bims-mikwok 2020-09-13 papers

bims-mikwok

Biomed News

on Mitochondrial quality control

Issue of 2020–09–13
sixteen papers selected by
Avinash N. Mukkala, University of Toronto

Assessment of mitochondrial physiology of murine white adipose tissue by mechanical permeabilization and lipid depletion.
Myoglobin promotes nitrite-dependent mitochondrial S-NITROSATION to mediate cytoprotection after hypoxia/reoxygenation.
SLC25A51 is a mammalian mitochondrial NAD+ transporter.
Regulators of calcineurin 1 deficiency attenuates tubulointerstitial fibrosis through improving mitochondrial fitness.
Propofol induces mitochondrial-associated protein LRPPRC and protects mitochondria against hypoxia in cardiac cells.
Two different axes CALCOCO2-RB1CC1 and OPTN-ATG9A initiate PRKN-mediated mitophagy.
Hyperbaric oxygen protects against myocardial ischemia‑reperfusion injury through inhibiting mitochondria dysfunction and autophagy.
Knockout of VDAC1 in H9c2 Cells Promotes Oxidative Stress-Induced Cell Apoptosis through Decreased Mitochondrial Hexokinase II Binding and Enhanced Glycolytic Stress.
Aging Promotes Mitochondria-Mediated Apoptosis in Rat Hearts.
Mitophagy impairment is involved in sevoflurane-induced cognitive dysfunction in aged rats.
ER-mitochondria contacts promote mtDNA nucleoids active transportation via mitochondrial dynamic tubulation.
The Third Coiled Coil Domain of Atg11 Is Required for Shaping Mitophagy Initiation Sites.
GFP fluorescence tagging alters dynamin-related protein 1 oligomerization dynamics and creates disassembly-refractory puncta to mediate mitochondrial fission.
Early intramyocardial implantation of exogenous mitochondria effectively preserved left ventricular function in doxorubicin-induced dilated cardiomyopathy rat.
Bases for Treating Skin Aging With Artificial Mitochondrial Transfer/Transplant (AMT/T).
Chronic intermittent hypoxia alters the dendritic mitochondrial structure and activity in the pre-Bötzinger complex of rats.

Anal Biochem. 2020 Sep 05. pii: S0003-2697(20)30467-X. [Epub ahead of print] 113935

Assessment of mitochondrial physiology of murine white adipose tissue by mechanical permeabilization and lipid depletion.

Natália C Romeiro, Caroline M Ferreira, Marcus F Oliveira.

  White adipose tissue (WAT) represents a major site of triacylglycerol energy storage and is directly associated with metabolic disorders. Mitochondria regulate cellular energy expenditure and are active in WAT. Although isolated mitochondria have been classically used to assess their functions, several artifacts can be introduced by this approach. Furthermore, important limitations exist in the available methods to determine mitochondrial physiology in permeabilized WAT. Here, we established and validated a method for functional evaluation of mice mesenteric WAT (mWAT) mitochondria by using MEchanical Permeabilization and LIpid DEpletion (MEPLIDE) coupled to high-resolution respirometry. We observed that mild stirring of mWAT for 20 minutes at room temperature with 4 % fatty acid-free albumin (FAF-BSA) followed by 50 min without FAF-BSA selectively permeabilized white adipocytes plasma membrane. In these conditions, mWAT mitochondria were intact, exhibiting succinate-induced respiratory rates that were sensitive to classical oxidative phosphorylation modulators. Finally, the respiratory capacity of mWAT in female mice was significantly higher than in males, an observation that agrees with reported data. Therefore, the functional assessment of mWAT mitochondria through MEPLIDE coupled to high resolution respirometry proposed here will contribute to a better understanding of WAT biology in several pathophysiological contexts.

Keywords:  Metabolism; adipose; bioenergetics; method; mitochondria; obesity; respiration

DOI:  https://doi.org/10.1016/j.ab.2020.113935
Nitric Oxide. 2020 Sep 03. pii: S1089-8603(20)30183-X. [Epub ahead of print]

Myoglobin promotes nitrite-dependent mitochondrial S-NITROSATION to mediate cytoprotection after hypoxia/reoxygenation.

Kelly Quesnelle, Danielle A Guimaraes, Krithika Rao, Anuradha Bharara Singh, Yinna Wang, Neil Hogg, Sruti Shiva.

