bims-mikwok 2020-11-22 papers

bims-mikwok

Biomed News

on Mitochondrial quality control

Issue of 2020–11–22
thirty-two papers selected by
Avinash N. Mukkala, University of Toronto

Alternative mitochondrial quality control mediated by extracellular release.
Ubiquitination of Intramitochondrial Proteins: Implications for Metabolic Adaptability.
Mitochondrial Safeguard: a stress response that offsets extreme fusion and protects respiratory function via flickering-induced Oma1 activation.
Functions of outer mitochondrial membrane proteins: mediating the crosstalk between mitochondrial dynamics and mitophagy.
Mitochondrial Ca2+ Dynamics in MCU Knockout C. elegans Worms.
Ubl4A is critical for mitochondrial fusion process under nutrient deprivation stress.
FUNDC1-dependent mitophagy induced by tPA protects neurons against cerebral ischemia-reperfusion injury.
The Nrf2 induction prevents ferroptosis in Friedreich's Ataxia.
E3 Ubiquitin Ligases in Neurological Diseases: Focus on Gigaxonin and Autophagy.
Mitochondrial oxidative stress induces rapid intermembrane space/matrix translocation of Apurinic/apyrimidinic endonuclease 1 protein through TIM23 complex.
USP22 Protects Against Myocardial Ischemia-Reperfusion Injury via the SIRT1-p53/SLC7A11-Dependent Inhibition of Ferroptosis-Induced Cardiomyocyte Death.
Macrophage Regnase-1 Deletion Deteriorates Liver Ischemia/Reperfusion Injury Through Regulation of Macrophage Polarization.
Mitochondrial respiratory supercomplexes in mammalian cells: structural versus functional role.
Glutamine depletion disrupts mitochondrial integrity and impairs C2C12 myoblast proliferation, differentiation, and the heat-shock response.
Mitochondrial stress and GDF15 in the pathophysiology of sepsis.
Hyperpolarized mitochondria accumulate in Drosophila Hipk-overexpressing cells to drive tumor-like growth.
Idiopathic inflammatory myopathy human derived cells retain their ability to increase mitochondrial function.
Mitochondrial Dynamics in Placenta-Derived Mesenchymal Stem Cells Regulate the Invasion Activity of Trophoblast.
Medicinal Chemistry Targeting Mitochondria: From New Vehicles and Pharmacophore Groups to Old Drugs with Mitochondrial Activity.
Methamphetamine induces cardiomyopathy by Sigmar1 inhibition-dependent impairment of mitochondrial dynamics and function.
Mfn2 Overexpression Attenuates Cardio-Cerebrovascular Ischemia-Reperfusion Injury Through Mitochondrial Fusion and Activation of the AMPK/Sirt3 Signaling.
CRISPR-Mediated Induction of Neuron-Enriched Mitochondrial Proteins Boosts Direct Glia-to-Neuron Conversion.
Mitochondrial Function and Metabolism of Cultured Skeletal Cells.
Oxidized LDL Disrupts Metabolism and Inhibits Macrophage Survival by Activating a miR-9/Drp1/Mitochondrial Fission Signaling Pathway.
Chronic Alcohol Exposure Induces Aberrant Mitochondrial Morphology and Inhibits Respiratory Capacity in the Medial Prefrontal Cortex of Mice.
Targeting cellular senescence based on inter-organelle communication, multi-level proteostasis and metabolic control.
Physiological Ca2+ Transients Versus Pathological Steady-State Ca2+ Elevation, Who Flips the ROS Coin in Skeletal Muscle Mitochondria.
Mitochondrial DNA lesions and copy number are strain dependent in endurance-trained mice.
Mitochondrial Ca2 + Is Related to Mitochondrial Activity and Dynamic Events in Mouse Oocytes.
Quantitative analysis of mitochondrial membrane potential heterogeneity in unsynchronized and synchronized cancer cells.
Induction of mitophagy in C2C12 cells by electrical pulse stimulation involves increasing the level of the mitochondrial receptor FUNDC1 through the AMPK-ULK1 pathway.
p62-Nrf2-p62 Mitophagy Regulatory Loop as a Target for Preventive Therapy of Neurodegenerative Diseases.

Autophagy. 2020 Nov 20.

Alternative mitochondrial quality control mediated by extracellular release.

Chi-Jing Choong, Tatsusada Okuno, Kensuke Ikenaka, Kousuke Baba, Hideki Hayakawa, Masato Koike, Mutsumi Yokota, Junko Doi, Keita Kakuda, Toshihide Takeuchi, Akiko Kuma, Shuhei Nakamura, Yoshitaka Nagai, Seiichi Nagano, Tamotsu Yoshimori, Hideki Mochizuki.

  Mitochondrial quality control, which is crucial for maintaining cellular homeostasis, has been considered to be achieved exclusively through mitophagy. Here we report an alternative mitochondrial quality control pathway mediated by extracellular mitochondria release. By performing time-lapse confocal imaging on a stable cell line with fluorescent-labeled mitochondria, we observed release of mitochondria from cells into the extracellular space. Correlative light-electron microscopy revealed that majority of the extracellular mitochondria are in free form and, on rare occasions, some are enclosed in membrane-surrounded vesicles. Rotenone- and carbonyl cyanide m-chlorophenylhydrazone-induced mitochondrial quality impairment promotes the extracellular release of depolarized mitochondria. Overexpression of PRKN (parkin RBR E3 ubiquitin protein ligase), which has a pivotal role in mitophagy regulation, suppresses the extracellular mitochondria release under basal and stress condition, whereas its knockdown exacerbates it. Correspondingly, overexpression of PRKN-independent mitophagy regulators, BNIP3 (BCL2 interacting protein 3) and BNIP3L/NIX (BCL2 interacting protein 3 like), suppress extracellular mitochondria release. Autophagy-deficient cell lines show elevated extracellular mitochondria release. These results imply that perturbation of mitophagy pathway prompts mitochondria expulsion. Presence of mitochondrial protein can also be detected in mouse sera. Sera of PRKN-deficient mice contain higher level of mitochondrial protein compared to that of wild-type mice. More importantly, fibroblasts and cerebrospinal fluid samples from Parkinson disease patients carrying loss-of-function PRKN mutations show increased extracellular mitochondria compared to control subjects, providing evidence in a clinical context. Taken together, our findings suggest that extracellular mitochondria release is a comparable yet distinct quality control pathway from conventional mitophagy.

Keywords:  Mitochondria; Parkinson disease; mitochondrial quality control; mitophagy; parkin

DOI:  https://doi.org/10.1080/15548627.2020.1848130
Biomolecules. 2020 Nov 16. pii: E1559. [Epub ahead of print]10(11):

Ubiquitination of Intramitochondrial Proteins: Implications for Metabolic Adaptability.

Prasad Sulkshane, Jonathan Ram, Michael H Glickman.

  Mitochondria are constantly subjected to stressful conditions due to their unique physiology and organization. The resulting damage leads to mitochondrial dysfunction, which underlies many pathophysiological conditions. Hence, constant surveillance is required to closely monitor mitochondrial health for sound maintenance of cellular metabolism and thus, for viability. In addition to internal mitochondrial chaperones and proteases, mitochondrial health is also governed by host cell protein quality control systems. The ubiquitin-proteasome system (UPS) and autophagy constitute the main pathways for removal of damaged or superfluous proteins in the cytosol, nucleus, and from certain organelles such as the Endoplasmic Reticulum (ER) and mitochondria. Although stress-induced ubiquitin-dependent degradation of mitochondrial outer membrane proteins has been widely studied, mechanisms of intramitochondrial protein ubiquitination has remained largely elusive due to the predominantly cytosolic nature of UPS components, separated from internal mitochondrial proteins by a double membrane. However, recent research has illuminated examples of intramitochondrial protein ubiquitination pathways and highlighted their importance under basal and stressful conditions. Owing to the dependence of mitochondria on the error-prone process of protein import from the cytosol, it is imperative that the cell eliminate any accumulated proteins in the event of mitochondrial protein import deficiency. Apparently, a significant portion of this activity involves ubiquitination in one way or another. In the present review article, following a brief introduction to mitochondrial protein quality control mechanisms, we discuss our recent understanding of intramitochondrial protein ubiquitination, its importance for basal function of mitochondria, metabolic implications, and possible therapeutic applications.

