bims-mikwok 2023-01-15 papers

bims-mikwok

Biomed News

on Mitochondrial quality control

Issue of 2023–01–15
eleven papers selected by
Avinash N. Mukkala, University of Toronto

Translation initiation of leaderless and polycistronic transcripts in mammalian mitochondria.
Mitochondrial transplantation against gentamicin-induced toxicity on rat renal proximal tubular cells: the higher activity of female rat mitochondria.
The mitophagy receptor BNIP3 is critical for the regulation of metabolic homeostasis and mitochondrial function in the nucleus pulposus cells of the intervertebral disc.
High-content phenotypic screen to identify small molecule enhancers of Parkin-dependent ubiquitination and mitophagy.
Monitoring Mitochondrial Protein Import Using Mitochondrial Targeting Sequence (MTS)-eGFP.
Targeting succinate metabolism to decrease brain injury upon mechanical thrombectomy treatment of ischemic stroke.
Intravenous Transplantation of an Ischemic-specific Peptide-TPP-mitochondrial Compound Alleviates Myocardial Ischemic Reperfusion Injury.
Pyruvate dehydrogenase kinase supports macrophage NLRP3 inflammasome activation during acute inflammation.
Mitochondrial Transfer Induced by Adipose-Derived Mesenchymal Stem Cell Transplantation Improves Cardiac Function in Rat Models of Ischemic Cardiomyopathy.
Disruption of mitochondrial dynamics triggers muscle inflammation through interorganellar contacts and mitochondrial DNA mislocation.
OxPhos defects cause hypermetabolism and reduce lifespan in cells and in patients with mitochondrial diseases.

Nucleic Acids Res. 2023 Jan 11. pii: gkac1233. [Epub ahead of print]

Translation initiation of leaderless and polycistronic transcripts in mammalian mitochondria.

Cristina Remes, Anas Khawaja, Sarah F Pearce, Adam M Dinan, Shreekara Gopalakrishna, Miriam Cipullo, Vasileios Kyriakidis, Jingdian Zhang, Xaquin Castro Dopico, Olessya Yukhnovets, Ilian Atanassov, Andrew E Firth, Barry Cooperman, Joanna Rorbach.

The synthesis of mitochondrial OXPHOS complexes is central to cellular metabolism, yet many molecular details of mitochondrial translation remain elusive. It has been commonly held view that translation initiation in human mitochondria proceeded in a manner similar to bacterial systems, with the mitoribosomal small subunit bound to the initiation factors, mtIF2 and mtIF3, along with initiator tRNA and an mRNA. However, unlike in bacteria, most human mitochondrial mRNAs lack 5' leader sequences that can mediate small subunit binding, raising the question of how leaderless mRNAs are recognized by mitoribosomes. By using novel in vitro mitochondrial translation initiation assays, alongside biochemical and genetic characterization of cellular knockouts of mitochondrial translation factors, we describe unique features of translation initiation in human mitochondria. We show that in vitro, leaderless mRNA transcripts can be loaded directly onto assembled 55S mitoribosomes, but not onto the mitoribosomal small subunit (28S), in a manner that requires initiator fMet-tRNAMet binding. In addition, we demonstrate that in human cells and in vitro, mtIF3 activity is not required for translation of leaderless mitochondrial transcripts but is essential for translation of ATP6 in the case of the bicistronic ATP8/ATP6 transcript. Furthermore, we show that mtIF2 is indispensable for mitochondrial protein synthesis. Our results demonstrate an important evolutionary divergence of the mitochondrial translation system and further our fundamental understanding of a process central to eukaryotic metabolism.

DOI: https://doi.org/10.1093/nar/gkac1233
In Vitro Cell Dev Biol Anim. 2023 Jan 11.

Mitochondrial transplantation against gentamicin-induced toxicity on rat renal proximal tubular cells: the higher activity of female rat mitochondria.