  It is well established that myoglobin supports mitochondrial respiration through the storage and transport of oxygen as well as through the scavenging of nitric oxide. However, during ischemia/reperfusion (I/R), myoglobin and mitochondria both propagate myocardial injury through the production of oxidants. Nitrite, an endogenous signaling molecule and dietary constituent, mediates potent cardioprotection after I/R and this effect relies on its interaction with both myoglobin and mitochondria. While independent mechanistic studies have demonstrated that nitrite-mediated cardioprotection requires the presence of myoglobin and the post-translational S-nitrosation of critical cysteine residues on mitochondrial complex I, it is unclear whether myoglobin directly catalyzes the S-nitrosation of complex I or whether mitochondrial-dependent nitrite reductase activity contributes to S-nitrosation. Herein, using purified myoglobin and isolated mitochondria, we characterize and directly compare the nitrite reductase activities of mitochondria and myoglobin and assess their contribution to mitochondrial S-nitrosation. We demonstrate that myoglobin is a significantly more efficient nitrite reductase than isolated mitochondria. Further, deoxygenated myoglobin catalyzes the nitrite-dependent S-nitrosation of mitochondrial proteins. This reaction is enhanced in the presence of oxidized (Fe3+) myoglobin and not significantly affected by inhibitors of mitochondrial respiration. Using a Chinese Hamster Ovary cell model stably transfected with human myoglobin, we show that both myoglobin and mitochondrial complex I expression are required for nitrite-dependent attenuation of cell death after anoxia/reoxygenation. These data expand the understanding of myoglobin's role both as a nitrite reductase to a mediator of S-nitrosation and as a regulator of mitochondrial function, and have implications for nitrite-mediated cardioprotection after I/R.

Keywords:  S-nitrosothiol; complex I; ischemia; mitochondria; myoglobin; nitrite

DOI:  https://doi.org/10.1016/j.niox.2020.08.005
Nature. 2020 Sep 09.

SLC25A51 is a mammalian mitochondrial NAD+ transporter.

Timothy S Luongo, Jared M Eller, Mu-Jie Lu, Marc Niere, Fabio Raith, Caroline Perry, Marc R Bornstein, Paul Oliphint, Lin Wang, Melanie R McReynolds, Marie E Migaud, Joshua D Rabinowitz, F Brad Johnson, Kai Johnsson, Mathias Ziegler, Xiaolu A Cambronne, Joseph A Baur.

Mitochondria require nicotinamide adenine dinucleotide (NAD+) in order to carry out the fundamental processes that fuel respiration and mediate cellular energy transduction. Mitochondrial NAD+ transporters have been identified in yeast and plants1,2 but their very existence is controversial in mammals3-5. Here we demonstrate that mammalian mitochondria are capable of taking up intact NAD+ and identify SLC25A51 (an essential6,7 mitochondrial protein of previously unknown function, also known as MCART1) as a mammalian mitochondrial NAD+ transporter. Loss of SLC25A51 decreases mitochondrial but not whole-cell NAD+ content, impairs mitochondrial respiration, and blocks the uptake of NAD+ into isolated mitochondria. Conversely, overexpression of SLC25A51 or a nearly identical paralog, SLC25A52, increases mitochondrial NAD+ levels and restores NAD+ uptake into yeast mitochondria lacking endogenous NAD+ transporters. Together, these findings identify SLC25A51 as the first transporter capable of importing NAD+ into mammalian mitochondria.

DOI: https://doi.org/10.1038/s41586-020-2741-7
FASEB J. 2020 Sep 07.

Regulators of calcineurin 1 deficiency attenuates tubulointerstitial fibrosis through improving mitochondrial fitness.

Xue-Yu Sang, Jing-Jie Xiao, Qing Liu, Rui Zhu, Jia-Jia Dai, Cheng Zhang, Hong Yu, Si-Jun Yang, Bai-Fang Zhang.