Keywords:  autophagy; metabolism; mitochondria; mitophagy; proteasome; protein import; protein quality control; proteolysis; ubiquitin

DOI:  https://doi.org/10.3390/biom10111559
EMBO J. 2020 Nov 17. e105074

Mitochondrial Safeguard: a stress response that offsets extreme fusion and protects respiratory function via flickering-induced Oma1 activation.

Daisuke Murata, Tatsuya Yamada, Takeshi Tokuyama, Kenta Arai, Pedro M Quirós, Carlos López-Otín, Miho Iijima, Hiromi Sesaki.

  The connectivity of mitochondria is regulated by a balance between fusion and division. Many human diseases are associated with excessive mitochondrial connectivity due to impaired Drp1, a dynamin-related GTPase that mediates division. Here, we report a mitochondrial stress response, named mitochondrial safeguard, that adjusts the balance of fusion and division in response to increased mitochondrial connectivity. In cells lacking Drp1, mitochondria undergo hyperfusion. However, hyperfusion does not completely connect mitochondria because Opa1 and mitofusin 1, two other dynamin-related GTPases that mediate fusion, become proteolytically inactivated. Pharmacological and genetic experiments show that the activity of Oma1, a metalloprotease that cleaves Opa1, is regulated by short pulses of the membrane depolarization without affecting the overall membrane potential in Drp1-knockout cells. Re-activation of Opa1 and Mitofusin 1 in Drp1-knockout cells further connects mitochondria beyond hyperfusion, termed extreme fusion, leading to bioenergetic deficits. These findings reveal an unforeseen safeguard mechanism that prevents extreme fusion of mitochondria, thereby maintaining mitochondrial function when the balance is shifted to excessive connectivity.

Keywords:  Drp1; Oma1; Opa1; mitochondrial fusion; mitofusin

DOI:  https://doi.org/10.15252/embj.2020105074
Cell Death Differ. 2020 Nov 18.

Functions of outer mitochondrial membrane proteins: mediating the crosstalk between mitochondrial dynamics and mitophagy.

Hongxu Xian, Yih-Cherng Liou.

Most cellular stress responses converge on the mitochondria. Consequently, the mitochondria must rapidly respond to maintain cellular homeostasis and physiological demands by fine-tuning a plethora of mitochondria-associated processes. The outer mitochondrial membrane (OMM) proteins are central to mediating mitochondrial dynamics, coupled with continuous fission and fusion. These OMM proteins also have vital roles in controlling mitochondrial quality and serving as mitophagic receptors for autophagosome enclosure during mitophagy. Mitochondrial fission segregates impaired mitochondria in smaller sizes from the mother mitochondria and may favor mitophagy for eliminating damaged mitochondria. Conversely, mitochondrial fusion mixes dysfunctional mitochondria with healthy ones to repair the damage by diluting the impaired components and consequently prevents mitochondrial clearance via mitophagy. Despite extensive research efforts into deciphering the interplay between fission-fusion and mitophagy, it is still not clear whether mitochondrial fission essentially precedes mitophagy. In this review, we summarize recent breakthroughs concerning OMM research, and dissect the functions of these proteins in mitophagy from their traditional roles in fission-fusion dynamics, in response to distinct context, at the intersection of the OMM platform. These insights into the OMM proteins in mechanistic researches would lead to new aspects of mitochondrial quality control and better understanding of mitochondrial homeostasis intimately tied to pathological impacts.

DOI: https://doi.org/10.1038/s41418-020-00657-z
Int J Mol Sci. 2020 Nov 16. pii: E8622. [Epub ahead of print]21(22):

Mitochondrial Ca2+ Dynamics in MCU Knockout C. elegans Worms.

Pilar Álvarez-Illera, Paloma García-Casas, Rosalba I Fonteriz, Mayte Montero, Javier Alvarez.

  Mitochondrial [Ca2+] plays an important role in the regulation of mitochondrial function, controlling ATP production and apoptosis triggered by mitochondrial Ca2+ overload. This regulation depends on Ca2+ entry into the mitochondria during cell activation processes, which is thought to occur through the mitochondrial Ca2+ uniporter (MCU). Here, we have studied the mitochondrial Ca2+ dynamics in control and MCU-defective C. elegans worms in vivo, by using worms expressing mitochondrially-targeted YC3.60 yellow cameleon in pharynx muscle. Our data show that the small mitochondrial Ca2+ oscillations that occur during normal physiological activity of the pharynx were very similar in both control and MCU-defective worms, except for some kinetic differences that could mostly be explained by changes in neuronal stimulation of the pharynx. However, direct pharynx muscle stimulation with carbachol triggered a large and prolonged increase in mitochondrial [Ca2+] that was much larger in control worms than in MCU-defective worms. This suggests that MCU is necessary for the fast mitochondrial Ca2+ uptake induced by large cell stimulations. However, low-amplitude mitochondrial Ca2+ oscillations occurring under more physiological conditions are independent of the MCU and use a different Ca2+ pathway.

Keywords:  C. elegans; MCU; calcium dynamics; knockout; mitochondria; mitochondrial calcium uniporter

DOI:  https://doi.org/10.3390/ijms21228622
PLoS One. 2020 ;15(11): e0242700

Ubl4A is critical for mitochondrial fusion process under nutrient deprivation stress.

Huaiyuan Zhang, Yu Zhao, Qi Yao, Zijing Ye, Adriana Mañas, Jialing Xiang.

Mitochondrial fusion and fission are dynamic processes regulated by the cellular microenvironment. Under nutrient starvation conditions, mitochondrial fusion is strengthened for energy conservation. We have previously shown that newborns of Ubl4A-deficient mice were more sensitive to starvation stress with a higher rate of mortality than their wild-type littermates. Ubl4A binds with the actin-related protein Arp2/3 complex to synergize the actin branching process. Here, we showed that deficiency in Ubl4A resulted in mitochondrial fragmentation and apoptosis. A defect in the fusion process was the main cause of the mitochondrial fragmentation and resulted from a shortage of primed Arp2/3 complex pool around the mitochondria in the Ubl4A-deficient cells compared to the wild-type cells. As a result, the mitochondrial fusion process was not undertaken quickly enough to sustain starvation stress-induced cell death. Consequently, fragmented mitochondria lost their membrane integrity and ROS was accumulated to trigger caspase 9-dependent apoptosis before autophagic rescue. Furthermore, the wild-type Ubl4A, but not the Arp2/3-binding deficient mutant, could rescue the starvation-induced mitochondrial fragmentation phenotype. These results suggest that Ubl4A promotes the mitochondrial fusion process via Arp2/3 complex during the initial response to nutrient deprivation for cell survival.

DOI: https://doi.org/10.1371/journal.pone.0242700
Redox Biol. 2020 Nov 07. pii: S2213-2317(20)30997-6. [Epub ahead of print]38 101792

FUNDC1-dependent mitophagy induced by tPA protects neurons against cerebral ischemia-reperfusion injury.

Ying Cai, Eryan Yang, Xiuhua Yao, Xuebin Zhang, Qixue Wang, Yunfei Wang, Ji Liu, Weijia Fan, Kaikai Yi, Chunsheng Kang, Jialing Wu.

  Autophagy of mitochondria, termed mitophagy, plays an important role in cerebral ischemia-reperfusion (IR) injury, but the mechanism is not yet clear. Tissue-type plasminogen activator (tPA) is the most important thrombolytic drug in the clinical treatment of ischemic stroke and has neuroprotective effects. Here, we explored the effects of tPA on neuronal apoptosis and mitophagy following IR. We found that knocking out the tPA gene significantly aggravated brain injury and increased neuronal apoptosis and mitochondrial damage. Exposure of neurons to tPA reduced injury severity and protected mitochondria. Further studies demonstrated that this protective effect of tPA was achieved via regulation of FUNDC1-mediated mitophagy. Furthermore, we found that tPA enhanced the expression level of FUNDC1 by activating the phosphorylation of AMPK. In summary, our results confirm that tPA exerts neuroprotective effects by increasing the phosphorylation of AMPK and the expression of FUNDC1, thereby inhibiting apoptosis and improving mitochondrial function.