Abdollah Arjmand, Saba Shiranirad, Fateme Ameritorzani, Farzaneh Kamranfar, Enayatollah Seydi, Jalal Pourahmad.

  Mitochondrial dysfunction is a fundamental mechanism leading to drug nephrotoxicity, such as gentamicin-induced nephrotoxicity. Mitochondrial therapy (mitotherapy) or exogenous mitochondria transplantation is a method that can be used to replace dysfunctional mitochondria with healthy mitochondria. This method can help in the treatment of diseases related to mitochondria. In this research, we studied the transplantation effect of freshly isolated mitochondria on the toxicity induced by gentamicin on renal proximal tubular cells (RPTCs). Furthermore, possible gender-related effects on supplying exogenous rat kidney mitochondria on gentamicin-induced RPTCs were investigated. At first, the normality and proper functioning of fresh mitochondria were assessed by measuring mitochondrial succinate dehydrogenase activity (SDH) and changes in mitochondrial membrane potential (MMP). Then, the protective effects of mitochondrial transplantation against gentamicin-induced mitochondrial toxicity were evaluated through parameters including lactate dehydrogenase (LDH) leakiness, reactive oxygen species (ROS) production, lipid peroxidation (LPO) content, reduced glutathione (GSH) level, extracellular oxidized glutathione (GSSG) level, ATP level, MMP collapse, and caspase-3 activity. According to the statistical analysis, transplanting the healthy mitochondria decreased the cytotoxicity, ROS production, MMP collapse, LPO content, GSSG levels, and caspase-3 activity caused by gentamicin in RPTCs. Also, it has caused an increase in the level of ATP and GSH in the RPTCs. Furthermore, higher preventive effects were observed for the female group. According to the current study, mitochondrial transplantation is a potent therapeutic method in xenobiotic-caused nephrotoxicity.

Keywords:  Gentamicin; Mitochondrial transplantation; Nephrotoxicity; Oxidative stress

DOI:  https://doi.org/10.1007/s11626-022-00743-1
Autophagy. 2023 Jan 10. 1-23

The mitophagy receptor BNIP3 is critical for the regulation of metabolic homeostasis and mitochondrial function in the nucleus pulposus cells of the intervertebral disc.

Vedavathi Madhu, Miriam Hernandez-Meadows, Paige K Boneski, Yunping Qiu, Anyonya R Guntur, Irwin J Kurland, Ruteja A Barve, Makarand V Risbud.

  The contribution of mitochondria to the metabolic function of hypoxic NP cells has been overlooked. We have shown that NP cells contain networked mitochondria and that mitochondrial translocation of BNIP3 mediates hypoxia-induced mitophagy. However, whether BNIP3 also plays a role in governing mitochondrial function and metabolism in hypoxic NP cells is not known. BNIP3 knockdown altered mitochondrial morphology, and number, and increased mitophagy. Interestingly, BNIP3 deficiency in NP cells reduced glycolytic capacity reflected by lower production of lactate/H+ and lower ATP production rate. Widely targeted metabolic profiling and flux analysis using 1-2-13C-glucose showed that the BNIP3 loss resulted in redirection of glycolytic flux into pentose phosphate and hexosamine biosynthesis as well as pyruvate resulting in increased TCA flux. An overall reduction in one-carbon metabolism was noted suggesting reduced biosynthesis. U13C-glutamine flux analysis showed preservation of glutamine utilization to maintain TCA intermediates. The transcriptomic analysis of the BNIP3-deficient cells showed dysregulation of cellular functions including membrane and cytoskeletal integrity, ECM-growth factor signaling, and protein quality control with an overall increase in themes related to angiogenesis and innate immune response. Importantly, we observed strong thematic similarities with the transcriptome of a subset of human degenerative samples. Last, we noted increased autophagic flux, decreased disc height index and aberrant COL10A1/collagen X expression, signs of early disc degeneration in young adult bnip3 knockout mice. These results suggested that in addition to mitophagy regulation, BNIP3 plays a role in maintaining mitochondrial function and metabolism, and dysregulation of mitochondrial homeostasis could promote disc degeneration.Abbreviations: ECAR extracellular acidification rate; HIF hypoxia inducible factor; MFA metabolic flux analysis; NP nucleus pulposus; OCR oxygen consumption rate; ShBnip3 short-hairpin Bnip3.

