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F-ATP synthase is a leading candidate as the mitochondrial permeability transition pore (PTP) but the mechanism(s) leading to channel formation remain undefined. Here, to shed light on the structural requirements for PTP formation, we test cells ablated for g, OSCP and b subunits, and ρ 0 cells lacking subunits a and A6L. Δg cells (that also lack subunit e) do not show PTP channel opening in intact cells or patch-clamped mitoplasts unless atractylate is added. Δb and ΔOSCP cells display currents insensitive to cyclosporin A but inhibited by bongkrekate, suggesting that the adenine nucleotide translocator (ANT) can contribute to channel formation in the absence of an assembled F-ATP synthase. Mitoplasts from ρ 0 mitochondria display PTP currents indistinguishable from their wild-type counterparts. In this work, we show that peripheral stalk subunits are essential to turn the F-ATP synthase into the PTP and that the ANT provides mitochondria with a distinct permeability pathway., (© 2021. The Author(s).)

Microglia act as sensors of injury in the brain, favoring its homeostasis. Their activation and polarization toward a proinflammatory phenotype are associated with injury and disease. These processes are linked to a metabolic reprogramming of the cells, characterized by high rates of glycolysis and suppressed oxidative phosphorylation. This metabolic switch can be reproduced in vitro by microglial stimulation with LPS plus IFN-γ. To understand the mechanisms regulating mitochondrial respiration abolishment, we examined potential alterations in mitochondrial features during this switch using rat primary microglia. Cells did not show any change in mitochondrial membrane potential, suggesting a limited impact in the mitochondrial viability. We provide evidence that reverse operation of F 0 F 1 -ATP synthase contributes to mitochondrial membrane potential. In addition, we studied the possible implication of mitochondrial dynamics in the metabolic switch using the mitochondrial division inhibitor-1 (Mdivi-1), which blocks dynamin-related protein 1 (Drp1)-dependent mitochondrial fission. Mdivi-1 significantly reduced the expression of proinflammatory markers in LPS plus IFN-γ-treated microglia. However, this inhibition did not lead to a recovery of the oxidative phosphorylation ablation by LPS plus IFN-γ or to a microglia repolarization. Altogether, these results suggest that Drp1-dependent mitochondrial fission, although potentially involved in microglial activation, does not play an essential role in metabolic reprogramming and repolarization of microglia., (Copyright © 2021 The Authors.)

Background and Aims: Inflammatory bowel diseases [IBD], comprising Crohn's disease [CD] and ulcerative colitis [UC], are chronic conditions characterized by severe dysregulation of innate and adaptive immunity resulting in the destruction of the intestinal mucosa. Natural killer [NK] cells play a pivotal role in the dynamic interaction between the innate and adaptive immune response. There is an increasing appreciation for the key role immunometabolism plays in the regulation of NK cell function, yet little remains known about the metabolic profile, cytokine secretion, and killing capacity of human NK cells during active IBD., Methods: Peripheral blood mononuclear cells were isolated from peripheral blood of patients with moderate to severely active IBD and healthy controls. NK cells were stained with a combination of cell surface receptors, intracellular cytokines, and proteins and analyzed by flow cytometry. For measurements of NK cell cytotoxicity, the calcein-AM release assay was performed. The metabolic profile was analyzed by an extracellular flux analyzer., Results: NK cells from IBD patients produce large quantities of pro-inflammatory cytokines, IL-17A and TNF-α ex vivo, but have limited killing capability. Furthermore, patient NK cells have reduced mitochondrial mass and oxidative phosphorylation. mTORC1, an important cell and metabolic regulator, demonstrated limited activity in both freshly isolated cells and cytokine-stimulated cells., Conclusions: Our results demonstrate that circulating NK cells of IBD patients have an unbalanced metabolic profile, with faulty mitochondria and reduced capacity to kill. These aberrations in NK cell metabolism may contribute to defective killing and thus the secondary infections and increased risk of cancer observed in IBD patients., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation.)

