular epithelial cell injury and disrupted mitochondrial dynamics and functions through promoting Mfn2 ubiquitination. SIRT3 knockdown led to Mfn2 ubiquitination by binding to UBC; while its overexpression alleviated tubular epithelial cell injury. Briefly, SIRT3 mediates Mfn2 ubiquitination to relieve IR-induced AKI. This investigation may offer new insights for the treatment of IR-induced AKI injury.
    Keywords:  Acute kidney injury; Ischemia reperfusion; Mfn2; Mitochondria; SIRT3; Ubiquitination
  10. Hepatology. 2021 Oct 08.
      Nrf2 is a master regulator of reactive oxygen species (ROS) and inflammation and has been implicated in both human and murine inflammatory disease models. We aimed to characterize the roles of macrophage-specific Nrf2 in liver ischemia-reperfusion injury (IRI). First, macrophage Nrf2 expression and liver injury in patients undergoing orthotopic liver transplant (OLT) or ischemia-related hepatectomy were analyzed. Subsequently, we created a myeloid-specific Nrf2-knockout (Nrf2M-KO ) strain to study the function and mechanism of macrophage Nrf2 in a murine liver IRI model. In human specimens, macrophage Nrf2 expression was significantly increased in liver tissues after transplantation or hepatectomy. Interestingly, lower Nrf2 expressions correlated with more severe liver injury postoperatively. In a mouse model, we found Nrf2M-KO mice showed worse hepatocellular damage than Nrf2-proficient controls based on serum biochemistry, pathology, ROS, and inflammation. In vitro, Nrf2 deficiency promoted innate immune activation and migration in macrophages upon TLR4 stimulation. Microarray profiling showed Nrf2 deletion caused markedly lower transcriptional levels of tissue inhibitor of metalloproteinase 3 (Timp3). ChIP-seq, PCR, and luciferase reporter assay further demonstrated Nrf2 bound to the promoter region of Timp3. Moreover, ADAM10/ROCK1 was specifically increased in Nrf2-deficient macrophages. Increasing Timp3 expression effectively inhibited ADAM10/ROCK1 expression and rescued the Nrf2M-KO -mediated inflammatory response upon TLR4 stimulation in vitro. Importantly, Timp3 overexpression, recombinant Timp3 protein, or ROCK1 knockdown rescued Nrf2M-KO -related liver IRI by inhibiting macrophage activation. In conclusion, macrophage Nrf2 mediates innate proinflammatory responses, attenuates liver IRI by binding to Timp3, and inhibits the RhoA/ROCK pathway, which provides a therapeutic target for clinical organ IRI.
    Keywords:  Hepatic ischemia/reperfusion injury; Macrophage; Nrf2; RhoA/ROCK pathway; Timp3
  11. Nature. 2021 Oct 06.
      The enzymes of the mitochondrial electron transport chain are key players of cell metabolism. Despite being active when isolated, in vivo they associate into supercomplexes1, whose precise role is debated. Supercomplexes CIII2CIV1-2 (refs. 2,3), CICIII2 (ref. 4) and CICIII2CIV (respirasome)5-10 exist in mammals, but in contrast to CICIII2 and the respirasome, to date the only known eukaryotic structures of CIII2CIV1-2 come from Saccharomyces cerevisiae11,12 and plants13, which have different organization. Here we present the first, to our knowledge, structures of mammalian (mouse and ovine) CIII2CIV and its assembly intermediates, in different conformations. We describe the assembly of CIII2CIV from the CIII2 precursor to the final CIII2CIV conformation, driven by the insertion of the N terminus of the assembly factor SCAF1 (ref. 14) deep into CIII2, while its C terminus is integrated into CIV. Our structures (which include CICIII2 and the respirasome) also confirm that SCAF1 is exclusively required for the assembly of CIII2CIV and has no role in the assembly of the respirasome. We show that CIII2 is asymmetric due to the presence of only one copy of subunit 9, which straddles both monomers and prevents the attachment of a second copy of SCAF1 to CIII2, explaining the presence of one copy of CIV in CIII2CIV in mammals. Finally, we show that CIII2 and CIV gain catalytic advantage when assembled into the supercomplex and propose a role for CIII2CIV in fine tuning the efficiency of electron transfer in the electron transport chain.