  Renal fibrosis is the common pathological process of various chronic kidney diseases (CKD). Recent studies indicate that mitochondrial fragmentation is closely associated with renal fibrosis in CKD. However, the molecular mechanisms leading to mitochondrial fragmentation remain to be elucidated. The present study investigated the role of regulators of calcineurin 1 (RCAN1) in mitochondrial fission and renal interstitial fibrosis using conditional knockout mice in which RCAN1 was genetically deleted in tubular epithelial cells (TECs). TEC-specific deletion of RCAN1 attenuated tubulointerstitial fibrosis and epithelial to mesenchymal transition (EMT)-like phenotype change after unilateral ureteral obstruction (UUO) and ischemia reperfusion injury (IRI) through suppressing TGF-β1/Smad3 signaling pathway. TEC-specific deletion of RCAN1 also reduced the tubular apoptosis after UUO by inhibiting cytochrome c/caspase-9 pathway. Ultrastructure analysis revealed a marked decrease in mitochondrial fragmentation in TECs of RCAN1-deficient mice in experimental CKD models. The expression of mitochondrial profission proteins dynamin-related protein 1 (Drp1) and mitochondrial fission factor (Mff) was also downregulated in obstructed kidney of TEC-specific RCAN1-deficient mice. Furthermore, TEC-specific deletion of RCAN1 attenuated the dysfunctional tubular autophagy by regulating PINK1/Parkin-induced mitophagy in CKD. RCAN1 knockdown and knockout similarly improved the mitochondrial quality control in HK-2 cells and primary cultured mouse tubular cells stimulated by TGF-β1. Put together, our data indicated that RCAN1 plays an important role in the progression of tubulointerstitial fibrosis through regulating the mitochondrial quality. Therefore, targeting RCAN1 may provide a potential therapeutic approach in CKD.

Keywords:  chronic kidney disease; mitochondrial fragmentation; tubular apoptosis

DOI:  https://doi.org/10.1096/fj.202000781RRR
PLoS One. 2020 ;15(9): e0238857

Propofol induces mitochondrial-associated protein LRPPRC and protects mitochondria against hypoxia in cardiac cells.

Qianlu Zhang, Shiwei Cai, Liping Guo, Guojun Zhao.

BACKGROUND: Hypoxia-induced oxidative stress is one of the main mechanisms of myocardial injury, which frequently results in cardiomyocyte death and precipitates life-threatening heart failure. Propofol (2,6-diisopropylphenol), which is used to sedate patients during surgery, was shown to strongly affect the regulation of physiological processes, including hypoxia-induced oxidative stress. However, the exact mechanism is still unclear.
METHODS: Expression of LRPPRC, SLIRP, and Bcl-2 after propofol treatment was measured by RT-qPCR and western blot analyses. The effects of propofol under hypoxia were determine by assessing mitochondrial homeostasis and mitochondrial function, including the ATP level and mitochondrial mass. Autophagy/mitophagy was measured by detecting the presence of LC3B, and autophagosomes were observed by transmission microscopy.
RESULTS: Propofol treatment inhibited cleaved caspase-9 and caspase-3, indicating its inhibitory roles in mitochondrial-related apoptosis. Propofol treatment also transcriptionally activated LRPPRC, a mitochondrial-associated protein that exerts multiple functions by maintaining mitochondrial homeostasis, in a manner dependent on the presence of hypoxia-induced factor (HIF)-1α transcriptional activity in H9C2 and primary rat cardiomyocytes. LRPPRC induced by propofol maintained the mitochondrial membrane potential (MMP) and promoted mitochondrial function, including ATP synthesis and transcriptional activity. Furthermore, LRPPRC induced by propofol contributes, at least partially, to the inhibition of apoptotic cell death induced by hypoxia.
CONCLUSION: Taken together, our results indicate that LRPPRC may have a protective antioxidant effect by maintaining mitochondrial homoeostasis induced by propofol and provide new insight into the protective mechanism of propofol against oxidative stress.

DOI: https://doi.org/10.1371/journal.pone.0238857
Autophagy. 2020 Sep 07. 1-3

Two different axes CALCOCO2-RB1CC1 and OPTN-ATG9A initiate PRKN-mediated mitophagy.

Koji Yamano, Richard J Youle.