Hypoxia-induced cardiomyocyte apoptosis is one of the leading causes of heart failure. Nuclear respiratory factor 1 (NRF-1) was suggested as a protector against cell apoptosis; However, the mechanism is not clear. Therefore, the aim of this study was to elucidate the role of NRF-1 in hypoxia-induced H9C2 cardiomyocyte apoptosis and to explore its effect on regulating the death receptor pathway and mitochondrial pathway. NRF-1 was overexpressed or knocked down in H9C2 cells, which were then exposed to a hypoxia condition for 0, 3, 6, 12, and 24 h. Changes in cell proliferation, cell viability, reactive oxygen species (ROS) generation, and mitochondrial membrane potential (MMP) were investigated. The activities of caspase-3, -8, and -9, apoptosis rate, and the gene and protein expression levels of the death receptor pathway and mitochondrial pathway were analyzed. Under hypoxia exposure, NRF-1 overexpression improved the proliferation and viability of H9C2 cells and decreased ROS generation, MMP loss, caspase activities, and the apoptosis rate. However, the NRF-1 knockdown group showed the opposite results. Additionally, NRF-1 upregulated the expression of antiapoptotic molecules involved in the death receptor and mitochondrial pathways, such as CASP8 and FADD-like apoptosis regulator, B-cell lymphoma 2, B-cell lymphoma-extra-large, and cytochrome C. Conversely, the expression of proapoptotic molecules, such as caspase-8, BH3-interacting domain death agonist, Bcl-2-associated X protein, caspase-9, and caspase-3 was downregulated by NRF-1 overexpression in hypoxia-induced H9C2 cells. These results suggest that NRF-1 functions as an antiapoptotic factor in the death receptor and mitochondrial pathways to mitigate hypoxia-induced apoptosis in H9C2 cardiomyocytes., (© 2021 International Federation for Cell Biology.)

Trypanosoma congolense is a principal agent causing livestock trypanosomiasis in Africa, costing developing economies billions of dollars and undermining food security. Only the diamidine diminazene and the phenanthridine isometamidium are regularly used, and resistance is widespread but poorly understood. We induced stable diminazene resistance in T. congolense strain IL3000 in vitro. There was no cross-resistance with the phenanthridine drugs, melaminophenyl arsenicals, oxaborole trypanocides, or with diamidine trypanocides, except the close analogs DB829 and DB75. Fluorescence microscopy showed that accumulation of DB75 was inhibited by folate. Uptake of [ 3 H]-diminazene was slow with low affinity and partly but reciprocally inhibited by folate and by competing diamidines. Expression of T. congolense folate transporters in diminazene-resistant Trypanosoma brucei brucei significantly sensitized the cells to diminazene and DB829, but not to oxaborole AN7973. However, [ 3 H]-diminazene transport studies, whole-genome sequencing, and RNA-seq found no major changes in diminazene uptake, folate transporter sequence, or expression. Instead, all resistant clones displayed a moderate reduction in the mitochondrial membrane potential Ψm. We conclude that diminazene uptake in T. congolense proceed via multiple low affinity mechanisms including folate transporters; while resistance is associated with a reduction in Ψm it is unclear whether this is the primary cause of the resistance., (© 2021 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Autophagy is believed to be essential for the maintenance of axonal homeostasis in neurons. However, whether autophagy is causally related to the axon degeneration in organophosphorus-induced delayed neuropathy (OPIDN) still remains unclear. This research was designed to investigate the role of autophagy in axon degeneration following tri-ortho-cresyl phosphate (TOCP) in an in vitro model. Differentiated wild-type and Atg7 -/- neuro-2a (N2a) cells were treated with TOCP for 24 h. Axonal degeneration in N2a cells was quantitatively analyzed; the key molecules responsible for axon degeneration and its upstream signaling pathway were determined by Western blotting and real-time PCR. The results found that Atg7 -/- cells exhibited a higher resistance to TOCP insult than wild-type cells. Further study revealed that TOCP caused a significant decrease in pro-survival factors NMNATs and SCG10 and a significant increase in pro-degenerative factor SARM1 in both cells. Notably, Atg7 -/- cells presented a higher level of pro-survival factors and a lower level of pro-degenerative factors than wild-type cells in the same setting of TOCP administration. Moreover, DLK-MAPK pathway was activated following TOCP. Altogether, our results suggest that autophagy is able to affect TOCP-induced axonal injury via regulating the balance between pro-survival and pro-degenerative factors, providing a promising avenue for the potential therapy for OPIDN patients., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature.)