Keywords:  CRISPR/Cas9; FUNDC1; Ischemia-reperfusion; Mitophagy; tPA

DOI:  https://doi.org/10.1016/j.redox.2020.101792
Redox Biol. 2020 Nov 05. pii: S2213-2317(20)30996-4. [Epub ahead of print]38 101791

The Nrf2 induction prevents ferroptosis in Friedreich's Ataxia.

Piergiorgio La Rosa, Sara Petrillo, Riccardo Turchi, Francesco Berardinelli, Tommaso Schirinzi, Gessica Vasco, Daniele Lettieri-Barbato, Maria Teresa Fiorenza, Enrico S Bertini, Katia Aquilano, Fiorella Piemonte.

  Ferroptosis is an iron-dependent cell death caused by impaired glutathione metabolism, lipid peroxidation and mitochondrial failure. Emerging evidences report a role for ferroptosis in Friedreich's Ataxia (FRDA), a neurodegenerative disease caused by the decreased expression of the mitochondrial protein frataxin. Nrf2 signalling is implicated in many molecular aspects of ferroptosis, by upstream regulating glutathione homeostasis, mitochondrial function and lipid metabolism. As Nrf2 is down-regulated in FRDA, targeting Nrf2-mediated ferroptosis in FRDA may be an attractive option to counteract neurodegeneration in such disease, thus paving the way to new therapeutic opportunities. In this study, we evaluated ferroptosis hallmarks in frataxin-silenced mouse myoblasts, in hearts of a frataxin Knockin/Knockout (KIKO) mouse model, in skin fibroblasts and blood of patients, particularly focusing on ferroptosis-driven gene expression, mitochondrial impairment and lipid peroxidation. The efficacy of Nrf2 inducers to neutralize ferroptosis has been also evaluated.

Keywords:  EPI-743; Ferroptosis; Friedreich ataxia; Lipid peroxides; Mitochondria; Nrf2; Redox imbalance; Sulforaphane

DOI:  https://doi.org/10.1016/j.redox.2020.101791
Front Physiol. 2020 ;11 1022

E3 Ubiquitin Ligases in Neurological Diseases: Focus on Gigaxonin and Autophagy.

Léa Lescouzères, Pascale Bomont.

  Ubiquitination is a dynamic post-translational modification that regulates the fate of proteins and therefore modulates a myriad of cellular functions. At the last step of this sophisticated enzymatic cascade, E3 ubiquitin ligases selectively direct ubiquitin attachment to specific substrates. Altogether, the ∼800 distinct E3 ligases, combined to the exquisite variety of ubiquitin chains and types that can be formed at multiple sites on thousands of different substrates confer to ubiquitination versatility and infinite possibilities to control biological functions. E3 ubiquitin ligases have been shown to regulate behaviors of proteins, from their activation, trafficking, subcellular distribution, interaction with other proteins, to their final degradation. Largely known for tagging proteins for their degradation by the proteasome, E3 ligases also direct ubiquitinated proteins and more largely cellular content (organelles, ribosomes, etc.) to destruction by autophagy. This multi-step machinery involves the creation of double membrane autophagosomes in which engulfed material is degraded after fusion with lysosomes. Cooperating in sustaining homeostasis, actors of ubiquitination, proteasome and autophagy pathways are impaired or mutated in wide range of human diseases. From initial discovery of pathogenic mutations in the E3 ligase encoding for E6-AP in Angelman syndrome and Parkin in juvenile forms of Parkinson disease, the number of E3 ligases identified as causal gene for neurological diseases has considerably increased within the last years. In this review, we provide an overview of these diseases, by classifying the E3 ubiquitin ligase types and categorizing the neurological signs. We focus on the Gigaxonin-E3 ligase, mutated in giant axonal neuropathy and present a comprehensive analysis of the spectrum of mutations and the recent biological models that permitted to uncover novel mechanisms of action. Then, we discuss the common functions shared by Gigaxonin and the other E3 ligases in cytoskeleton architecture, cell signaling and autophagy. In particular, we emphasize their pivotal roles in controlling multiple steps of the autophagy pathway. In light of the various targets and extending functions sustained by a single E3 ligase, we finally discuss the challenge in understanding the complex pathological cascade underlying disease and in designing therapeutic approaches that can apprehend this complexity.

Keywords:  E3 ligase; Gigaxonin; autophagy; cell signaling; cytoskeleton; neurodegenerative disease; neurodevelopmental disease; ubiquitin

DOI:  https://doi.org/10.3389/fphys.2020.01022
J Mol Biol. 2020 Nov 13. pii: S0022-2836(20)30631-8. [Epub ahead of print]

Mitochondrial oxidative stress induces rapid intermembrane space/matrix translocation of Apurinic/apyrimidinic endonuclease 1 protein through TIM23 complex.

Arianna Barchiesi, Veronica Bazzani, Vanessa Tolotto, Praveenraj Elancheliyan, Michał Wasilewski, Agnieszka Chacinska, Carlo Vascotto.

  Mitochondria are essential cellular organelles that import the majority of proteins to sustain their function in cellular metabolism and homeostasis. Due to their role in oxidative phosphorylation, mitochondria are constantly affected by oxidative stress. Stability of mitochondrial DNA (mtDNA) is essential for mitochondrial physiology and cellular well-being and for this reasons mtDNA lesions have to be rapidly recognized and repaired. Base excision repair (BER) is the main pathway responsible for repair non-helix distorting base lesions both into the nucleus and in mitochondria. Apurinic/Apyrimidinic Endonuclease 1 (APE1) is a key component of BER pathway and the only protein that can recognize and process an abasic (AP) site. Comprehensions of the mechanisms regulating APE1 intracellular trafficking are still fragmentary. In this study we focused our attention on the mitochondrial form of APE1 protein and how oxidative stress induce its translocation to maintain mtDNA integrity. Our data proved that: (i) the rise of mitochondrial ROS determines a very rapid translocation of APE1 from the intermembrane space (IMS) into the matrix; and (ii) TIM23/PAM machinery complex is responsible for the matrix translocation of APE1. Moreover, our data support the hypothesis that the IMS, were the majority of APE1 resides, could represent a sort of storage site for the protein.

Keywords:  Apurinic/apyrimidinic endonuclease 1; mitochondria; mitochondrial DNA; oxidative stress; translocase of the inner membrane

DOI:  https://doi.org/10.1016/j.jmb.2020.11.012
Front Physiol. 2020 ;11 551318

USP22 Protects Against Myocardial Ischemia-Reperfusion Injury via the SIRT1-p53/SLC7A11-Dependent Inhibition of Ferroptosis-Induced Cardiomyocyte Death.

Shuxian Ma, Linyan Sun, Wenhao Wu, Jiangli Wu, Zhangnan Sun, Jianjun Ren.

  Myocardial ischemia-reperfusion (MI/R) injury is characterized by iron deposition and reactive oxygen species production, which can induce ferroptosis. Ferroptosis has also been proposed to promote cardiomyocyte death. The current study sought to define the mechanism governing cardiomyocyte death in MI/R injury. An animal model of MI/R was established by ligation and perfusion of the left anterior descending coronary artery, and a cellular model of IR was constructed in cardiomyocytes. ChIP assay was then conducted to determine the interaction among USP22, SIRT1, p53, and SLC7A11. Loss- and gain-of-function assays were also conducted to determine the in vivo and in vitro roles of USP22, SIRT1, and SLC7A11. The infarct size and pathological changes of myocardial tissue were observed using TCC and hematoxylin-eosin staining, and the levels of cardiac function- and myocardial injury-related factors of rats were determined. Cardiomyocyte viability and apoptosis were evaluated in vitro, followed by detection of ferroptosis-related indicators (glutathione (GSH), reactive oxygen species, lipid peroxidation, and iron accumulation). USP22, SIRT1, and SLC7A11 expressions were found to be down-regulated, whereas p53 was highly expressed during MI/R injury. USP22, SIRT1, or SLC7A11 overexpression reduced the infarct size and ameliorated pathological conditions, cardiac function, as evidenced by reduced maximum pressure, ejection fraction, maximum pressure rate, and myocardial injury characterized by lower creatine phosphokinase and lactate dehydrogenase levels in vivo. Moreover, USP22, SIRT1, or SLC7A11 elevation contributed to enhanced cardiomyocyte viability and attenuated ferroptosis-induced cell death in vitro, accompanied by increased GSH levels, as well as decreased reactive oxygen species production, lipid peroxidation, and iron accumulation. Together, these results demonstrate that USP22 overexpression could inhibit ferroptosis-induced cardiomyocyte death to protect against MI/R injury via the SIRT1/p53/SLC7A11 association.