Keywords:  BNIP3; disc degeneration; hypoxia; intervertebral disc; metabolism; mitochondria; mitophagy; nucleus pulposus

DOI:  https://doi.org/10.1080/15548627.2022.2162245
SLAS Discov. 2023 Jan 03. pii: S2472-5552(22)13716-0. [Epub ahead of print]

High-content phenotypic screen to identify small molecule enhancers of Parkin-dependent ubiquitination and mitophagy.

Roberta Tufi, Emily H Clark, Tamaki Hoshikawa, Christiana Tsagkaraki, Jack Stanley, Kunitoshi Takeda, James M Staddon, Thomas Briston.

  Mitochondrial dysfunction and aberrant mitochondrial homeostasis are key aspects of Parkinson's disease (PD) pathophysiology. Mutations in PINK1 and Parkin proteins lead to autosomal recessive PD, suggesting that defective mitochondrial clearance via mitophagy is key in PD etiology. Accelerating the identification and/or removal of dysfunctional mitochondria could therefore provide a disease-modifying approach to treatment. To that end, we performed a high-content phenotypic screen (HCS) of ∼125,000 small molecules to identify compounds that positively modulate mitochondrial accumulation of the PINK1-Parkin-dependent mitophagy initiation marker p-Ser65-Ub in Parkin haploinsufficiency (Parkin +/R275W) human fibroblasts. Following confirmatory counter-screening and orthogonal assays, we selected compounds of interest that enhance mitophagy-related biochemical and functional endpoints in patient-derived fibroblasts. Identification of inhibitors of the ubiquitin-specific peptidase and negative regulator of mitophagy USP30 within our hits further validated our approach. The compounds identified provide a novel starting point for further investigation and optimisation.

Keywords:  PINK1; Parkin; Parkinson's disease; USP30; high-content screening; mitophagy

DOI:  https://doi.org/10.1016/j.slasd.2022.12.004
Bio Protoc. 2022 Dec 20. pii: e4578. [Epub ahead of print]12(24):

Monitoring Mitochondrial Protein Import Using Mitochondrial Targeting Sequence (MTS)-eGFP.

Jonas B Michaelis, Süleyman Bozkurt, Jasmin A Schäfer, Christian Münch.

  Mitochondria are cellular organelles essential for the function and survival of eukaryotic cells. Nearly all mitochondrial proteins are nuclear-encoded and require mitochondrial import upon their synthesis in the cytosol. Various approaches have been described to study mitochondrial protein import, such as monitoring the entry of radiolabeled proteins into purified mitochondria or quantifying newly synthesized proteins within mitochondria by proteomics. Here, we provide a detailed protocol for a commonly used and straightforward assay that quantitatively examines mitochondrial protein import by monitoring the co-localization of mitochondrially targeted enhanced green fluorescent protein (eGFP) with the mitochondrial fluorescence dye MitoTracker TM Deep Red FM by live cell imaging. We describe the preparation and use of a stable mammalian cell line inducibly expressing a mitochondrial targeting sequence (MTS)-eGFP, followed by quantitative image analysis using an open-source ImageJ-based plugin. This inducible expression system avoids the need for transient transfection while enabling titration of MTS-eGFP expression and thereby avoiding protein folding stress. Overall, the assay provides a simple and robust approach to assess mitochondrial import capacity of cells in various disease-related settings. This protocol was validated in: Mol Cell (2021), DOI: 10.1016/j.molcel.2021.11.004 Graphical abstract.