Diabetic retinopathy (DR) has been considered to involve mitochondrial alterations and be related to the nucleotide-binding oligomerization domain-like receptors 3 (NLRP3) inflammasome activation. The voltage-dependent anion channel 1 (VDAC1) protein is one of the key proteins that regulates the metabolic and energetic functions of the mitochondria. To explore the involvement of VDAC1 in mitophagy regulation of NLRP3 inflammasome activation under high-glucose (HG) conditions, this study examined expressions of VDAC1, mitochondrial function and mitophagy-related proteins, and NLRP3 inflammasome-related proteins in human retinal capillary endothelial cells (HRCECs) cultured with 30 mM of glucose in the presence or absence of mitophagy inhibitor (Mdivi-1) using Western blot. Mitochondrial membrane potential and mitochondrial reactive oxygen species (mtROS) were detected using flow cytometry. GFP-tagged pAdTrack-VDAC1 adenovirus was used to overexpress VDAC1. Cell biological behaviors, including proliferation, migration, tubule formation, and apoptosis, were also observed. Our results showed that when compared to the normal glucose and high mannitol groups, increased amounts of mitochondrial fragments, reduced mitochondrial membrane potential, increased expression of mitochondrial fission protein Drp 1, decreased expression of mitochondrial fusion protein Mfn 2, accumulation of mtROS, and activation of the NLRP3 inflammasome were observed in the HG group. Meanwhile, HG markedly reduced the protein expressions of PINK1, Parkin and VDAC1. Inhibition of mitophagy reduced PINK1 expression, enhanced NLRP3 expression, but failed to alter VDAC1. VDAC1 overexpression promoted PINK1 expression, inhibited NLRP3 activation and changed the cell biological behaviors under HG conditions. These findings demonstrate that VDAC1-mediated mitophagy plays a crucial role in regulating NLRP3 inflammasome activation in retinal capillary endothelial cells under HG conditions, suggesting that VDAC1 may be a potential target for preventing or treating DR., (Copyright © 2021. Published by Elsevier Ltd.)

Ischemia is a major cause of kidney damage. Proximal tubular epithelial cells (PTECs) are highly susceptible to ischemic insults that frequently cause acute kidney injury (AKI), a potentially life-threatening condition with high mortality. Accumulating evidence has identified altered mitochondrial function as a central pathologic feature of AKI. The mitochondrial NAD + -dependent enzyme sirtuin 5 (SIRT5) is a key regulator of mitochondrial form and function, but its role in ischemic renal injury (IRI) is unknown. SIRT5 expression was increased in murine PTECs after IRI in vivo and in human PTECs (hPTECs) exposed to an oxygen/nutrient deprivation (OND) model of IRI in vitro. SIRT5-depletion impaired ATP production, reduced mitochondrial membrane potential, and provoked mitochondrial fragmentation in hPTECs. Moreover, SIRT5 RNAi exacerbated OND-induced mitochondrial bioenergetic dysfunction and swelling, and increased degradation by mitophagy. These findings suggest SIRT5 is required for normal mitochondrial function in hPTECs and indicate a potentially important role for the enzyme in the regulation of mitochondrial biology in ischemia., (© 2021. The Author(s).)

Autophagy plays a double-edged sword for cancer; particularly, mitophagy plays important roles in the selective degradation of damaged mitochondria. However, whether mitophagy is involved in killing effects of tumor cells by ionizing radiation (IR) and its underlying mechanism remain elusive. The purpose is to evaluate the effects of mitochondrial ROS (mROS) on autophagy after IR; furthermore, we hypothesized that KillerRed (KR) targeting mitochondria could induce mROS generation, subsequent mitochondrial depolarization, accumulation of Pink1, and recruitment of PARK2 to promote the mitophagy. Thereby, we would achieve a new strategy to enhance mROS accumulation and clarify the roles and mechanisms of radiosensitization by KR and IR. Our data demonstrated that IR might cause autophagy of both MCF-7 and HeLa cells, which is related to mitochondria and mROS, and the ROS scavenger N-acetylcysteine (NAC) could reduce the effects. Based on the theory, mitochondrial targeting vector sterile α - and HEAT/armadillo motif-containing protein 1- (Sarm1-) mtKR has been successfully constructed, and we found that ROS levels have significantly increased after light exposure. Furthermore, mitochondrial depolarization of HeLa cells was triggered, such as the decrease of Na + K + ATPase, Ca 2+ Mg 2+ ATPase, and mitochondrial respiratory complex I and III activities, and mitochondrial membrane potential (MMP) has significantly decreased, and voltage-dependent anion channel 1 (VDAC1) protein has significantly increased in the mitochondria. Additionally, HeLa cell proliferation was obviously inhibited, and the cell autophagic rates dramatically increased, which referred to the regulation of the Pink1/PARK2 pathway. These results indicated that mitophagy induced by mROS can initiate the sensitization of cancer cells to IR and might be regulated by the Pink1/PARK2 pathway., Competing Interests: The authors declare that there is no conflict of interests regarding the publication of this paper., (Copyright © 2021 Lei Yu et al.)