Keywords:  cardiomyocyte death; ferroptosis; myocardial ischemia–reperfusion injury; p53; sirtuin-1; solute carrier family 7 member 11; ubiquitin specific peptidase 22

DOI:  https://doi.org/10.3389/fphys.2020.551318
Front Physiol. 2020 ;11 582347

Macrophage Regnase-1 Deletion Deteriorates Liver Ischemia/Reperfusion Injury Through Regulation of Macrophage Polarization.

Ai Xiaoming, Jia Wenbo, Wang Jinyi, Wu Bin, Hu Chunyang, Chen Qi, Kong Lianbao.

   Background: Regnase-1 (MCPIP) has been identified as an anti-inflammatory agent, but little is known about its influence on liver ischemia/reperfusion (I/R) injury. Macrophages can evolve biphasic responses and differentiate into remarkable polarizations, contributing greatly to the uncontrolled inflammatory cascades during liver I/R injury. Therefore, the aim of this study was to explore whether regnase-1 participated in liver I/R via manipulating macrophage polarization.
Materials and methods: C57BL/6 mice were randomly divided into five groups: Sham, I/R, Clodronate, Clo + BMDM, and Clo + LV MCPIP BMDM. A liver I/R model was established, and histopathological and immunostaining examinations were performed for the liver specimens; double immunofluorescence staining was used to localize MCPIP in the liver. Primary hepatocytes were isolated to simulate a hypoxia and reoxygenation (H/R) model in vitro. Bone marrow-derived macrophages (BMDM) were extracted and subjected to lentiviral transduction to knockdown MCPIP expression. BMDM with or without MCPIP deletion were exposed to H/R supernatants, and the polarized states were measured by flow cytometry. RT-PCR analysis and Western blot were also conducted.
Results: Compared to those in the Sham group, liver functions and Suzuki's scores were deteriorated in the I/R group, which were reversed in the Clodronate group. The increased expression of regnase-1 in the I/R group diminished with pretreatment of clodronate liposomes. Subsequent double immunofluorescence staining established the localization of regnase-1 in macrophages in the liver. The insulted lesions in the Clodronate group became progressively aggravated with adoptive transfer of BMDM in the Clo + BMDM group, and they were further exacerbated with the transfusion of BMDM with MCPIP knockdown in the Clo + LV MCPIP BMDM group. Gene expressions of M1 and M2 markers were detected by RT-PCR, suggesting that MCPIP knockdown tended to favor the M1 transformation. Subsequently, ex vivo flow cytometrical detection showed that, upon stimulation by H/R supernatants, LV-MCPIP BMDM posed a higher ratio of M1/M2 than BMDM. Finally, we found that MCPIP participated in macrophage M1/M2 polarization through the NF-κB, C/EBPβ, and PPARγ signaling pathways during liver I/R.
Conclusion: Our study confirms that regnase-1 plays a critical role in liver I/R via regulation of macrophage polarization and, thus, might offer a potential therapeutic target.

Keywords:  ischemia/reperfusion injury; liver; macrophage; polarization; regnase-1 (MCPIP)

DOI:  https://doi.org/10.3389/fphys.2020.582347
J Mol Med (Berl). 2020 Nov 17.

Mitochondrial respiratory supercomplexes in mammalian cells: structural versus functional role.

Sabzali Javadov, Sehwan Jang, Xavier R Chapa-Dubocq, Zaza Khuchua, Amadou Ks Camara.

  Mitochondria are recognized as the main source of ATP to meet the energy demands of the cell. ATP production occurs by oxidative phosphorylation when electrons are transported through the electron transport chain (ETC) complexes and develop the proton motive force across the inner mitochondrial membrane that is used for ATP synthesis. Studies since the 1960s have been concentrated on the two models of structural organization of ETC complexes known as "solid-state" and "fluid-state" models. However, advanced new techniques such as blue-native gel electrophoresis, mass spectroscopy, and cryogenic electron microscopy for analysis of macromolecular protein complexes provided new data in favor of the solid-state model. According to this model, individual ETC complexes are assembled into macromolecular structures known as respiratory supercomplexes (SCs). A large number of studies over the last 20 years proposed the potential role of SCs to facilitate substrate channeling, maintain the integrity of individual ETC complexes, reduce electron leakage and production of reactive oxygen species, and prevent excessive and random aggregation of proteins in the inner mitochondrial membrane. However, many other studies have challenged the proposed functional role of SCs. Recently, a third model known as the "plasticity" model was proposed that partly reconciles both "solid-state" and "fluid-state" models. According to the "plasticity" model, respiratory SCs can co-exist with the individual ETC complexes. To date, the physiological role of SCs remains unknown, although several studies using tissue samples of patients or animal/cell models of human diseases revealed an associative link between functional changes and the disintegration of SC assembly. This review summarizes and discusses previous studies on the mechanisms and regulation of SC assembly under physiological and pathological conditions.

Keywords:  Electron transport chain complexes; Human diseases; Inner mitochondrial membrane; Mitochondria; Respiratory Supercomplexes

DOI:  https://doi.org/10.1007/s00109-020-02004-8
Nutr Res. 2020 Sep 19. pii: S0271-5317(20)30529-7. [Epub ahead of print]

Glutamine depletion disrupts mitochondrial integrity and impairs C2C12 myoblast proliferation, differentiation, and the heat-shock response.

Jacob Dohl, Maria Elizabeth Pereira Passos, Jonathan Foldi, Yifan Chen, Tania Pithon-Curi, Rui Curi, Renata Gorjao, Patricia A Deuster, Tianzheng Yu.

  Glutamine and glucose are both oxidized in the mitochondria to supply the majority of usable energy for processes of cellular function. Low levels of plasma and skeletal muscle glutamine are associated with severe illness. We hypothesized that glutamine deficiency would disrupt mitochondrial integrity and impair cell function. C2C12 mouse myoblasts were cultured in control media supplemented with 5.6 mmol/L glucose and 2 mmol/L glutamine, glutamine depletion (Gln-) or glucose depletion (Glc-) media. We compared mitochondrial morphology and function, as well as cell proliferation, myogenic differentiation, and heat-shock response in these cells. Glc- cells exhibited slightly elongated mitochondrial networks and increased mitochondrial mass, with normal membrane potential (ΔΨm). Mitochondria in Gln- cells became hyperfused and swollen, which were accompanied by severe disruption of cristae and decreases in ΔΨm, mitochondrial mass, the inner mitochondrial membrane remodeling protein OPA1, electron transport chain complex IV protein expression, and markers of mitochondrial biogenesis and bioenergetics. In addition, Gln- increased the autophagy marker LC3B-II on the mitochondrial membrane. Notably, basal mitochondrial respiration was increased in Glc- cells as compared to control cells, whereas maximal respiration remained unchanged. In contrast, basal respiration, maximal respiration and reserve capacity were all decreased in Gln- cells. Consistent with the aforementioned mitochondrial deficits, Gln- cells had lower growth rates and myogenic differentiation, as well as a higher rate of cell death under heat stress conditions than Glc- and control cells. We conclude that glutamine is essential for mitochondrial integrity and function; glutamine depletion impairs myoblast proliferation, differentiation, and the heat-shock response.

Keywords:  mitochondrial bioenergetics; mitochondrial biogenesis; mitochondrial fission; mitochondrial fusion; mitophagy; nutrient depletion; oxidative phosphorylation

DOI:  https://doi.org/10.1016/j.nutres.2020.09.006
Arch Biochem Biophys. 2020 Nov 11. pii: S0003-9861(20)30677-9. [Epub ahead of print]696 108668

Mitochondrial stress and GDF15 in the pathophysiology of sepsis.

