bims-mitdyn 2022-01-02 papers

bims-mitdyn

Biomed News

on Mitochondrial dynamics: mechanisms

Issue of 2022–01–02
eight papers selected by
Edmond Chan, Queen’s University, School of Medicine

SIRT2 regulates mitochondrial dynamics and reprogramming via MEK1-ERK-DRP1 and AKT1-DRP1 axes.
NAD kinase sustains lipogenesis and mitochondrial metabolismthrough fatty acid synthesis.
Defining the interactome of the human mitochondrial ribosome identifies SMIM4 and TMEM223 as respiratory chain assembly factors.
Mitochondria in human neutrophils mediate killing of Staphylococcus aureus.
Targeted up-regulation of Drp1 in dorsal horn attenuates neuropathic pain hypersensitivity by increasing mitochondrial fission.
p53 regulates skeletal muscle mitophagy and mitochondrial quality control following denervation-induced muscle disuse.
Immunofluorescence-Based Measurement of Autophagosome Formation During Mitophagy.
Induction and Detection of Mitophagy.

Cell Rep. 2021 Dec 28. pii: S2211-1247(21)01651-X. [Epub ahead of print]37(13): 110155

SIRT2 regulates mitochondrial dynamics and reprogramming via MEK1-ERK-DRP1 and AKT1-DRP1 axes.

Young Cha, Taewoo Kim, Jeha Jeon, Yongwoo Jang, Patrick B Kim, Claudia Lopes, Pierre Leblanc, Bruce M Cohen, Kwang-Soo Kim.

  During somatic reprogramming, cellular energy metabolism fundamentally switches from predominantly mitochondrial oxidative phosphorylation toward glycolysis. This metabolic reprogramming, also called the Warburg effect, is critical for the induction of pluripotency, but its molecular mechanisms remain poorly defined. Notably, SIRT2 is consistently downregulated during the reprogramming process and regulates glycolytic switch. Here, we report that downregulation of SIRT2 increases acetylation of mitogen-activated protein kinase (MAPK) kinase-1 (MEK1) at Lys175, resulting in activation of extracellular signal-regulated kinases (ERKs) and subsequent activation of the pro-fission factor dynamin-related protein 1 (DRP1). In parallel, downregulation of SIRT2 hyperacetylates the serine/threonine protein kinase AKT1 at Lys20 in a non-canonical way, activating DRP1 and metabolic reprogramming. Together, our study identified two axes, SIRT2-MEK1-ERK-DRP1 and SIRT2-AKT1-DRP1, that critically link mitochondrial dynamics and oxidative phosphorylation to the somatic reprogramming process. These upstream signals, together with SIRT2's role in glycolytic switching, may underlie the Warburg effect observed in human somatic cell reprogramming.

Keywords:  AKT1; DRP1; MEK1-ERK axis; OXPHOS; SIRT2; Warburg-like effect; human somatic cell reprogramming; induced pluripotent stem cells; metabolic reprogramming; mitochondrial remodeling

DOI:  https://doi.org/10.1016/j.celrep.2021.110155
Cell Rep. 2021 Dec 28. pii: S2211-1247(21)01653-3. [Epub ahead of print]37(13): 110157

NAD kinase sustains lipogenesis and mitochondrial metabolismthrough fatty acid synthesis.

Mengyao Xu, Long Ding, Jingjing Liang, Xiao Yang, Yuan Liu, Yingchun Wang, Mei Ding, Xun Huang.

  Lipid storage in fat tissue is important for energy homeostasis and cellular functions. Through RNAi screening in Drosophila fat body, we found that knockdown of a Drosophila NAD kinase (NADK), which phosphorylates NAD to synthesize NADP de novo, causes lipid storage defects. NADK sustains lipogenesis by maintaining the pool of NADPH. Promoting NADPH production rescues the lipid storage defect in the fat body of NADK RNAi animals. Furthermore, NADK and fatty acid synthase 1 (FASN1) regulate mitochondrial mass and function by altering the levels of acetyl-CoA and fatty acids. Reducing the level of acetyl-CoA or increasing the synthesis of cardiolipin (CL), a mitochondrion-specific phospholipid, partially rescues the mitochondrial defects of NADK RNAi. Therefore, NADK- and FASN1-mediated fatty acid synthesis coordinates lipid storage and mitochondrial function.

Keywords:  Drosophila; FASN; NADK; lipogenesis; mitochondrial metabolism

DOI:  https://doi.org/10.1016/j.celrep.2021.110157
Elife. 2021 Dec 31. pii: e68213. [Epub ahead of print]10

Defining the interactome of the human mitochondrial ribosome identifies SMIM4 and TMEM223 as respiratory chain assembly factors.

Sven Dennerlein, Sabine Poerschke, Silke Oeljeklaus, Cong Wang, Ricarda Richter-Dennerlein, Johannes Sattmann, Diana Bauermeister, Elisa Hanitsch, Stefan Stoldt, Thomas Langer, Stefan Jakobs, Bettina Warscheid, Peter Rehling.