The phenylpyrazole fipronil is an insecticide that inhibits γ -amino-butyric acid (GABA) ionotropic receptors in the central nervous system. Experimental evidence suggests that fipronil acts as a neurotoxin and it is implicated in neurodegenerative diseases; however, the mechanisms of neurotoxicity are not fully elucidated. The objective of this study was to quantify mechanisms of fipronil-induced neurotoxicity in dopamine cells. Rat primary immortalized mesencephalic dopaminergic cells (N27) were treated with fipronil (0.25 up to 500 μM depending on the assay). We measured endpoints related to mitochondrial bioenergetics, mitophagy, mitochondrial membrane potential, and ATP production in addition to discerning transcriptome responses to the pesticide. Fipronil reduced cell viability at 500 μM after 24 h exposure and caspase 3/7 activity was significant increased after 6 and 12 h by 250 and 500 μM fipronil. Subsequent endpoints were thus assessed at concentrations that were below cytotoxicity. We measured oxidative respiration of N27 cells following a 24 h exposure to one dose of either 0.25, 2.5, 25, or 50 μM fipronil. Oxygen consumption rates (OCR) were not different between vehicle-control and 0.25 or 2.5 μM fipronil treatments, but there was a ∼40-60 % reduction in basal respiration, as well as reduced oligomycin-induced ATP production at 50 μM. The reduction in OCR is hypothesized to be related to lower mitochondrial mass due to mitophagy. Mitochondrial membrane potential was also sensitive to fipronil, and it was compromised at concentrations of 2.5 μM and above. To further elucidate the mechanisms linked to neurotoxicity, we conducted transcriptomics in dopamine cells following treatment with 25 μM fipronil. Fipronil suppressed transcriptional networks associated with mitochondria (damage, depolarization, permeability, and fission), consistent with its effects on mitochondrial membrane potential. Altered gene networks also included those related to Alzheimer disease, inflammatory disease, nerve fiber degeneration, and neurofibrillary tangles. This study clarifies molecular targets of fipronil-induced neurotoxicity and supports, through multiple lines of evidence, that fipronil acts as a mitochondrial toxicant in dopamine cells. This is relevant to neurodegenerative diseases like Parkinson's disease as exposure to fipronil is associated with the progressive loss of nigrostriatal dopaminergic neurons in rodents., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Purpose : To investigate the protective effects of nicotinamide riboside (NR) on oxidative damage in hydrogen peroxide (H 2 O 2 )-exposed human lens epithelial cell lines (SRA01/04) and the possible mechanisms underlying its protective effects. Materials and methods : SRA01/04 cells were divided into three groups: the control (CON) group, model (H 2 O 2 ) group and treatment (NR+H 2 O 2 ) group. Superoxide dismutase (SOD), catalase (CAT) and total glutathione (GSH) levels were detected to evaluate oxidative damage induced by different concentrations of H 2 O 2 in SRA01/04 cells. After SRA01/04 cells were treated with NR and/or H 2 O 2 , cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Hoechst staining, cell apoptosis was analysed using flow cytometry, reactive oxygen species (ROS) were measured with the DCFH-DA probe, and mitochondria were stained with MitoTracker to measure the mitochondrial membrane potential (MMP). In addition, western blotting was performed to detect the levels of proteins associated with apoptosis and related signalling pathways. Results : H 2 O 2 induced oxidative damage in SRA01/04 cells by inhibiting the activity of SOD and CAT and reducing total GSH levels. Treatment of SRA01/04 cells with NR significantly increased cell viability and reduced cell apoptosis and ROS generation, whereas SOD and CAT activities and total GSH and MMP levels were improved by the NR treatment in an H 2 O 2 -exposed cell model. Furthermore, NR significantly inhibited the activation of the MAPK pathway but promoted activation of the JAK2/Stat3 pathway compared with the model group. Conclusions : NR may alleviate oxidative damage by targeting the MAPK and JAK2/Stat3 pathways in H 2 O 2 -treated SRA01/04 cells. NR may represent anovel drug for preventing or treating cataracts.