Yasunori Fujita, Masafumi Ito, Ikuroh Ohsawa.

  Mitochondria are multifunctional organelles that regulate diverse cellular processes. Mitochondrial stress, including stress generated by electron transport chain defects and impaired mitochondrial proteostasis, is intimately involved in various diseases and pathological conditions. Sepsis is a life-threatening condition that occurs when an imbalanced host response to infection leads to organ dysfunction. Metabolic disturbances and impaired immune responses are implicated in the pathogenesis and development of sepsis. Given that mitochondria play central roles in cellular metabolism, mitochondrial stress is predicted to be involved in the pathological mechanism of sepsis. Under mitochondrial stress, cells activate stress response systems to maintain homeostasis. This mitochondrial stress response transcriptionally activates genes involved in cell survival and death. Mitochondrial stress also induces the release of distinctive secretory proteins from cells. Recently, we showed that growth differentiation factor 15 (GDF15) is a major secretory protein induced by mitochondrial dysfunction. In this article, we provide a brief overview of mitochondrial stress response and GDF15, and discuss the potential role of GDF15 in the pathophysiology of sepsis.

Keywords:  GDF15; Inflammation; Integrated stress response; Mitochondrial stress; Sepsis

DOI:  https://doi.org/10.1016/j.abb.2020.108668
J Cell Sci. 2020 Nov 16. pii: jcs.250944. [Epub ahead of print]

Hyperpolarized mitochondria accumulate in Drosophila Hipk-overexpressing cells to drive tumor-like growth.

Kenneth Kin Lam Wong, Jenny Zhe Liao, Claire R Y Shih, Nicholas Harden, Esther M Verheyen.

  Both functional and dysfunctional mitochondria are known to underlie tumor progression. Here, we establish use of the proto-oncogene Drosophila Homeodomain-interacting protein kinase (Hipk) as a new tool to address this paradox. We find that, in Hipk-overexpressing tumor-like cells, mitochondria accumulate and switch from fragmented to highly fused interconnected morphologies. Moreover, elevated Hipk promotes mitochondrial membrane hyperpolarization. These mitochondrial changes are at least in part driven by the upregulation of Myc. Furthermore, we show that the altered mitochondrial energetics, but not morphology, is required for Hipk tumor-like growth as knockdown of pdsw (NDUFB10 in mammals; a Complex I subunit) abrogates the growth. Knockdown of ATPsynβ (a Complex V subunit), which produces higher levels of reactive oxygen species (ROS) than pdsw knockdown, instead synergizes with Hipk to potentiate JNK activation and the downstream induction of Matrix metalloproteinases. Accordingly, ATPsynβ knockdown suppresses Hipk tumor-like growth only when ROS scavengers are co-expressed. Altogether, our work presents an in vivo tumor model featuring the accumulation of hyperfused and hyperpolarized mitochondria, and reveals respiratory Complex subunit-dependent, opposing effects on tumorigenic outcomes.

Keywords:  Drosophila; Energetics; Hipk; Mitochondria; Myc; ROS

DOI:  https://doi.org/10.1242/jcs.250944
PLoS One. 2020 ;15(11): e0242443

Idiopathic inflammatory myopathy human derived cells retain their ability to increase mitochondrial function.

Carla Basualto-Alarcón, Félix A Urra, María Francisca Bozán, Fabián Jaña, Alejandra Trangulao, Jorge A Bevilacqua, J César Cárdenas.

Idiopathic Inflammatory Myopathies (IIMs) have been studied within the framework of autoimmune diseases where skeletal muscle appears to have a passive role in the illness. However, persiting weakness even after resolving inflammation raises questions about the role that skeletal muscle plays by itself in these diseases. "Non-immune mediated" hypotheses have arisen to consider inner skeletal muscle cell processes as trigger factors in the clinical manifestations of IIMs. Alterations in oxidative phosphorylation, ATP production, calcium handling, autophagy, endoplasmic reticulum stress, among others, have been proposed as alternative cellular pathophysiological mechanisms. In this study, we used skeletal muscle-derived cells, from healthy controls and IIM patients to determine mitochondrial function and mitochondrial ability to adapt to a metabolic stress when deprived of glucose. We hypothesized that mitochondria would be dysfunctional in IIM samples, which was partially true in normal glucose rich growing medium as determined by oxygen consumption rate. However, in the glucose-free and galactose supplemented condition, a medium that forced mitochondria to function, IIM cells increased their respiration, reaching values matching normal derived cells. Unexpectedly, cell death significantly increased in IIM cells under this condition. Our findings show that mitochondria in IIM is functional and the decrease respiration observed is part of an adaptative response to improve survival. The increased metabolic function obtained after forcing IIM cells to rely on mitochondrial synthesized ATP is detrimental to the cell's viability. Thus, therapeutic interventions that activate mitochondria, could be detrimental in IIM cell physiology, and must be avoided in patients with IIM.

DOI: https://doi.org/10.1371/journal.pone.0242443
Int J Mol Sci. 2020 Nov 14. pii: E8599. [Epub ahead of print]21(22):

Mitochondrial Dynamics in Placenta-Derived Mesenchymal Stem Cells Regulate the Invasion Activity of Trophoblast.

Jin Seok, Sujin Jun, Jung Ok Lee, Gi Jin Kim.

  Mitochondrial dynamics are involved in many cellular events, including the proliferation, differentiation, and invasion/migration of normal as well as cancerous cells. Human placenta-derived mesenchymal stem cells (PD-MSCs) were known to regulate the invasion activity of trophoblasts. However, the effects of PD-MSCs on mitochondrial function in trophoblasts are still insufficiently understood. Therefore, the objectives of this study are to analyze the factors related to mitochondrial function and investigate the correlation between trophoblast invasion and mitophagy via PD-MSC cocultivation. We assess invasion ability and mitochondrial function in invasive trophoblasts according to PD-MSC cocultivation by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and extracellular flux (XF) assay. Under PD-MSCs co-cultivation, invasion activity of a trophoblast is increased via activation of the Rho signaling pathway as well as Matrix metalloproteinases (MMPs). Additionally, the expression of mitochondrial function (e.g., reactive oxygen species (ROS), calcium, and adenosine triphosphate (ATP) synthesis) in trophoblasts are increased via PD-MSCs co-cultivation. Finally, PD-MSCs regulate mitochondrial autophagy factors in invasive trophoblasts via regulating the balance between PTEN-induced putative kinase 1 (PINK1) and parkin RBR E3 ubiquitin protein ligase (PARKIN) expression. Taken together, these results demonstrate that PD-MSCs enhance the invasion ability of trophoblasts via altering mitochondrial dynamics. These results support the fundamental mechanism of trophoblast invasion via mitochondrial function and provide a new stem cell therapy for infertility.

Keywords:  invasion; mitochondrial dynamics; mitophagy; placenta-derived mesenchymal stem cells; trophoblast

DOI:  https://doi.org/10.3390/ijms21228599
Int J Mol Sci. 2020 Nov 18. pii: E8684. [Epub ahead of print]21(22):

Medicinal Chemistry Targeting Mitochondria: From New Vehicles and Pharmacophore Groups to Old Drugs with Mitochondrial Activity.

Mabel Catalán, Ivonne Olmedo, Jennifer Faúndez, José A Jara.