Keywords:   Live cell imaging ; Microscopy ; Mitochondria ; Mitochondrial protein import ; Protein translocation

DOI:  https://doi.org/10.21769/BioProtoc.4578
Redox Biol. 2023 Feb;pii: S2213-2317(23)00001-0. [Epub ahead of print]59 102600

Targeting succinate metabolism to decrease brain injury upon mechanical thrombectomy treatment of ischemic stroke.

Amin Mottahedin, Hiran A Prag, Andreas Dannhorn, Richard Mair, Christina Schmidt, Ming Yang, Annabel Sorby-Adams, Jordan J Lee, Nils Burger, Duvaraka Kulaveerasingam, Margaret M Huang, Stefano Pluchino, Luca Peruzzotti-Jametti, Richard Goodwin, Christian Frezza, Michael P Murphy, Thomas Krieg.

Current treatments for acute ischemic stroke aim to reinstate a normal perfusion in the ischemic territory but can also cause significant ischemia-reperfusion (IR) injury. Previous data in experimental models of stroke show that ischemia leads to the accumulation of succinate, and, upon reperfusion, the accumulated succinate is rapidly oxidized by succinate dehydrogenase (SDH) to drive superoxide production at mitochondrial complex I. Despite this process initiating IR injury and causing further tissue damage, the potential of targeting succinate metabolism to minimize IR injury remains unexplored. Using both quantitative and untargeted high-resolution metabolomics, we show a time-dependent accumulation of succinate in both human and mouse brain exposed to ischemia ex vivo. In a mouse model of ischemic stroke/mechanical thrombectomy mass spectrometry imaging (MSI) shows that succinate accumulation is confined to the ischemic region, and that the accumulated succinate is rapidly oxidized upon reperfusion. Targeting succinate oxidation by systemic infusion of the SDH inhibitor malonate upon reperfusion leads to a dose-dependent decrease in acute brain injury. Together these findings support targeting succinate metabolism upon reperfusion to decrease IR injury as a valuable adjunct to mechanical thrombectomy in ischemic stroke.

DOI: https://doi.org/10.1016/j.redox.2023.102600
ACS Nano. 2023 Jan 10.

Intravenous Transplantation of an Ischemic-specific Peptide-TPP-mitochondrial Compound Alleviates Myocardial Ischemic Reperfusion Injury.

Xiaolei Sun, Hang Chen, Rifeng Gao, Yanan Qu, Ya Huang, Ning Zhang, Shiyu Hu, Fan Fan, Yunzeng Zou, Kai Hu, Zhaoyang Chen, Junbo Ge, Aijun Sun.

  It is known that mitochondrial dysfunction is a critical factor involved in myocardial ischemia-reperfusion injury. Mitochondrial transplantation has been suggested as an effective therapeutic strategy to protect against myocardial ischemia-reperfusion injury. However, its clinical translation remains limited because it requires the local injection of mitochondria into the myocardium. Here, a polypeptide, CSTSMLKAC (PEP), bound to triphenylphosphonium cations (TPP+) effectively binds mitochondria to form a PEP-TPP-mitochondrial compound. Further investigation of this compound has revealed that the ischemia-sensing properties of PEP promote its translocation into the ischemic myocardium. Additionally, the targeting peptide, PEP, readily dissociates from the PEP-TPP-mitochondrial compound, allowing for the transplanted mitochondria to be efficiently internalized by cardiomyocytes or transferred to cardiomyocytes by endothelial cells. Mitochondrial transplantation promotes cardiomyocyte energetics and mechanical contraction, subsequently reducing cellular apoptosis, macrophage infiltration, and the pro-inflammatory response, all of which lead to attenuation of ischemia-reperfusion injury. Thus, this study provides promising evidence that the PEP-TPP-mitochondrial compound effectively promotes intravenous mitochondrial transplantation into the ischemic myocardium and subsequently ameliorates myocardial ischemia-reperfusion injury.