Perfluorooctanoic acid (PFOA) is an abundant per- and polyfluoroalkyl substance (PFAS) detected in both indoor and outdoor environments. While studies suggest exposure concerns for humans, studies investigating PFOA-induced neurotoxicity are lacking. To address this gap, we exposed differentiated human SH-SY5Y cells to PFOA (0.1 μM up to 500 μM) at different time points (4, 24, 48, and 72 h) and measured cell viability, Casp3/7 activity, ATP levels, ATP synthase enzyme activity, mitochondrial membrane potential, reactive oxygen species (ROS), oxygen consumption rates for mitochondrial stress test (XFe24 Flux analyzer), glucose utilization, and global metabolome profiles to assess the potential for PFOA-induced neurotoxicity. Treatment with 10 or 100 μM PFOA did not compromise cell viability nor induce cytotoxicity to SH-SY5Y cells over a 48-hour exposure period. However, >250 μM PFOA compromised cell viability, induced cytotoxicity, and induced caspase 3/7 activity at 48 h. ATP levels were reduced in cells treated with 400 μM PFOA for 24 and 48 h, and with 100 μM PFOA and higher at 72 h. ATP synthase activity was inhibited by 250 μM PFOA but was unchanged by PFOA treatment at 200 μM or less. Conversely, mitochondrial membrane potential was reduced by >10 μM PFOA after 24 h. Total ROS was increased with 100 μM PFOA and higher after 4 h of exposure. Several mitochondria-related endpoints (basal respiration, ATP production, maximum respiration) were negatively affected at 250 μM PFOA at both 24- and 48-hour exposure, but were unaltered at concentrations of 100 μM PFOA or less. One exception was mitochondrial spare capacity, which was reduced by 100 μM PFOA after 24-hour exposure. Similarly, glycolysis, glycolytic capacity, and glycolytic reserve of SH-SY5Y cells were not altered by 10 nor 100 μM PFOA. Nontargeted metabolomics was conducted in cells treated with either 10 or 100 μM PFOA for 48 h, as these two concentrations were not cytotoxic and 28 metabolites differed among treatments. Notable was that 10 μM PFOA had little effect on the SH-SY5Y metabolome, and the metabolic profile was not statistically different from media nor solvent controls. On the other hand, 100 μM PFOA shifted the metabolic signature of the neuronal cells, leading to reduced abundance of ATP-related metabolites (adenine, nicotinamide), neurotransmitter precursors (DL-tryptophan, l-tyrosine), and metabolites that protect mitochondria during oxidative stress (betaine, orotic acid, and l-acetyl carnitine). We hypothesize that this metabolic signature may be associated with the reduced mitochondrial membrane potential observed at lower PFOA concentrations. Metabolic shifts appear to precede compromised cell viability, cytotoxicity, and apoptosis. This study generates mechanistic knowledge regarding PFOA-induced neurotoxicity, focusing on mitochondrial oxidative respiration and the neuronal metabolome., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Mitochondrial dysfunction is now considered an important sign of neuronal death during cerebral ischemia/reperfusion (I/R) injury. Studies have shown that the transfer of mitochondria from astrocytes to injured neurons contributes to endogenous neuroprotection after stroke. Basic and clinical studies have shown that mild hypothermia exerts a clear protective effect on neurons after cerebral ischemic injury, but the role of mild hypothermia in this endogenous neuroprotective mechanism remains unclear. Here, we established a neuronal cell oxygen-glucose deprivation (OGD)/reoxygenation (OGD/R)-induced injury model and explored the effect of mild hypothermia on the transfer of mitochondria from astrocytes to injured neurons. Astrocytes in the hypothermia group (33 °C) released more functional mitochondria into the extracellular medium than those in the normal temperature group (37 °C). Compared with cells in the normal temperature group, OGD-injured neuronal cells in the mild hypothermia group exhibited an increased intracellular ATP content, mitochondrial membrane potential (MMP) and cellular viability and a decreased death rate after the addition of astrocyte-derived conditioned medium. Based on the results of this study, mild hypothermia promotes endogenous neuroprotective effects through a mechanism related to functional mitochondria released from astrocytes into the extracellular space and transferred into injured neurons., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Oxidative stress and mitochondrial dysfunction are involved in cerebral ischemia/reperfusion injury-induced neuronal apoptosis. Mitophagy is the main method to eliminate dysfunctional mitochondria. Apelin-36, a type of neuropeptide, has been reported to exert protective effects in cerebral I/R (I/R) injury, but its precise mechanisms remain to be elucidated. To study the effects of Apelin-36 on oxidative stress and mitochondrial dysfunction in cerebral I/R injury, the oxygen-glucose deprivation/reperfusion (OGD/R) model with 6 h of ischemia and 6 h of reperfusion was established in HT22 cells. Results demonstrated that Apelin-36 protected against OGD/R injury by improving cell viability, decreasing the apoptotic cells ratio and increasing the ratio of Bcl-2/Bax. In addition, Apelin-36 treatment inhibited oxidative stress by downregulating the level of reactive oxygen species (ROS) and malondialdehyde (MDA) as well as the expression of inducible nitric oxide synthase (iNOS). And Apelin-36 also activated the level of superoxide dismutase (SOD) and glutathione (GSH). Mitochondrial apoptosis was also alleviated with Apelin-36 treatment detected by the mitochondrial membrane potential (MMP) and the expression of Cytochrome c (Cyt c), Cleaved caspase-9, and Cleaved caspase-3. Furthermore, the SIRT1-mediated PINK1/Parkin-dependent mitophagy was activated by Apelin-36 treatment with the downregulation of p62 and upregulation of LC3B-II and Beclin1. Both EX527 and Cyclosporine A (CsA), which are inhibitors of SIRT1 and mitophagy, markedly alleviated the inhibition of oxidative stress and mitochondrial dysfunction caused by Apelin-36. These findings suggest that SIRT1-mediated PINK1/Parkin-dependent mitophagy is involved in the neuroprotective effects of Apelin-36 on OGD/R-induced oxidative stress and mitochondrial dysfunction.