  PINK1 and PRKN, proteins mutated in Parkinson disease, selectively amplify ubiquitin signals on damaged mitochondria for elimination via mitophagy. Because all five macroautophagy/autophagy receptors in mammals possess domains binding to ubiquitin and Atg8-family proteins, they were thought to recruit Atg8-family protein labeled phagophores from a cytosolic pool. However, our recent findings show that, in addition to Atg8-family protein binding, two of the receptors CALCOCO2 and OPTN interact with RB1CC1 and ATG9A, respectively, indicating that two different axes, CALCOCO2-RB1CC1 and OPTN-ATG9A, can initiate de novo biogenesis of autophagic membranes on ubiquitin-coated damaged mitochondria. These results explain the critical roles of the autophagy receptors CALCOCO2 and OPTN in mitochondrial degradation, and their abilities to simultaneously bind multiple autophagy core proteins propose a new function, i.e. a scaffold to build multivalent interactions for the orchestrated assembly of autophagy proteins near the ubiquitinated cargo.
ABBREVIATIONS: ATG: autophagy-related; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CRABP2: cellular retinoic acid binding protein 2; LIR: MAP1LC3/LC3-interacting region; MAP1LC3: microtubule associated protein 1 light chain 3; NBR1: NBR1 autophagy cargo receptor; OPTN: optineurin; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SNIPER: specific and nongenetic IAP-dependent protein eraser; SQSTM1/p62: sequestosome 1; ULK: unc-51 like autophagy activating kinase.

Keywords:  Mitochondria; PINK1; Parkin; Parkinson’s disease; ubiquitin

DOI:  https://doi.org/10.1080/15548627.2020.1815457
Mol Med Rep. 2020 Sep 09.

Hyperbaric oxygen protects against myocardial ischemia‑reperfusion injury through inhibiting mitochondria dysfunction and autophagy.

Wan Chen, Liwen Lv, Zhihuan Nong, Xiaoyu Chen, Xiaorong Pan, Chunxia Chen.

Our previous study demonstrated that hyperbaric oxygen (HBO) improves heart function predominantly through reducing oxygen stress, modulating energy metabolism and inhibiting cell apoptosis. The present study aimed to investigate the protective effects of HBO on mitochondrial function and autophagy using rats with a ligated left anterior descending artery. The cardioprotective effects of HBO were mainly evaluated using ELISA, fluorescent probes, transmission electron microscopy and reverse transcription‑quantitative PCR (RT‑qPCR). HBO pretreatment for 14 days (once a day) using a 0.25 MPa chamber improved mitochondrial morphology and decreased the number of autophagic vesicles, as observed using a transmission electron microscope. HBO pretreatment significantly increased the levels of ATP, ADP, energy charge and the opening of the mitochondrial permeability transition pore, but decreased the levels of AMP, cytochrome c and reactive oxygen species. Moreover, HBO pretreatment significantly increased the gene or protein expression levels of eIF4E‑binding protein 1, mammalian target of rapamycin (mTOR), mitochondrial DNA, NADH dehydrogenase subunit 1, mitofusin 1 and mitofusin 2, whereas it decreased the gene or protein expression levels of autophagy‑related 5 (Atg5), cytochrome c, dynamin‑related protein 1 and p53, as determined using RT‑qPCR or immunohistochemistry. In conclusion, HBO treatment was observed to protect cardiomyocytes during myocardial ischemia‑reperfusion injury (MIRI) by preventing mitochondrial dysfunction and inhibiting autophagy. Thus, these results provide novel evidence to support the use of HBO as a potential agent for the mitigation of MIRI.

DOI: https://doi.org/10.3892/mmr.2020.11497
Cell Physiol Biochem. 2020 Sep 09. 54(5): 853-874

Knockout of VDAC1 in H9c2 Cells Promotes Oxidative Stress-Induced Cell Apoptosis through Decreased Mitochondrial Hexokinase II Binding and Enhanced Glycolytic Stress.

Meiying Yang, Jie Sun, David F Stowe, Emad Tajkhorshid, Wai-Meng Kwok, Amadou K S Camara.