Keywords:  PEP−TPP−mitochondrial compound; cardiomyocytes; mitochondrial transplantation; myocardial ischemia−reperfusion injury; targeting peptide

DOI:  https://doi.org/10.1021/acsnano.2c05286
Cell Rep. 2023 Jan 09. pii: S2211-1247(22)01842-3. [Epub ahead of print]42(1): 111941

Pyruvate dehydrogenase kinase supports macrophage NLRP3 inflammasome activation during acute inflammation.

Allison K Meyers, Zhan Wang, Wenzheng Han, Qingxia Zhao, Manal Zabalawi, Likun Duan, Juan Liu, Qianyi Zhang, Rajesh K Manne, Felipe Lorenzo, Matthew A Quinn, Qianqian Song, Daping Fan, Hui-Kuan Lin, Cristina M Furdui, Jason W Locasale, Charles E McCall, Xuewei Zhu.

  Activating the macrophage NLRP3 inflammasome can promote excessive inflammation with severe cell and tissue damage and organ dysfunction. Here, we show that pharmacological or genetic inhibition of pyruvate dehydrogenase kinase (PDHK) significantly attenuates NLRP3 inflammasome activation in murine and human macrophages and septic mice by lowering caspase-1 cleavage and interleukin-1β (IL-1β) secretion. Inhibiting PDHK reverses NLRP3 inflammasome-induced metabolic reprogramming, enhances autophagy, promotes mitochondrial fusion over fission, preserves crista ultrastructure, and attenuates mitochondrial reactive oxygen species (ROS) production. The suppressive effect of PDHK inhibition on the NLRP3 inflammasome is independent of its canonical role as a pyruvate dehydrogenase regulator. Our study suggestsa non-canonical role of mitochondrial PDHK in promoting mitochondrial stress and supporting NLRP3 inflammasome activation during acute inflammation.

Keywords:  CP: Immunology; NLRP3 inflammasome; autophagy; cristae; immunometabolism; macrophages; metabolic flux; mitochondria; mitochondrial fission and fusion; pyruvate dehydrogenase kinase; sepsis

DOI:  https://doi.org/10.1016/j.celrep.2022.111941
Cell Transplant. 2023 Jan-Dec;32:32 9636897221148457

Mitochondrial Transfer Induced by Adipose-Derived Mesenchymal Stem Cell Transplantation Improves Cardiac Function in Rat Models of Ischemic Cardiomyopathy.

Daisuke Mori, Shigeru Miyagawa, Takuji Kawamura, Daisuke Yoshioka, Hiroki Hata, Takayoshi Ueno, Koichi Toda, Toru Kuratani, Miwa Oota, Kotoe Kawai, Hayato Kurata, Hiroyuki Nishida, Akima Harada, Toshihiko Toyofuku, Yoshiki Sawa.

  Although mesenchymal stem cell transplantation has been successful in the treatment of ischemic cardiomyopathy, the underlying mechanisms remain unclear. Herein, we investigated whether mitochondrial transfer could explain the success of cell therapy in ischemic cardiomyopathy. Mitochondrial transfer in co-cultures of human adipose-derived mesenchymal stem cells and rat cardiomyocytes maintained under hypoxic conditions was examined. Functional recovery was monitored in a rat model of myocardial infarction following human adipose-derived mesenchymal stem cell transplantation. We observed mitochondrial transfer in vitro, which required the formation of cell-to-cell contacts and synergistically enhanced energy metabolism. Rat cardiomyocytes exhibited mitochondrial transfer 3 days following human adipose-derived mesenchymal stem cell transplantation to the ischemic heart surface post-myocardial infarction. We detected donor mitochondrial DNA in the recipient myocardium concomitant with a significant improvement in cardiac function. Mitochondrial transfer is vital for successful cell transplantation therapies and improves treatment outcomes in ischemic cardiomyopathy.