Mitochondria respond to many cellular functions and act as central hubs in innate immunity against viruses. This response is notably due to their role in the activation of interferon (IFN) signaling pathways through the activity of MAVS (mitochondrial antiviral signaling protein) present at the mitochondrial surface. Here, we report that the BHRF1 protein, a BCL2 homolog encoded by Epstein-Barr virus (EBV), inhibits IFNB/IFN-β induction by targeting the mitochondria. Indeed, we have demonstrated that BHRF1 expression modifies mitochondrial dynamics and stimulates DNM1L/Drp1-mediated mitochondrial fission. Concomitantly, we have shown that BHRF1 is pro-autophagic because it stimulates the autophagic flux by interacting with BECN1/Beclin 1. In response to the BHRF1-induced mitochondrial fission and macroautophagy/autophagy stimulation, BHRF1 drives mitochondrial network reorganization to form juxtanuclear mitochondrial aggregates known as mito-aggresomes. Mitophagy is a cellular process, which can specifically sequester and degrade mitochondria. Our confocal studies uncovered that numerous mitochondria are present in autophagosomes and acidic compartments using BHRF1-expressing cells. Moreover, mito-aggresome formation allows the induction of mitophagy and the accumulation of PINK1 at the mitochondria. As BHRF1 modulates the mitochondrial fate, we explored the effect of BHRF1 on innate immunity and showed that BHRF1 expression could prevent IFNB induction. Indeed, BHRF1 inhibits the IFNB promoter activation and blocks the nuclear translocation of IRF3 (interferon regulatory factor 3). Thus, we concluded that BHRF1 can counteract innate immunity activation by inducing fission of the mitochondria to facilitate their sequestration in mitophagosomes for degradation. Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; BCL2: BCL2 apoptosis regulator; CARD: caspase recruitment domain; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CI: compaction index; CQ: chloroquine; DAPI: 4',6-diamidino-2-phenylindole, dihydrochloride; DDX58/RIG-I: DExD/H-box helicase 58; DNM1L/Drp1: dynamin 1 like; EBSS: Earle's balanced salt solution; EBV: Epstein-Barr virus; ER: endoplasmic reticulum; EV: empty vector; GFP: green fluorescent protein; HEK: human embryonic kidney; IFN: interferon; IgG: immunoglobulin G; IRF3: interferon regulatory factor 3; LDHA: lactate dehydrogenase A; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; MMP: mitochondrial membrane potential; MOM: mitochondrial outer membrane; PINK1: PTEN induced kinase 1; RFP: red fluorescent protein; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TOMM20: translocase of outer mitochondrial membrane 20; VDAC: voltage dependent anion channel.