   BACKGROUND/AIMS: The role of VDAC1, the most abundant mitochondrial outer membrane protein, in cell death depends on cell types and stimuli. Both silencing and upregulation of VDAC1 in various type of cancer cell lines can stimulate apoptosis. In contrast, in mouse embryonic stem (MES) cells and mouse embryonic fibroblasts (MEFs), the roles of VDAC1 knockout (VDAC1-/-) in apoptotic cell death are contradictory. The contribution and underlying mechanism of VDAC1-/- in oxidative stress-induced cell death in cardiac cells has not been established. We hypothesized that VDAC1 is an essential regulator of oxidative stress-induced cell death in H9c2 cells.
METHODS: We knocked out VDAC1 in this rat cardiomyoblast cell line with CRISPR-Cas9 genome editing technique to produce VDAC1-/- H9c2 cells, and determined if VDAC1 is critical in promoting cell death via oxidative stress induced by tert-butylhydroperoxide (tBHP), an organic peroxide, or rotenone (ROT), an inhibitor of mitochondrial complex I by measuring cell viability with MTT assay, cell death with TUNEL stain and LDH release. The mitochondrial and glycolytic stress were examined by measuring O2 consumption rate (OCR) and extracellular acidification rate (ECAR) with a Seahorse XFp analyzer.
RESULTS: We found that under control conditions, VDAC1-/- did not affect H9c2 cell proliferation or mitochondrial respiration. However, compared to the wildtype (WT) cells, exposure to either tBHP or ROT enhanced the production of ROS, ECAR, and the proton (H+) production rate (PPR) from glycolysis, as well as promoted apoptotic cell death in VDAC1-/- H9c2 cells. VDAC1-/- H9c2 cells also exhibited markedly reduced mitochondria-bound hexokinase II (HKII) and Bax. Restoration of VDAC1 in VDAC1-/- H9c2 cells reinstated mitochondria-bound HKII and concomitantly decreased tBHP and ROT-induced ROS production and cell death. Interestingly, mitochondrial respiration remained the same after tBHP treatment in VDAC1-/- and WT H9c2 cells.
CONCLUSION: Our results suggest that VDAC1-/- in H9c2 cells enhances oxidative stress-mediated cell apoptosis that is directly linked to the reduction of mitochondria-bound HKII and concomitantly associated with enhanced ROS production, ECAR, and PPR.

Keywords:  VDAC1 knockout; Oxidative stress; Mitochondria-bound hexokinase II; Extracellular acidification; Cell death/apoptosis; Bax

DOI:  https://doi.org/10.33594/000000274
Life (Basel). 2020 Sep 05. pii: E178. [Epub ahead of print]10(9):

Aging Promotes Mitochondria-Mediated Apoptosis in Rat Hearts.

Mi-Hyun No, Youngju Choi, Jinkyung Cho, Jun-Won Heo, Eun-Jeong Cho, Dong-Ho Park, Ju-Hee Kang, Chang-Ju Kim, Dae Yun Seo, Jin Han, Hyo-Bum Kwak.

  Aging represents a major risk for developing cardiac disease, including heart failure. The gradual deterioration of cell quality control with aging leads to cell death, a phenomenon associated with mitochondrial dysfunction in the heart. Apoptosis is an important quality control process and a necessary phenomenon for maintaining homeostasis and normal function of the heart. However, the mechanism of mitochondria-mediated apoptosis in aged hearts remains poorly understood. Here, we used male Fischer 344 rats of various ages, representing very young (1 month), young (4 months), middle-aged (12 months), and old (20 months) rats, to determine whether mitochondria-mediated apoptotic signals and apoptosis in the left ventricle of the heart are altered notably with aging. As the rats aged, the extramyocyte space and myocyte cross-sectional area in their left ventricle muscle increased, while the number of myocytes decreased. Additionally, mitochondrion-mediated apoptotic signals and apoptosis increased remarkably during aging. Therefore, our results demonstrate that aging promotes remarkable morphological changes and increases the degree of mitochondrion-mediated apoptosis in the left ventricle of rat hearts.

Keywords:  Bcl-2 family; aging heart; mitochondria; programmed cell death

DOI:  https://doi.org/10.3390/life10090178
Aging (Albany NY). 2020 Sep 09. 12

Mitophagy impairment is involved in sevoflurane-induced cognitive dysfunction in aged rats.

Yeru Chen, Piao Zhang, Xianyi Lin, Huan Zhang, Jiamin Miao, Youfa Zhou, Gang Chen.