  Interest in tumor cell mitochondria as a pharmacological target has been rekindled in recent years. This attention is due in part to new publications documenting heterogenous characteristics of solid tumors, including anoxic and hypoxic zones that foster cellular populations with differentiating metabolic characteristics. These populations include tumor-initiating or cancer stem cells, which have a strong capacity to adapt to reduced oxygen availability, switching rapidly between glycolysis and oxidative phosphorylation as sources of energy and metabolites. Additionally, this cell subpopulation shows high chemo- and radioresistance and a high capacity for tumor repopulation. Interestingly, it has been shown that inhibiting mitochondrial function in tumor cells affects glycolysis pathways, cell bioenergy, and cell viability. Therefore, mitochondrial inhibition may be a viable strategy for eradicating cancer stem cells. In this context, medicinal chemistry research over the last decade has synthesized and characterized "vehicles" capable of transporting novel or existing pharmacophores to mitochondrial tumor cells, based on mechanisms that exploit the physicochemical properties of the vehicles and the inherent properties of the mitochondria. The pharmacophores, some of which have been isolated from plants and others, which were synthesized in the lab, are diverse in chemical nature. Some of these molecules are active, while others are prodrugs that have been evaluated alone or linked to mitochondria-targeted agents. Finally, researchers have recently described drugs with well-proven safety and efficacy that may exert a mitochondria-specific inhibitory effect in tumor cells through noncanonical mechanisms. The effectiveness of these molecules may be improved by linking them to mitochondrial carrier molecules. These promising pharmacological agents should be evaluated alone and in combination with classic chemotherapeutic drugs in clinical studies.

Keywords:  cancer bioenergy; cancer metabolism; cancer stem cells; delocalized lipophilic cations; drugs; mitocans; pharmacophores groups; targeting mitochondria

DOI:  https://doi.org/10.3390/ijms21228684
Commun Biol. 2020 Nov 17. 3(1): 682

Methamphetamine induces cardiomyopathy by Sigmar1 inhibition-dependent impairment of mitochondrial dynamics and function.

Chowdhury S Abdullah, Richa Aishwarya, Shafiul Alam, Mahboob Morshed, Naznin Sultana Remex, Sadia Nitu, Gopi K Kolluru, James Traylor, Sumitra Miriyala, Manikandan Panchatcharam, Brandon Hartman, Judy King, Mohammad Alfrad Nobel Bhuiyan, Sunitha Chandran, Matthew D Woolard, Xiuping Yu, Nicholas E Goeders, Paari Dominic, Connie L Arnold, Karen Stokes, Christopher G Kevil, A Wayne Orr, Md Shenuarin Bhuiyan.

Methamphetamine-associated cardiomyopathy is the leading cause of death linked with illicit drug use. Here we show that Sigmar1 is a therapeutic target for methamphetamine-associated cardiomyopathy and defined the molecular mechanisms using autopsy samples of human hearts, and a mouse model of "binge and crash" methamphetamine administration. Sigmar1 expression is significantly decreased in the hearts of human methamphetamine users and those of "binge and crash" methamphetamine-treated mice. The hearts of methamphetamine users also show signs of cardiomyopathy, including cellular injury, fibrosis, and enlargement of the heart. In addition, mice expose to "binge and crash" methamphetamine develop cardiac hypertrophy, fibrotic remodeling, and mitochondrial dysfunction leading to contractile dysfunction. Methamphetamine treatment inhibits Sigmar1, resulting in inactivation of the cAMP response element-binding protein (CREB), decreased expression of mitochondrial fission 1 protein (FIS1), and ultimately alteration of mitochondrial dynamics and function. Therefore, Sigmar1 is a viable therapeutic agent for protection against methamphetamine-associated cardiomyopathy.

DOI: https://doi.org/10.1038/s42003-020-01408-z
Front Cell Dev Biol. 2020 ;8 598078

Mfn2 Overexpression Attenuates Cardio-Cerebrovascular Ischemia-Reperfusion Injury Through Mitochondrial Fusion and Activation of the AMPK/Sirt3 Signaling.

Min Liu, Xiaoyang Li, Dezhi Huang.

  Mitochondria are potential targets for the treatment of cardio-cerebrovascular ischemia-reperfusion (I/R) injury. However, the role of the mitofusin 2 (Mfn2) protein in regulating mitochondrial fusion and cell survival has not been investigated. In the present study, an adenovirus-mediated Mfn2 overexpression assay was performed to understand the effects of Mfn2 on mitochondrial function and cell damage during cardio-cerebrovascular I/R injury. After exposure to I/R injury in vitro, the transcription and expression of Mfn2 were significantly downregulated, which correlated with decreased cell viability and increased apoptosis. By contrast, overexpression of Mfn2 significantly repressed I/R-mediated cell death through modulation of glucose metabolism and oxidative stress. Furthermore, Mfn2 overexpression improved mitochondrial fusion in cells, an effect that was followed by increased mitochondrial membrane potential, improved mitophagy, and inhibition of mitochondria-mediated apoptosis. Our data also demonstrated that Mfn2 overexpression was associated with activation of the AMPK/Sirt3 signaling pathway. Inhibition of the AMPK/Sirt3 pathway abolished the protective effects of Mfn2 on I/R-induced cell injury arising from mitochondrial damage. Our results indicate that Mfn2 protects against cardio-cerebrovascular I/R injury by augmenting mitochondrial fusion and activating the AMPK/Sirt3 signaling pathway.

Keywords:  AMPK/Sirt3 signaling pathway; apoptosis; cardio-cerebrovascular ischemia–reperfusion (I/R) injury; mitochondrial fusion; mitofusin 2 (Mfn2)

DOI:  https://doi.org/10.3389/fcell.2020.598078
Cell Stem Cell. 2020 Nov 09. pii: S1934-5909(20)30509-9. [Epub ahead of print]

CRISPR-Mediated Induction of Neuron-Enriched Mitochondrial Proteins Boosts Direct Glia-to-Neuron Conversion.

Gianluca L Russo, Giovanna Sonsalla, Poornemaa Natarajan, Christopher T Breunig, Giorgia Bulli, Juliane Merl-Pham, Sabine Schmitt, Jessica Giehrl-Schwab, Florian Giesert, Martin Jastroch, Hans Zischka, Wolfgang Wurst, Stefan H Stricker, Stefanie M Hauck, Giacomo Masserdotti, Magdalena Götz.

  Astrocyte-to-neuron conversion is a promising avenue for neuronal replacement therapy. Neurons are particularly dependent on mitochondrial function, but how well mitochondria adapt to the new fate is unknown. Here, we determined the comprehensive mitochondrial proteome of cortical astrocytes and neurons, identifying about 150 significantly enriched mitochondrial proteins for each cell type, including transporters, metabolic enzymes, and cell-type-specific antioxidants. Monitoring their transition during reprogramming revealed late and only partial adaptation to the neuronal identity. Early dCas9-mediated activation of genes encoding mitochondrial proteins significantly improved conversion efficiency, particularly for neuron-enriched but not astrocyte-enriched antioxidant proteins. For example, Sod1 not only improves the survival of the converted neurons but also elicits a faster conversion pace, indicating that mitochondrial proteins act as enablers and drivers in this process. Transcriptional engineering of mitochondrial proteins with other functions improved reprogramming as well, demonstrating a broader role of mitochondrial proteins during fate conversion.

Keywords:  CRISPR-a; antioxidant; direct reprogramming; metabolism; mitochondria; proteome

DOI:  https://doi.org/10.1016/j.stem.2020.10.015
Methods Mol Biol. 2021 ;2230 437-447

Mitochondrial Function and Metabolism of Cultured Skeletal Cells.

Li Tian, Clifford J Rosen, Anyonya R Guntur.

  Measuring cellular metabolism accurately is necessary to understand bioenergetic pathways in cells. The major ATP generating pathways in cells are oxidative phosphorylation and glycolysis. We have recently analyzed and published bioenergetic pathways active in osteoblasts undergoing differentiation in response to various substrates. Based on those studies, here we provide step-by-step procedures to isolate, culture, plate and run a seahorse assay for measuring cellular metabolism. Furthermore, we provide an example of oxygen consumption and extracellular acidification rate traces obtained from MC3T3E1-C4 cells using the XFe96 seahorse analyzer. One of the limitations of studying bioenergetics in bone cells is the current lack of techniques to analyze bioenergetics in vivo in live animals. There are currently techniques that have been developed using third harmonic generation to study osteocytes using three-photon microscopy along with metabolic changes using endogenous two-photon excited fluorescence. However, these sophisticated techniques are not widely available. The relative ease with which one can obtain data pertaining to metabolic parameters using the XF technology makes it a very attractive technique to utilize on a monolayer of adherent cells.