Damaged mitochondria need to be cleared to maintain the quality of the mitochondrial pool. Here, we report mitocytosis, a migrasome-mediated mitochondrial quality-control process. We found that, upon exposure to mild mitochondrial stresses, damaged mitochondria are transported into migrasomes and subsequently disposed of from migrating cells. Mechanistically, mitocytosis requires positioning of damaged mitochondria at the cell periphery, which occurs because damaged mitochondria avoid binding to inward motor proteins. Functionally, mitocytosis plays an important role in maintaining mitochondrial quality. Enhanced mitocytosis protects cells from mitochondrial stressor-induced loss of mitochondrial membrane potential (MMP) and mitochondrial respiration; conversely, blocking mitocytosis causes loss of MMP and mitochondrial respiration under normal conditions. Physiologically, we demonstrate that mitocytosis is required for maintaining MMP and viability in neutrophils in vivo. We propose that mitocytosis is an important mitochondrial quality-control process in migrating cells, which couples mitochondrial homeostasis with cell migration., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Cisplatin is an efficacious anticancer agent, but its use is limited by ototoxicity and resultant irreversible sensorineural hearing loss. Cisplatin ototoxicity is associated with cochlear cell oxidative stress and mitochondrial damage. However, mitophagy is vital for maintaining mitochondrial quality and cellular metabolism. Accordingly, we investigated the role of mitophagy in regulating cisplatin-induced ototoxicity using the auditory cell line HEI-OC1. In this study, HEI-OC1 cells were treated with either cisplatin alone (10  μ M, 0, 8, 16, and 24 h); cisplatin (10  μ M, 24 h) post transfection with small-interfering (si)RNAs targeting mitophagy-associated mRNAs; cisplatin (10  μ M, 24 h) succeeding pretreatment with the mitophagy suppressor, 3-methyladenine (3-MA; 5 or 10 mM, 6 h); or cisplatin (30  μ M, 24 h) following pretreatment with the mitophagy promoter, carbonyl cyanide m-chlorophenylhydrazone (CCCP; 1 or 2  μ M, 2 h). The viability of cells, expression of mitophagy marker, and mitochondrial functions were then assessed in these cells. Cell viability was determined by a water-soluble tetrazolium assay; expression of mitophagy-associated proteins PINK1 , Parkin , BNIP3 , FUNDC1 , p62, and LC3B was analyzed by Western blotting, mitochondrial membrane potential by flow cytometry, intracellular ATP by spectrophotometry, and mitochondrial degradation by dual staining for mitochondria and autophagosomes or lysosomes. Our results showed that cisplatin gradually reduced the viable cell number over time, induced mitochondrial depolarization, decreased intracellular ATP concentration, and enhanced the expression of PINK1 , Parkin , BNIP3 , p62, and LC3B. In addition, Parkin and BNIP3 knockdown accelerated cisplatin-induced loss of cell viability, mitochondrial membrane potential, mitophagosome/lysosome formation, and reduction in intracellular ATP production. Pretreatment with 3-MA aggravated the cisplatin-induced cytotoxicity, while that with CCCP reversed this effect. Overall, our findings indicate that mitophagy protects HEI-OC1 cells against cisplatin-induced cell death. Consequently, we strongly believe that targeted promotion of mitophagy may confer protection against cisplatin-induced ototoxicity., Competing Interests: None of the authors has a conflict of interest to declare., (Copyright © 2021 Sung Il Cho et al.)

Brain is not homogenous and neurons from various brain regions are known to have different vulnerabilities to mitochondrial mutations and mitochondrial toxins. However, it is not clear if this vulnerability is connected to different energy metabolism in specific brain regions. Here, using live-cell imaging, we compared mitochondrial membrane potential and nicotinamide adenine dinucleotide (NADH) redox balance in acute rat brain slices in different brain regions and further detailed the mitochondrial metabolism in primary neurons and astrocytes from rat cortex, midbrain and cerebellum. We have found that mitochondrial membrane potential is higher in brain slices from the hippocampus and brain stem. In primary co-cultures, mitochondrial membrane potential in astrocytes was lower than in neurons, whereas in midbrain cells it was higher than in cortex and cerebellum. The rate of NADH production and mitochondrial NADH pool were highest in acute slices from midbrain and midbrain primary neurons and astrocytes. Although the level of adenosine tri phosphate (ATP) was similar among primary neurons and astrocytes from cortex, midbrain and cerebellum, the rate of ATP consumption was highest in midbrain cells that lead to faster neuronal and astrocytic collapse in response to inhibitors of ATP production. Thus, midbrain neurons and astrocytes have a higher metabolic rate and ATP consumption that makes them more vulnerable to energy deprivation., (© 2020 International Society for Neurochemistry.)

Mitochondrial diseases impair oxidative phosphorylation and ATP production, while effective treatment is still lacking. Defective complex III is associated with a highly variable clinical spectrum. We show that pyocyanin, a bacterial redox cycler, can replace the redox functions of complex III, acting as an electron shunt. Sub-μM pyocyanin was harmless, restored respiration and increased ATP production in fibroblasts from five patients harboring pathogenic mutations in TTC19, BCS1L or LYRM7, involved in assembly/stabilization of complex III. Pyocyanin normalized the mitochondrial membrane potential, and mildly increased ROS production and biogenesis. These in vitro effects were confirmed in both Drosophila TTC19KO and in Danio rerio TTC19KD , as administration of low concentrations of pyocyanin significantly ameliorated movement proficiency. Importantly, daily administration of pyocyanin for two months was not toxic in control mice. Our results point to utilization of redox cyclers for therapy of complex III disorders.

