bims-mikwok Biomed News
on Mitochondrial quality control
Issue of 2022‒04‒17
ten papers selected by
Avinash N. Mukkala
University of Toronto

  1. Cells. 2022 Mar 28. pii: 1132. [Epub ahead of print]11(7):
      The elimination of intracellular components by autophagy maintains metabolic homeostasis and is a quality-control pathway that enables organelle regeneration. Mitophagy is a type of selective autophagy that regulates mitochondrial turnover, and the dysregulation of mitophagy has been implicated in the pathogenesis of liver diseases. However, the detailed molecular mechanism underlying mitophagy regulation in liver cells remains unclear, and the small molecules that may potentially modulate hepatic mitophagy are still unavailable. Here, we report that baicalein, a flavonoid extracted from Scutellaria baicalensis, induces the entire autophagy that proceeds through the autolysosome maturation stage in human hepatoma cells. In addition, baicalein-induced autophagy is demonstrated to target mitochondria for degradation. Further studies show that baicalein triggers the translocation of Parkin and TBK1 to mitochondria to induce mitophagy. Moreover, the phosphorylation of TBK1 at Ser172 and ubiquitin at Ser65 is shown to trigger mitophagy in baicalein-treated cells. Furthermore, two specific autophagy cargo receptors, NDP52 and OPTN, that function in baicalein-activated mitophagy are identified. Taken together, these findings not only delineate the molecular process of Parkin-dependent mitophagy in liver cells, but also reveal baicalein as a novel inducer of hepatic mitophagy.
    Keywords:  autophagy; baicalein; cargo receptor; mitophagy; parkin; selective autophagy
  2. Redox Biol. 2022 Apr 06. pii: S2213-2317(22)00076-3. [Epub ahead of print]52 102304
      As essential regulators of mitochondrial quality control, mitochondrial dynamics and mitophagy play key roles in maintenance of metabolic health and cellular homeostasis. Here we show that knockdown of the membrane-inserted scaffolding and structural protein caveolin-1 (Cav-1) and expression of tyrosine 14 phospho-defective Cav-1 mutant (Y14F), as opposed to phospho-mimicking Y14D, altered mitochondrial morphology, and increased mitochondrial matrix mixing, mitochondrial fusion and fission dynamics as well as mitophagy in MDA-MB-231 triple negative breast cancer cells. Further, we found that interaction of Cav-1 with mitochondrial fusion/fission machinery Mitofusin 2 (Mfn2) and Dynamin related protein 1 (Drp1) was enhanced by Y14D mutant indicating Cav-1 Y14 phosphorylation prevented Mfn2 and Drp1 translocation to mitochondria. Moreover, limiting mitochondrial recruitment of Mfn2 diminished formation of the PINK1/Mfn2/Parkin complex required for initiation of mitophagy resulting in accumulation of damaged mitochondria and ROS (mtROS). Thus, these studies indicate that phospho-Cav-1 may be an important switch mechanism in cancer cell survival which could lead to novel strategies for complementing cancer therapies.
    Keywords:  Cav-1; Dynamin-related protein 1; Mitochondrial dynamics; Mitofusin 2; Mitophagy; mtROS
  3. Cells. 2022 Apr 01. pii: 1193. [Epub ahead of print]11(7):
      Hypoxia-ischemia (HI) leads to immature brain injury mediated by mitochondrial stress. If damaged mitochondria cannot be repaired, mitochondrial permeabilization ensues, leading to cell death. Non-optimal turnover of mitochondria is critical as it affects short and long term structural and functional recovery and brain development. Therefore, disposal of deficient mitochondria via mitophagy and their replacement through biogenesis is needed. We utilized mt-Keima reporter mice to quantify mitochondrial morphology (fission, fusion) and mitophagy and their mechanisms in primary neurons after Oxygen Glucose Deprivation (OGD) and in brain sections after neonatal HI. Molecular mechanisms of PARK2-dependent and -independent pathways of mitophagy were investigated in vivo by PCR and Western blotting. Mitochondrial morphology and mitophagy were investigated using live cell microscopy. In primary neurons, we found a primary fission wave immediately after OGD with a significant increase in mitophagy followed by a secondary phase of fission at 24 h following recovery. Following HI, mitophagy was upregulated immediately after HI followed by a second wave at 7 days. Western blotting suggests that both PINK1/Parkin-dependent and -independent mechanisms, including NIX and FUNDC1, were upregulated immediately after HI, whereas a PINK1/Parkin mechanism predominated 7 days after HI. We hypothesize that excessive mitophagy in the early phase is a pathologic response which may contribute to secondary energy depletion, whereas secondary mitophagy may be involved in post-HI regeneration and repair.