Keywords:  Bone marrow stromal cells; Calvarial osteoblasts; Extracellular acidification rate; Glycolysis; MC3T3E1C4 preosteoblast oxidative phosphorylation; Oxygen consumption rate

DOI:  https://doi.org/10.1007/978-1-0716-1028-2_27
Oxid Med Cell Longev. 2020 ;2020 8848930

Oxidized LDL Disrupts Metabolism and Inhibits Macrophage Survival by Activating a miR-9/Drp1/Mitochondrial Fission Signaling Pathway.

Ting Xin, Chengzhi Lu, Jing Zhang, Jiaxin Wen, Shuangbin Yan, Chao Li, Feng Zhang, Jin Zhang.

Mitochondrial dysfunction is associated with macrophage damage, but the role of mitochondrial fission in macrophage cholesterol metabolism is not fully understood. In this study, we explored the influences of miR-9 and mitochondrial fission on macrophage viability and cholesterol metabolism. Macrophages were incubated with oxidized low-density lipoprotein (ox-LDL) in vitro, after which mitochondrial fission, cell viability, and cholesterol metabolism were examined using qPCR, ELISAs, and immunofluorescence. ox-LDL treatment significantly increased Drp1-associated mitochondrial fission. Transfection of Drp1 siRNA significantly reduced cell death, attenuated oxidative stress, and inhibited inflammatory responses in ox-LDL-treated macrophages. Interestingly, inhibition of Drp1-related mitochondrial fission also improved cholesterol metabolism by balancing the transcription of cholesterol influx/efflux enzymes. We also found that miR-9 was downregulated in ox-LDL-treated macrophages, and administration of a miR-9 mimic decreased Drp1 transcription and mitochondrial fission, as well as its effects. These results indicate that signaling via the novel miR-9/Drp1/mitochondrial fission axis is a key determinant of macrophage viability and cholesterol metabolism.

DOI: https://doi.org/10.1155/2020/8848930
Front Neurosci. 2020 ;14 561173

Chronic Alcohol Exposure Induces Aberrant Mitochondrial Morphology and Inhibits Respiratory Capacity in the Medial Prefrontal Cortex of Mice.

Pei Shang, Daniel Lindberg, Phillip Starski, Lee Peyton, Sa-Ik Hong, Sun Choi, Doo-Sup Choi.

  Alcohol use disorder (AUD) is characterized as a chronic, relapsing disease with a pattern of excessive drinking despite negative consequences to an individual's life. Severe chronic alcohol use impairs the function of the medial prefrontal cortex (mPFC), which contributes to alcohol-induced cognitive and executive dysfunction. The mPFC contains more mitochondria compared to other cortical areas, which suggests mitochondrial damage may occur in AUD and trigger subsequent behavior change. Here, we identified morphological and functional changes in mitochondria in the mPFC in C57BL6/J mice after 8 h of withdrawal from chronic intermittent alcohol (CIA) exposure. Three-dimensional serial block-face scanning electron microscopy (SBFSEM) reconstruction revealed that CIA exposure elongated mPFC mitochondria and formed mitochondria-on-a-string (MOAS). Furthermore, alcohol significantly affected mitochondrial bioenergetics, including oxidative phosphorylation and electron transport, with inhibited aerobic respiration in mPFC mitochondria after CIA exposure. We also found decreased expression of fusion (mitofusin 2, Mfn2) and increased fission (mitochondrial fission 1 protein, Fis1) proteins in the mPFC of alcohol-treated mice. In sum, our study suggests that CIA exposure impairs mitochondrial dynamics and function in the mPFC.

Keywords:  alcohol use disorder; fission; fusion; medial prefrontal cortex; mitochondria; morphology; respiratory capacity

DOI:  https://doi.org/10.3389/fnins.2020.561173
FEBS J. 2020 Nov 17.

Targeting cellular senescence based on inter-organelle communication, multi-level proteostasis and metabolic control.

Maria Cavinato, Corina T Madreiter-Sokolowski, Sabrina Büttner, Markus Schosserer, Werner Zwerschke, Sophia Wedel, Johannes Grillari, Wolfgang F Graier, Pidder Jansen-Dürr.

  Cellular senescence, a stable cell division arrest caused by severe damage and stress, is a hallmark of aging in vertebrates including humans. With progressing age, senescent cells accumulate in a variety of mammalian tissues, where they contribute to tissue aging, identifying cellular senescence as a major target to delay or prevent aging. There is an increasing demand for the discovery of new classes of small molecules which would either avoid or postpone cellular senescence by selectively eliminating senescent cells from the body (i.e. "senolytics") or inactivating/switching damage-inducing properties of senescent cells (i.e. "senostatics/senomorphics"), such as the senescence-associated secretory phenotype. Whereas compounds with senolytic or senostatic activity have already been described, their efficacy and specificity has not been fully established for clinical use yet. Here, we review mechanisms of senescence that are related to mitochondria and their inter-organelle communication, and the involvement of proteostasis networks and metabolic control in the senescent phenotype. These cellular functions are associated with cellular senescence in in vitro and in vivo models but have not been fully exploited for the search of new compounds to counteract senescence yet. Therefore, we explore possibilities to target these mechanisms as new opportunities to selectively eliminate and/or disable senescent cells with the aim of tissue rejuvenation. We assume that this research will provide new compounds from the chemical space which act as mimetics of caloric restriction, modulators of calcium signaling and mitochondrial physiology, or as proteostasis optimizers, bearing the potential to counteract cellular senescence, thereby allowing healthy aging.

Keywords:  Autophagy; RNA modification; calcium signaling homeostasis; caloric restriction(CR) mimetic; interorganellar connectivity; lysosome; mitochondria; mitophagy; proteostasis; senescence; translational control

DOI:  https://doi.org/10.1111/febs.15631
Front Physiol. 2020 ;11 595800

Physiological Ca2+ Transients Versus Pathological Steady-State Ca2+ Elevation, Who Flips the ROS Coin in Skeletal Muscle Mitochondria.

Ang Li, Jianxun Yi, Xuejun Li, Jingsong Zhou.

  Mitochondria are both the primary provider of ATP and the pivotal regulator of cell death, which are essential for physiological muscle activities. Ca2+ plays a multifaceted role in mitochondrial function. During muscle contraction, Ca2+ influx into mitochondria activates multiple enzymes related to tricarboxylic acid (TCA) cycle and oxidative phosphorylation, resulting in increased ATP synthesis to meet the energy demand. Pathophysiological conditions such as skeletal muscle denervation or unloading also lead to elevated Ca2+ levels inside mitochondria. However, the outcomes of this steady-state elevation of mitochondrial Ca2+ level include exacerbated reactive oxygen species (ROS) generation, sensitized opening of mitochondrial permeability transition pore (mPTP), induction of programmed cell death, and ultimately muscle atrophy. Previously, both acute and long-term endurance exercises have been reported to activate certain signaling pathways to counteract ROS production. Meanwhile, electrical stimulation is known to help prevent apoptosis and alleviate muscle atrophy in denervated animal models and patients with motor impairment. There are various mechanistic studies that focus on the excitation-transcription coupling framework to understand the beneficial role of exercise and electrical stimulation. Interestingly, a recent study has revealed an unexpected role of rapid mitochondrial Ca2+ transients in keeping mPTP at a closed state with reduced mitochondrial ROS production. This discovery motivated us to contribute this review article to inspire further discussion about the potential mechanisms underlying differential outcomes of physiological mitochondrial Ca2+ transients and pathological mitochondrial Ca2+ elevation in skeletal muscle ROS production.

Keywords:  electric field stimulation; mitochondrial Ca2+ homeostasis; mitochondrial ROS; mitoflash; skeletal muscle; transitory mPTP opening

DOI:  https://doi.org/10.3389/fphys.2020.595800
Physiol Rep. 2020 Nov;8(21): e14605

Mitochondrial DNA lesions and copy number are strain dependent in endurance-trained mice.

Heather L Vellers, Michael P Massett, Josh J Avila, Seung Kyum Kim, Jacqui M Marzec, Janine H Santos, J Timothy Lightfoot, Steven R Kleeberger.