  Postoperative cognitive dysfunction (POCD) is frequently observed in elderly patients following anesthesia, but its pathophysiological mechanisms have not been fully elucidated. Sevoflurane was reported to repress autophagy in aged rat neurons; however, the role of mitophagy, which is crucial for the control of mitochondrial quality and neuronal health, in sevoflurane-induced POCD in aged rats remains undetermined. Therefore, this study investigated whether mitophagy impairment is involved in sevoflurane-induced cognitive dysfunction. We found sevoflurane treatment inhibited mitochondrial respiration and mitophagic flux, changes in mitochondria morphology, impaired lysosomal acidification, and increased Tomm20 and deceased LAMP1 accumulation were observed in H4 cell and aged rat models. Rapamycin counteracted ROS induced by sevoflurane, restored mitophagy and improved mitochondrial function. Furthermore, rapamycin ameliorated the cognitive deficits observed in aged rats given sevoflurane anesthesia as determined by the Morris water maze test; this improvement was associated with an increased number of dendritic spines and pyramidal neurons. Overexpression of PARK2, but not mutant PARK2 lacking enzyme activity, in H4 cells decreased ROS and Tomm20 accumulation and reversed mitophagy dysfunction after sevoflurane treatment. These findings suggest that mitophagy dysfunction could be a mechanism underlying sevoflurane-induced POCD and that activating mitophagy may provide a new strategy to rescue cognitive deficits.

Keywords:  aged rat; mitophagy; postoperative cognitive dysfunction; rapamycin; sevoflurane

DOI:  https://doi.org/10.18632/aging.103673
Nat Commun. 2020 09 08. 11(1): 4471

ER-mitochondria contacts promote mtDNA nucleoids active transportation via mitochondrial dynamic tubulation.

Jinshan Qin, Yuting Guo, Boxin Xue, Peng Shi, Yang Chen, Qian Peter Su, Huiwen Hao, Shujuan Zhao, Congying Wu, Li Yu, Dong Li, Yujie Sun.

A human cell contains hundreds to thousands of mitochondrial DNA (mtDNA) packaged into nucleoids. Currently, the segregation and allocation of nucleoids are thought to be passively determined by mitochondrial fusion and division. Here we provide evidence, using live-cell super-resolution imaging, that nucleoids can be actively transported via KIF5B-driven mitochondrial dynamic tubulation (MDT) activities that predominantly occur at the ER-mitochondria contact sites (EMCS). We further demonstrate that a mitochondrial inner membrane protein complex MICOS links nucleoids to Miro1, a KIF5B receptor on mitochondria, at the EMCS. We show that such active transportation is a mechanism essential for the proper distribution of nucleoids in the peripheral zone of the cell. Together, our work identifies an active transportation mechanism of nucleoids, with EMCS serving as a key platform for the interplay of nucleoids, MICOS, Miro1, and KIF5B to coordinate nucleoids segregation and transportation.

DOI: https://doi.org/10.1038/s41467-020-18202-4
J Mol Biol. 2020 Sep 04. pii: S0022-2836(20)30528-3. [Epub ahead of print]

The Third Coiled Coil Domain of Atg11 Is Required for Shaping Mitophagy Initiation Sites.

Hannah K Margolis, Sarah Katzenell, Kelsie A Leary, Michael J Ragusa.

  Selective autophagy is the capture of specific cytosolic contents in double membrane vesicles that subsequently fuse with the vacuole or lysosome, thereby delivering cargo for degradation. Selective autophagy receptors (SARs) mark the cargo for degradation and, in yeast, recruit Atg11, the scaffolding protein for selective autophagy initiation. The mitochondrial protein Atg32 is the yeast SAR that mediates mitophagy, the selective autophagic capture of mitochondria. Atg11-Atg32 interactions concentrate Atg32 into puncta that are thought to represent sites of mitophagy initiation. However, it is unclear how Atg11 concentrates Atg32 to generate mitophagy initiation sites. We show here that the coiled coil 3 (CC3) domain of Atg11 is required for concentrating Atg32 into puncta. We determined the structure of the majority of the CC3, demonstrating that the CC3 forms a parallel homodimer whose dimer interface is formed by a small number of hydrophobic residues. We further show that the CC3 interface is not required for Atg11 dimerization but is required for shaping Atg32 into functional mitophagy initiation sites and for delivery of mitochondria to the vacuole. Our findings suggest that Atg11 self-interactions help concentrate SARs as a necessary precondition for cargo capture.

Keywords:  Atg11; Autophagy; Crystal structure; Mitophagy

DOI:  https://doi.org/10.1016/j.jmb.2020.08.025
Sci Rep. 2020 Sep 08. 10(1): 14777

GFP fluorescence tagging alters dynamin-related protein 1 oligomerization dynamics and creates disassembly-refractory puncta to mediate mitochondrial fission.