    Keywords:  metabolism; mitochondria; mitochondrial fission; mitophagy; neonatal brain injury; neonatal hypoxia–ischemia; reactive oxygen species
  4. Mitochondrion. 2022 Apr 06. pii: S1567-7249(22)00028-9. [Epub ahead of print]64 145-155
      We developed a thermal-gelling, erodible hydrogel system for localized delivery of viable mitochondria in vivo, as well as labeled transplanted mitochondria with specific dyes and/or genetically modified mitochondria tagged with red fluorescence protein (RFP). We also employed cell lines to optimize a hydrogel composed of methylcellulose and hyaluronic acid designed to preserve bioenergetics while facilitating mitochondrial release. We further investigated how transplantation of allogeneic or xenogeneic mitochondria into respective cell lines affects host cellular metabolism, as measured by MTS assay. We found that 70% of mitochondria are released from the hydrogel within 20 min at 37 °C, that the respiratory capacity of hydrogel-released mitochondria over 60 min was greater than those without gel, and that MTR-labeling of mitochondria is not indelible. RFP-tagged transgenic mitochondria isolated from modified SH-SY5Y human neuroblastoma cells showed effective uptake into both naïve SH-SY5Y cells and rat PC-12 cells, notably when released from hydrogel. The hydrogel both protected the mitochondria at physiological conditions in vitro while solidifying and diffusing within 60 min locally in situ. To assess metabolic effects, both cell lines were transplanted with different concentrations of SH-SY5Y or PC-12 cell line-derived mitochondria and all resulted in significant increases in metabolism at 6- and 24-hour after transplantation. Alternatively, transplanted mitochondria at highest concentration from rat brain and spinal cord tissues reduced metabolic activities after 24-hour. Along with hydrogel refinements, we are further investigating whether such metabolic changes are due to alterations in cell proliferation or the number of exogenous mitochondria incorporated into individual host cells.
    Keywords:  Hydrogel; MTR; MTS; Metabolism; Mitochondrial transplantation; Spinal cord
  5. Cell Death Dis. 2022 Apr 11. 13(4): 334
      Autophagy-mediated mitochondrial degradation plays pivotal roles in both the acquisition and maintenance of pluripotency, but the molecular mechanisms that link autophagy-mediated mitochondrial homeostasis to pluripotency regulation are unclear. Here, we identified that the mitophagy receptor BNIP3 regulates pluripotency. In mouse ESCs, depletion of BNIP3 caused accumulation of aberrant mitochondria accompanied by decreased mitochondrial membrane potential, increased production of reactive oxygen species (ROS), and reduced ATP generation, which led to compromised self-renewal and differentiation. Impairment of mitophagy by knockdown of BNIP3 inhibited mitochondrial clearance during pluripotency induction, resulting in decreased reprogramming efficiency. These defects were rescued by reacquisition of wild-type but not LIR-deficient BNIP3 expression. Taken together, our findings highlight a critical role of BNIP3-mediated mitophagy in the induction and maintenance of pluripotency.