Keywords:  adipose derived mesenchymal stem cell; epicardial cell transplantation; gap junction; ischemic cardiomyopathy; mitochondrial transfer

DOI:  https://doi.org/10.1177/09636897221148457
Nat Commun. 2023 Jan 06. 14(1): 108

Disruption of mitochondrial dynamics triggers muscle inflammation through interorganellar contacts and mitochondrial DNA mislocation.

Andrea Irazoki, Isabel Gordaliza-Alaguero, Emma Frank, Nikolaos Nikiforos Giakoumakis, Jordi Seco, Manuel Palacín, Anna Gumà, Lykke Sylow, David Sebastián, Antonio Zorzano.

Some forms of mitochondrial dysfunction induce sterile inflammation through mitochondrial DNA recognition by intracellular DNA sensors. However, the involvement of mitochondrial dynamics in mitigating such processes and their impact on muscle fitness remain unaddressed. Here we report that opposite mitochondrial morphologies induce distinct inflammatory signatures, caused by differential activation of DNA sensors TLR9 or cGAS. In the context of mitochondrial fragmentation, we demonstrate that mitochondria-endosome contacts mediated by the endosomal protein Rab5C are required in TLR9 activation in cells. Skeletal muscle mitochondrial fragmentation promotes TLR9-dependent inflammation, muscle atrophy, reduced physical performance and enhanced IL6 response to exercise, which improved upon chronic anti-inflammatory treatment. Taken together, our data demonstrate that mitochondrial dynamics is key in preventing sterile inflammatory responses, which precede the development of muscle atrophy and impaired physical performance. Thus, we propose the targeting of mitochondrial dynamics as an approach to treating disorders characterized by chronic inflammation and mitochondrial dysfunction.

DOI: https://doi.org/10.1038/s41467-022-35732-1
Commun Biol. 2023 Jan 12. 6(1): 22

OxPhos defects cause hypermetabolism and reduce lifespan in cells and in patients with mitochondrial diseases.

Gabriel Sturm, Kalpita R Karan, Anna S Monzel, Balaji Santhanam, Tanja Taivassalo, Céline Bris, Sarah A Ware, Marissa Cross, Atif Towheed, Albert Higgins-Chen, Meagan J McManus, Andres Cardenas, Jue Lin, Elissa S Epel, Shamima Rahman, John Vissing, Bruno Grassi, Morgan Levine, Steve Horvath, Ronald G Haller, Guy Lenaers, Douglas C Wallace, Marie-Pierre St-Onge, Saeed Tavazoie, Vincent Procaccio, Brett A Kaufman, Erin L Seifert, Michio Hirano, Martin Picard.

Patients with primary mitochondrial oxidative phosphorylation (OxPhos) defects present with fatigue and multi-system disorders, are often lean, and die prematurely, but the mechanistic basis for this clinical picture remains unclear. By integrating data from 17 cohorts of patients with mitochondrial diseases (n = 690) we find evidence that these disorders increase resting energy expenditure, a state termed hypermetabolism. We examine this phenomenon longitudinally in patient-derived fibroblasts from multiple donors. Genetically or pharmacologically disrupting OxPhos approximately doubles cellular energy expenditure. This cell-autonomous state of hypermetabolism occurs despite near-normal OxPhos coupling efficiency, excluding uncoupling as a general mechanism. Instead, hypermetabolism is associated with mitochondrial DNA instability, activation of the integrated stress response (ISR), and increased extracellular secretion of age-related cytokines and metabokines including GDF15. In parallel, OxPhos defects accelerate telomere erosion and epigenetic aging per cell division, consistent with evidence that excess energy expenditure accelerates biological aging. To explore potential mechanisms for these effects, we generate a longitudinal RNASeq and DNA methylation resource dataset, which reveals conserved, energetically demanding, genome-wide recalibrations. Taken together, these findings highlight the need to understand how OxPhos defects influence the energetic cost of living, and the link between hypermetabolism and aging in cells and patients with mitochondrial diseases.

DOI: https://doi.org/10.1038/s42003-022-04303-x