Felipe Montecinos-Franjola, Brianna L Bauer, Jason A Mears, Rajesh Ramachandran.

Green fluorescent protein (GFP)-tagging is the prevalent strategy to monitor protein dynamics in living cells. However, the consequences of appending the bulky GFP moiety to the protein of interest are rarely investigated. Here, using a powerful combination of quantitative fluorescence spectroscopic and imaging techniques, we have examined the oligomerization dynamics of the GFP-tagged mitochondrial fission GTPase dynamin-related protein 1 (Drp1) both in vitro and in vivo. We find that GFP-tagged Drp1 exhibits impaired oligomerization equilibria in solution that corresponds to a greatly diminished cooperative GTPase activity in comparison to native Drp1. Consequently, GFP-tagged Drp1 constitutes aberrantly stable, GTP-resistant supramolecular assemblies both in vitro and in vivo, neither of which reflects a more dynamic native Drp1 oligomerization state. Indeed, GFP-tagged Drp1 is detected more frequently per unit length over mitochondria in Drp1-null mouse embryonic fibroblasts (MEFs) compared to wild-type (wt) MEFs, indicating that the drastically reduced GTP turnover restricts oligomer disassembly from the mitochondrial surface relative to mixed oligomers comprising native and GFP-tagged Drp1. Yet, GFP-tagged Drp1 retains the capacity to mediate membrane constriction in vitro and mitochondrial division in vivo. These findings suggest that instead of robust assembly-disassembly dynamics, persistent Drp1 higher-order oligomerization over membranes is sufficient for mitochondrial fission.

DOI: https://doi.org/10.1038/s41598-020-71655-x
Am J Transl Res. 2020 ;12(8): 4612-4627

Early intramyocardial implantation of exogenous mitochondria effectively preserved left ventricular function in doxorubicin-induced dilated cardiomyopathy rat.

Hon-Kan Yip, Pei-Lin Shao, Christopher Glenn Wallace, Jiunn-Jye Sheu, Pei-Hsun Sung, Mel S Lee.

This study tested the hypothesis that early implantation of mitochondria (Mito) into left myocardium could effectively protect heart against doxorubicin/12 mg/kg-induced dilated cardiomyopathy (DCM) in rat. Adult-male SD rats (n = 18) were equally categorized into group 1 (sham control), group 2 (DCM) and group 3 [DCM + Mito (500 μg/rat intramyocardial injection by day-21 after DCM induction)] and euthanized by day 60. In vitro studies showed that exogenously-transferred Mito was abundantly identified in H9C2 cells. The q-PCR showed significant increase in relative number of mitDNA in Mito-transferred H9C2 cells than in control group (P<0.001). The mRNA-gene and protein expressions of NRF1/NRF2/Tfam/PGC-1α/ERRα/Mfn2 were significantly increased in low-dose Mito-transferred and more significantly increased in high-dose Mito-transferred H29C2 cells than in control group (all P<0.01). Day-60 left-ventricular-ejection-fraction (LVEF) was significantly lower in group 2 than in groups 1 and 3, and significantly lower in group 3 than in group 1 (P<0.0001). The ratios of lung and heart weights to tibial length and myocardial histopathological findings of fibrotic area/myocardial injured score/γ-H2AX+ cells exhibited an opposite pattern to LVEF among the three groups (all P<0.0001). The myocardial protein expressions of oxidative-stress (NOX-1/NOX-2/oxidized protein/p22phox), autophagic (beclin-1/Atg-5/ratio of CL3B-II/CL3B-I), and apoptotic/mitochondrial-damaged (cleaved-caspase-3/mitochondrial Bax/cleaved-PARP/cytosolic-cytochrome-C/DRP1/cyclophilin D1) biomarkers exhibited an opposite pattern, whereas the protein expressions of mitochondrial integrity (mitochondrial-cytochrome-C/mitochondrial-complex I/II/III/IV and Mfn2/PGC-1) exhibited an identical pattern to LVEF among the groups (all P<0.001). In conclusion, early Mito therapy effectively preserved LVEF and myocardial integrity in DCM rat.