  6. Mitochondrion. 2022 Apr 06. pii: S1567-7249(22)00027-7. [Epub ahead of print]64 136-144
      In studies with human participants, exosome-based biospecimens can facilitate unique biomarker assessments. As exosome cargos can include mitochondrial components, there is interest in using exosomes to inform the status of an individual's mitochondria. Here, we evaluated whether targeted pharmacologic manipulations could influence the quantity of exosomes shed by cells, and whether these manipulations could impact their mitochondrial cargos. We treated human SH-SY5Y cells with bafilomycin A1, which interferes with general autophagy and mitophagy by inhibiting lysosome acidification and lysosome-autophagosome fusion; deferiprone (DFP), which enhances receptor-mediated mitophagy; or both. Exosome fractions from treated cells were harvested from the cell medium and analyzed for content including mitochondria-derived components. We found bafilomycin increased particle yields, and a combination of bafilomycin plus DFP consistently increased particle yields and mitochondria-associated content. Specifically, the exosome fractions from the bafilomycin plus DFP-treated cells contained more mitochondrial DNA (mtDNA), mtDNA-derived mRNA transcripts, and citrate synthase protein. Our data suggest pharmacologic manipulations that enhance mitophagy initiation, while inhibiting the lysosomal digestion of autophagosomes and multivesicular bodies, could potentially enhance the sensitivity of exosome-based biomarker assays intended to inform the status of an individual's mitochondria.
    Keywords:  Biomarker; Exosome; Mitochondria; Mitochondrial DNA; Mitophagy
  7. Cell Rep. 2022 Apr 12. pii: S2211-1247(22)00387-4. [Epub ahead of print]39(2): 110635
      Circadian genes such as Clock, Bmal1, Cryptochrome1/2, and Period1/2/3 constitute the precise circadian system. ClockΔ19 is a commonly used mouse model harboring a circadian clock gene mutation, which lacks the EXON-19-encoded 51 amino acids. Previous reports have shown that ClockΔ19 mice have severe metabolic abnormalities. Here, we report that the mitochondria of ClockΔ19 mice exhibit excessive fission and dysfunction. We also demonstrate that CLOCK binds to the RNA-binding protein PUF60 through its EXON 19. Further, we find that PUF60 directly maintains mitochondrial homeostasis through regulating Drp1 mRNA stability, while the association with CLOCK can competitively inhibit this function. In ClockΔ19 mice, CLOCKΔ19 releases PUF60, leading to enhanced Drp1 mRNA stability and persistent mitochondrial fission. Our results reveal a direct post-transcriptional role of CLOCK in regulating mitochondrial homeostasis via Drp1 mRNA stability and that the loss of EXON 19 of CLOCK in ClockΔ19 mice leads to severe mitochondrial homeostasis disorders.
    Keywords:  CP: Metabolism; CP: Molecular biology; Clock; Drp1; PUF60; mRNA stability; mitochondrial fission
  8. Biosystems. 2022 Apr 09. pii: S0303-2647(22)00069-7. [Epub ahead of print] 104679
      Theoretical biophysical model is reported for mitochondrial swelling (MS) dynamics in vivo. This newly developed model is based on the detailed biophysical model of MS dynamics in vitro, where mechanical properties of the inner mitochondrial membrane (IMM) were taken into account. The present model of MS dynamics in vivo is capable of analyzing MS dynamic transition from the reversible (physiological) to the irreversible (pathological) mode. This model was used to describe myocytes, assuming 1000 mitochondria distributed homogeneously over the sarcoplasm. Solute transport through the myocyte membrane was described by simplified phenomenological mechanisms of solute uptake and release. Biophysical processes occurring in mitochondria within cells were similar to those included in the earlier reported in vitro biophysical model of MS dynamics. Additionally, in vivo MS dynamics was simulated in different initial conditions, with results different from those of the in vitro model. Note that the presently reported model is the first attempt to develop a detailed biophysical model for the analysis of MS dynamics in vivo, capable of reproducing the transition between reversible and irreversible MS dynamics.