  In this pilot work, we selected two inbred strains that respond well to endurance training (ET) (FVB/NJ, and SJL/J strains), and two strains that respond poorly (BALB/cByJ and NZW/LacJ), to determine the effect of a standardized ET treadmill program on mitochondrial and nuclear DNA (nucDNA) integrity, and mitochondrial DNA (mtDNA) copy number. DNA was isolated from plantaris muscles (n = 37) and a gene-specific quantitative PCR-based assay was used to measure DNA lesions and mtDNA copy number. Mean mtDNA lesions were not different within strains in the sedentary or exercise-trained states. However, mtDNA lesions were significantly higher in trained low-responding NZW/LacJ mice (0.24 ± 0.06 mtDNA lesions/10 Kb) compared to high-responding strains (mtDNA lesions/10 Kb: FVB/NJ = 0.11 ± 0.01, p = .049; SJL/J = 0.04 ± 0.02; p = .003). ET did not alter mean mtDNA copy numbers for any strain, although both sedentary and trained FVB/NJ mice had significantly higher mtDNA copies (99,890 ± 4,884 mtDNA copies) compared to low-responding strains (mtDNA copies: BALB/cByJ = 69,744 ± 4,675; NZW/LacJ = 65,687 ± 5,180; p < .001). ET did not change nucDNA lesions for any strain, however, SJL/J had the lowest mean nucDNA lesions (3.5 ± 0.14 nucDNA lesions/6.5 Kb) compared to all other strains (nucDNA lesions/6.5 Kb: FVB/NJ = 4.4 ± 0.11; BALB/cByJ = 4.7 ± 0.09; NZW/LacJ = 4.4 ± 0.11; p < .0001). Our results demonstrate strain differences in plantaris muscle mtDNA lesions in ET mice and, independent of condition, differences in mean mtDNA copy and nucDNA lesions between strains.

Keywords:  exercise training; interstrain variation; mtDNA copy number; mtDNA lesions

DOI:  https://doi.org/10.14814/phy2.14605
Front Cell Dev Biol. 2020 ;8 585932

Mitochondrial Ca2 + Is Related to Mitochondrial Activity and Dynamic Events in Mouse Oocytes.

Feng Wang, Tie-Gang Meng, Jian Li, Yi Hou, Shi-Ming Luo, Heide Schatten, Qing-Yuan Sun, Xiang-Hong Ou.

  Mitochondrial energy insufficiency is strongly associated with oocyte activation disorders. Ca2+, especially that in the mitochondrial matrix, plays a pivotal role in mitochondrial energy supplementation, but the underlying mechanisms are still only poorly understood. An encoded mitochondrial matrix Ca2+ probe (Mt-GCaMP6s) was introduced to observe mitochondrial Ca2+ ([Ca2+]m) dynamic changes during oocyte maturation and activation. We found that active mitochondria surrounding the nucleus showed a higher [Ca2+]m than those distributed in the cortex during oocyte maturation. During oocyte partheno-activation, the patterns of Ca2+ dynamic changes were synchronous in the cytoplasm and mitochondria. Such higher concentration of mitochondrial matrix Ca2+ was closely related to the distribution of mitochondrial calcium uptake (MICU) protein. We further showed that higher [Ca2+]m mitochondria around the chromosomes in oocytes might have a potential role in stimulating mitochondrial energy for calmodulin-responsive oocyte spindle formation, while synchronizing Ca2+ functions in the cytoplasm and nuclear area are important for oocyte activation.

Keywords:  Ca2+ oscillations; mitochondrial Ca2+; oocyte activation; oocyte maturation; oocyte metabolism

DOI:  https://doi.org/10.3389/fcell.2020.585932
FASEB J. 2020 Nov 16.

Quantitative analysis of mitochondrial membrane potential heterogeneity in unsynchronized and synchronized cancer cells.

Amandine Rovini, Kareem Heslop, Elizabeth G Hunt, Morgan E Morris, Diana Fang, Monika Gooz, Akos A Gerencser, Eduardo N Maldonado.

  Mitochondrial membrane potential (ΔΨm) is a global indicator of mitochondrial function. Previous reports on heterogeneity of ΔΨm were qualitative or semiquantitative. Here, we quantified intercellular differences in ΔΨm in unsynchronized human cancer cells, cells synchronized in G1, S, and G2, and human fibroblasts. We assessed ΔΨm using a two-pronged microscopy approach to measure relative fluorescence of tetramethylrhodamine methyl ester (TMRM) and absolute values of ΔΨm. We showed that ΔΨm is more heterogeneous in cancer cells compared to fibroblasts, and it is maintained throughout the cell cycle. The effect of chemical inhibition of the respiratory chain and ATP synthesis differed between basal, low and high ΔΨm cells. Overall, our results showed that intercellular heterogeneity of ΔΨm is mainly modulated by intramitochondrial factors, it is independent of the ΔΨm indicator and it is not correlated with intercellular heterogeneity of plasma membrane potential or the phases of the cell cycle.

Keywords:  HepG2 cells; TMRM; cancer; cell cycle; fibroblasts; heterogeneity; mitochondria; mitochondrial membrane potential; plasma membrane potential

DOI:  https://doi.org/10.1096/fj.202001693R
Am J Transl Res. 2020 ;12(10): 6879-6894

Induction of mitophagy in C2C12 cells by electrical pulse stimulation involves increasing the level of the mitochondrial receptor FUNDC1 through the AMPK-ULK1 pathway.

Jiuxiang Gao, Liang Yu, Zhen Wang, Ruiyuan Wang, Xiaoran Liu.

The mitochondrial receptor protein FUN14 domain-containing-1 (FUNDC1) can induce mitophagy under hypoxic conditions, as well as playing important roles in normal metabolism and intracellular homeostasis. Exercise not only elevates mitochondrial biosynthesis, but also exerts a significant impact on mitochondrial fission, integration and mitophagy. However, it is still not clear whether FUNDC1 plays a regulatory role in this context. Electrical pulse stimulation (EPS) of cultured myotubes is widely used as an in vitro model of muscle contraction. We simulated the contraction of C2C12 myotubes by EPS (15 V, 1 Hz, 2 ms, 1 h) to examine the role of FUNDC1 in mitophagy. EPS was found to induce mitophagy by activating the AMPK-ULK1 pathway to an even greater extent than AICAR and FUNDC1 is involved in the associated mitophagy. However, when AMPK is inhibited, other pathways may regulate mitophagy. Our findings indicate that mitophagy helps maintain the normal functions of mitochondria. EPS of C2C12 myotubes results in contraction, induction of mitophagy and potential activation of the AMPK-ULK1 pathway that promotes the expression of FUNDC1.

Keywords: AMPK-ULK1 pathway; FUNDC1; electrical pulse stimulation; mitophagy
Brain Sci. 2020 Nov 12. pii: E847. [Epub ahead of print]10(11):

p62-Nrf2-p62 Mitophagy Regulatory Loop as a Target for Preventive Therapy of Neurodegenerative Diseases.

Artem P Gureev, Irina S Sadovnikova, Natalia N Starkov, Anatoly A Starkov, Vasily N Popov.

  Turnover of the mitochondrial pool due to coordinated processes of mitochondrial biogenesis and mitophagy is an important process in maintaining mitochondrial stability. An important role in this process is played by the Nrf2/ARE signaling pathway, which is involved in the regulation of the expression of genes responsible for oxidative stress protection, regulation of mitochondrial biogenesis, and mitophagy. The p62 protein is a multifunctional cytoplasmic protein that functions as a selective mitophagy receptor for the degradation of ubiquitinated substrates. There is evidence that p62 can positively regulate Nrf2 by binding to its negative regulator, Keap1. However, there is also strong evidence that Nrf2 up-regulates p62 expression. Thereby, a regulatory loop is formed between two important signaling pathways, which may be an important target for drugs aimed at treating neurodegeneration. Constitutive activation of p62 in parallel with Nrf2 would most likely result in the activation of mTORC1-mediated signaling pathways that are associated with the development of malignant neoplasms. The purpose of this review is to describe the p62-Nrf2-p62 regulatory loop and to evaluate its role in the regulation of mitophagy under various physiological conditions.

Keywords:  Nrf2; mitochondria; mitophagy; neurodegenerative disease; p62; regulatory loop

DOI:  https://doi.org/10.3390/brainsci10110847