Keywords: Mitochondria; dilated cardiomyopathy; doxorubicin; molecular-cellular perturbations
Front Bioeng Biotechnol. 2020 ;8 919

Bases for Treating Skin Aging With Artificial Mitochondrial Transfer/Transplant (AMT/T).

Micaela Balcázar, Stalin Cañizares, Tatiana Borja, Patricia Pontón, Sirivanh Bisiou, Eva Carabasse, Angela Bacilieri, Celia Canavese, Ramiro F Diaz, Francisco Cabrera, Andrés Caicedo.

  The perception of mitochondria as only the powerhouse of the cell has dramatically changed in the last decade. It is now accepted that in addition to being essential intracellularly, mitochondria can promote cellular repair when transferred from healthy to damaged cells. The artificial mitochondria transfer/transplant (AMT/T) group of techniques emulate this naturally occurring process and have been used to develop therapies to treat a range of diseases including cardiac and neurodegenerative. Mitochondria accumulate damage with time, resulting in cellular senescence. Skin cells and its mitochondria are profoundly affected by ultraviolet radiation and other factors that induce premature and accelerated aging. In this article, we propose the basis to use AMT/T to treat skin aging by transferring healthy mitochondria to senescent cells, possibly revitalizing them. We provide insightful information about how skin structure, components, and cells could age rapidly depending on the amount of damage received. Arguments are shown in favor of the use of AMT/T to treat aging skin and its cells, among them the possibility to stop free radical production, add new genetic material, and provide an energetic boost to help cells prolong their viability over time. This article intends to present one of the many aspects in which mitochondria could be used as a universal treatment for cell and tissue damage and aging.

Keywords:  MitoCeption; aging; artificial mitochondria transfer transplant (AMT/T); mitochondria; regenerative medicine; senescence; skin

DOI:  https://doi.org/10.3389/fbioe.2020.00919
FASEB J. 2020 Sep 10.

Chronic intermittent hypoxia alters the dendritic mitochondrial structure and activity in the pre-Bötzinger complex of rats.

Jun-Jun Kang, Man-Lung Fung, Kun Zhang, Chun-Sing Lam, Sheng-Xi Wu, Xiao-Feng Huang, Shou-Jing Yang, Margaret T T Wong-Riley, Ying-Ying Liu.

  Mitochondrial bioenergetics is dynamically coupled with neuronal activities, which are altered by hypoxia-induced respiratory neuroplasticity. Here we report structural features of postsynaptic mitochondria in the pre-Bötzinger complex (pre-BötC) of rats treated with chronic intermittent hypoxia (CIH) simulating a severe condition of obstructive sleep apnea. The subcellular changes in dendritic mitochondria and histochemistry of cytochrome c oxidase (CO) activity were examined in pre-BötC neurons localized by immunoreactivity of neurokinin 1 receptors. Assays of mitochondrial electron transport chain (ETC) complex I, IV, V activities, and membrane potential were performed in the ventrolateral medulla containing the pre-BötC region. We found significant decreases in the mean length and area of dendritic mitochondria in the pre-BötC of CIH rats, when compared to the normoxic control and hypoxic group with daily acute intermittent hypoxia (dAIH) that evokes robust synaptic plasticity. Notably, these morphological alterations were mainly observed in the mitochondria in close proximity to the synapses. In addition, the proportion of mitochondria presented with enlarged compartments and filamentous cytoskeletal elements in the CIH group was less than the control and dAIH groups. Intriguingly, these distinct characteristics of structural adaptability were observed in the mitochondria within spatially restricted dendritic spines. Furthermore, the proportion of moderately to darkly CO-reactive mitochondria was reduced in the CIH group, indicating reduced mitochondrial activity. Consistently, mitochondrial ETC enzyme activities and membrane potential were lowered in the CIH group. These findings suggest that hypoxia-induced respiratory plasticity was characterized by spatially confined mitochondrial alterations within postsynaptic spines in the pre-BötC neurons. In contrast to the robust plasticity evoked by dAIH preconditioning, a severe CIH challenge may weaken the local mitochondrial bioenergetics that the fuel postsynaptic activities of the respiratory motor drive.

Keywords:  intermittent hypoxia; neuroplasticity; postsynaptic; respiration; ultrastructure·chronic

DOI:  https://doi.org/10.1096/fj.201902141R