    Keywords:  Biophysical model in vivo; Irreversible mitochondrial swelling; Matrix solute dynamics; Mitochondrial swelling; Reversible mitochondrial swelling; Solute dynamics
  9. EMBO J. 2022 Apr 12. e109390
      Mitophagy removes defective mitochondria via lysosomal elimination. Increased mitophagy coincides with metabolic reprogramming, yet it remains unknown whether mitophagy is a cause or consequence of such state changes. The signalling pathways that integrate with mitophagy to sustain cell and tissue integrity also remain poorly defined. We performed temporal metabolomics on mammalian cells treated with deferiprone, a therapeutic iron chelator that stimulates PINK1/PARKIN-independent mitophagy. Iron depletion profoundly rewired the metabolome, hallmarked by remodelling of lipid metabolism within minutes of treatment. DGAT1-dependent lipid droplet biosynthesis occurred several hours before mitochondrial clearance, with lipid droplets bordering mitochondria upon iron chelation. We demonstrate that DGAT1 inhibition restricts mitophagy in vitro, with impaired lysosomal homeostasis and cell viability. Importantly, genetic depletion of DGAT1 in vivo significantly impaired neuronal mitophagy and locomotor function in Drosophila. Our data define iron depletion as a potent signal that rapidly reshapes metabolism and establishes an unexpected synergy between lipid homeostasis and mitophagy that safeguards cell and tissue integrity.
    Keywords:  DGAT1; iron; lipid droplet; metabolism; mitophagy
  10. Autophagy. 2022 Apr 13. 1-19
      Externalization of the phospholipid cardiolipin (CL) to the outer mitochondrial membrane has been proposed to act as a mitophagy trigger. CL would act as a signal for binding the LC3 macroautophagy/autophagy proteins. As yet, the behavior of the LC3-subfamily members has not been directly compared in a detailed way. In the present contribution, an analysis of LC3A, LC3B and LC3C interaction with CL-containing model membranes, and of their ability to translocate to mitochondria, is described. Binding of LC3A to CL was stronger than that of LC3B; both proteins showed a similar ability to colocalize with mitochondria upon induction of CL externalization in SH-SY5Y cells. Besides, the double silencing of LC3A and LC3B proteins was seen to decrease CCCP-induced mitophagy. Residues 14 and 18 located in the N-terminal region of LC3A were shown to be important for its recognition of damaged mitochondria during rotenone- or CCCP-induced mitophagy. Moreover, the in vitro results suggested a possible role of LC3A, but not of LC3B, in oxidized-CL recognition as a counterweight to excessive apoptosis activation. In the case of LC3C, even if this protein showed a stronger CL binding than LC3B or LC3A, the interaction was less specific, and colocalization of LC3C with mitochondria was not rotenone dependent. These results suggest that, at variance with LC3A, LC3C does not participate in cargo recognition during CL-mediated-mitophagy. The data support the notion that the various LC3-subfamily members might play different roles during autophagy initiation, identifying LC3A as a novel stakeholder in CL-mediated mitophagy. Abbreviations: ACTB/β-actin: actin beta; Atg8: autophagy-related 8; CL: cardiolipin; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; DMSO: dimethyl sulfoxide; DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DTT: DL-dithiothreitol; FKBP8: FKBP prolyl isomerase 8; GABARAP: GABA type A receptor associated protein; GABARAPL1: GABA type A receptor associated protein like 1; GABARAPL2: GABA type A receptor associated protein like 2; GFP: green fluorescent protein; IMM: inner mitochondrial membrane; LUV/LUVs: large unilamellar vesicle/s; MAP1LC3A/LC3A: microtubule associated protein 1 light chain 3 alpha; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAP1LC3C/LC3C: microtubule associated protein 1 light chain 3 gamma; NME4/NDPK-D/Nm23-H4: NME/NM23 nucleoside diphosphate kinase 4; O/A: oligomycin A + antimycin A; OMM: outer mitochondrial membrane; PA: phosphatidic acid; PC: phosphatidylcholine; PG: phosphatidylglycerol; PINK1: PTEN induced putative kinase 1; PtdIns4P: phosphatidylinositol-4-phosphate; Rho-PE: lissamine rhodamine phosphatidylethanolamine; SUV/SUVs: small unilamellar vesicle/s.
    Keywords:  Atg8; LC3/GABARAP-protein family; autophagosome; autophagy cargo recognition; lipid oxidation; lipid-protein interaction; membrane curvature; mitochondria; negatively charged phospholipids