  Human mitochondria express a genome that encodes thirteen core subunits of the oxidative phosphorylation system (OXPHOS). These proteins insert into the inner membrane co-translationally. Therefore, mitochondrial ribosomes engage with the OXA1L-insertase and membrane-associated proteins, which support membrane insertion of translation products and early assembly steps into OXPHOS complexes. To identify ribosome-associated biogenesis factors for the OXPHOS system, we purified ribosomes and associated proteins from mitochondria. We identified TMEM223 as a ribosome-associated protein involved in complex IV biogenesis. TMEM223 stimulates the translation of COX1 mRNA and is a constituent of early COX1 assembly intermediates. Moreover, we show that SMIM4 together with C12ORF73 interacts with newly synthesized cytochrome b to support initial steps of complex III biogenesis in complex with UQCC1 and UQCC2. Our analyses define the interactome of the human mitochondrial ribosome and reveal novel assembly factors for complex III and IV biogenesis that link early assembly stages to the translation machinery.

Keywords:  assembly; biochemistry; cell biology; chemical biology; mitochondria; oxidative phosphorylation; ribosome; translation

DOI:  https://doi.org/10.7554/eLife.68213
Redox Biol. 2021 Dec 24. pii: S2213-2317(21)00385-2. [Epub ahead of print]49 102225

Mitochondria in human neutrophils mediate killing of Staphylococcus aureus.

Kimberly J Dunham-Snary, Bas Gj Surewaard, Jeffrey D Mewburn, Rachel Et Bentley, Ashley Y Martin, Oliver Jones, Ruaa Al-Qazazi, Patricia Ad Lima, Paul Kubes, Stephen L Archer.

   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

DOI:  https://doi.org/10.1016/j.redox.2021.102225
Redox Biol. 2021 Dec 20. pii: S2213-2317(21)00376-1. [Epub ahead of print]49 102216

Targeted up-regulation of Drp1 in dorsal horn attenuates neuropathic pain hypersensitivity by increasing mitochondrial fission.

Kun-Long Zhang, Shu-Jiao Li, Xue-Yin Pu, Fei-Fei Wu, Hui Liu, Rui-Qing Wang, Bo-Zhi Liu, Ze Li, Kai-Feng Li, Nian-Song Qian, Yan-Ling Yang, Hua Yuan, Ya-Yun Wang.

  Mitochondria play an essential role in pathophysiology of both inflammatory and neuropathic pain (NP), but the mechanisms are not yet clear. Dynamin-related protein 1 (Drp1) is broadly expressed in the central nervous system and plays a role in the induction of mitochondrial fission process. Spared nerve injury (SNI), due to the dysfunction of the neurons within the spinal dorsal horn (SDH), is the most common NP model. We explored the neuroprotective role of Drp1 within SDH in SNI. SNI mice showed pain behavior and anxiety-like behavior, which was associated with elevation of Drp1, as well as increased density of mitochondria in SDH. Ultrastructural analysis showed SNI induced damaged mitochondria into smaller perimeter and area, tending to be circular. Characteristics of vacuole in the mitochondria further showed SNI induced the increased number of vacuole, widened vac-perimeter and vac-area. Stable overexpression of Drp1 via AAV under the control of the Drp1 promoter by intraspinal injection (Drp1 OE) attenuated abnormal gait and alleviated pain hypersensitivity of SNI mice. Mitochondrial ultrastructure analysis showed that the increased density of mitochondria induced by SNI was recovered by Drp1 OE which, however, did not change mitochondrial morphology and vacuole parameters within SDH. Contrary to Drp1 OE, down-regulation of Drp1 in the SDH by AAV-Drp1 shRNA (Drp1 RNAi) did not alter painful behavior induced by SNI. Ultrastructural analysis showed the treatment by combination of SNI and Drp1 RNAi (SNI + Drp1 RNAi) amplified the damages of mitochondria with the decreased distribution density, increased perimeter and area, as well as larger circularity tending to be more circular. Vacuole data showed SNI + Drp1 RNAi increased vacuole density, perimeter and area within the SDH mitochondria. Our results illustrate that mitochondria within the SDH are sensitive to NP, and targeted mitochondrial Drp1 overexpression attenuates pain hypersensitivity. Drp1 offers a novel therapeutic target for pain treatment.

Keywords:  Drp1; Mitochondria; Pain; SNI; Spinal dorsal horn

DOI:  https://doi.org/10.1016/j.redox.2021.102216
J Biol Chem. 2021 Dec 24. pii: S0021-9258(21)01350-8. [Epub ahead of print] 101540

p53 regulates skeletal muscle mitophagy and mitochondrial quality control following denervation-induced muscle disuse.

Jonathan M Memme, Ashley N Oliveira, David A Hood.

  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)

DOI:  https://doi.org/10.1016/j.jbc.2021.101540
Methods Mol Biol. 2022 ;2445 207-226

Immunofluorescence-Based Measurement of Autophagosome Formation During Mitophagy.

Benjamin S Padman, Michael Lazarou.

  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)

DOI:  https://doi.org/10.1007/978-1-0716-2071-7_13
Methods Mol Biol. 2022 ;2445 227-239

Induction and Detection of Mitophagy.

Maria A Yapryntseva, Boris Zhivotovsky, Vladimir Gogvadze.

  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

DOI:  https://doi.org/10.1007/978-1-0716-2071-7_14