Objective: To study the biological effects of resveratrol on the growth, electrophysiology, and mitochondrial function of human granulosa cells (h-GCs)., Design: Preclinical study., Setting: Electrophysiology laboratory and in vitro fertilization unit., Patient(s): This study included h-GCs from seven infertile women undergoing assisted reproductive techniques., Intervention(s): Human ovarian Granulosa Cell Tumor (GCT) cell line COV434 and h-GCs obtained after oocyte retrieval were cultured in the absence or presence of resveratrol., Main Outcome Measure(s): Granulosa cells were evaluated for cell viability and mitochondrial activity. Electrophysiological recordings and evaluation of potassium current (IKur) and Ca 2+ concentration were also performed., Result(s): Resveratrol induced mitochondrial activity in a bell-shaped, dose-effect-dependent manner. Specifically, resveratrol treatment (3 μM, 48 hours) increased ATP production and cell viability and promoted the induction of cellular differentiation. These biological changes were associated with mitochondrial biogenesis. Electrophysiological recordings showed that resveratrol reduced the functional expression of an ultra rapid activating, slow inactivating, delayed rectifier potassium current (IKur) that is associated with a plasma membrane depolarization and that promotes an increase in intracellular Ca 2 + . CONCLUSION(S): The effects of resveratrol on potassium current and mitochondrial biogenesis in h-GCs could explain the beneficial effects of this polyphenol on the physiology of the female reproductive system. These findings suggest there are therapeutic implications of resveratrol in a clinical setting., (Copyright © 2020 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Malignant tumors of the central nervous system (CNS) are among the types of cancer with the poorest prognosis and glioma is the commonest primary CNS tumor. A mitochondrial DNA (mtDNA)‑depleted cell line C6ρ0 was generated from C6 glioma cells after long‑term exposure to ethidium bromide and 2',3'‑dideoxycytidine in order to determine the effect of mtDNA damage on cell proliferation and pathological changes in glioma cells. Single cell clones were isolated and identified after 42 days of incubation. Repopulated cybrids were formed when the clonal C6ρ0 cells were fused with rat platelets and no difference was observed in their growth in a selective medium without uridine and pyruvate compared with the growth of the parent C6 cells. Disruption of mtDNA resulted in changes in mitochondrial morphology, decreased cell proliferation, reduced intracellular reactive oxygen species and intracellular ATP, along with decreased mtDNA and mitochondrial membrane potential in C6ρ0 cells compared with the C6 cells. Taken together, C6ρ0 cells without mtDNA were established for the first time and their characteristics were compared with parent cells. This C6ρ0 cell line could be used to explore the contribution of mitochondrial dysfunction and mtDNA mutations in the pathogenesis of glioma.

Cardiac hypertrophy (CH) is characterized by an increase in cardiomyocyte size, and is the most common cause of cardiac-related sudden death. A decrease in gap junction (GJ) coupling and mitochondrial dysfunction are important features of CH, but the mechanisms of decreased coupling and energy impairment are poorly understood. It has been reported that GJA1-20k has a strong tropism for mitochondria and is required for the trafficking of connexin 43 (Cx43) to cell-cell borders. In this study, we investigated the effects of GJA1-20k on Cx43 GJ coupling and mitochondrial function in the pathogenesis of CH. We performed hematoxylin-eosin (HE) and Masson staining, and observed significant CH in 18-week-old male spontaneously hypertensive rats (SHRs) compared to age-matched normotensive Wistar-Kyoto (WKY) rats. In cardiomyocytes from SHRs, the levels of Cx43 at the intercalated disc (ID) and the expression of GJA1-20k were significantly reduced, whereas JAK-STAT signaling was activated. Furthermore, the SHR rats displayed suppressed mitochondrial GJA1-20k and mitochondrial biogenesis. Administration of valsartan (10 mg· [Formula: see text] d -1 , i.g., for 8 weeks) prevented all of these changes. In neonatal rat cardiomyocytes (NRCMs), overexpression of GJA1-20k attenuated Ang II-induced cardiomyocyte hypertrophy and caused elevated levels of GJ coupling at the cell-cell borders. Pretreatment of NRCMs with the Jak2 inhibitor AG490 (10 µM) blocked Ang II-induced reduction in GJA1-20k expression and Cx43 gap junction formation; knockdown of Jak2 in NRCMs significantly lessened Ang II-induced cardiomyocyte hypertrophy and normalized GJA1-20k expression and Cx43 gap junction formation. Overexpression of GJA1-20k improved mitochondrial membrane potential and respiration and lowered ROS production in Ang II-induced cardiomyocyte hypertrophy. These results demonstrate the importance of GJA1-20k in regulating gap junction formation and mitochondrial function in Ang II-induced cardiomyocyte hypertrophy, thus providing a novel therapeutic strategy for patients with cardiomyocyte hypertrophy.

Mitochondrial DNA (mtDNA) encodes protein subunits and translational machinery required for oxidative phosphorylation (OXPHOS). Using repurposed whole-exome sequencing data, in the present study we demonstrate that pathogenic mtDNA mutations arise in tumours at a rate comparable to those in the most common cancer driver genes. We identify OXPHOS complexes as critical determinants shaping somatic mtDNA mutation patterns across tumour lineages. Loss-of-function mutations accumulate at an elevated rate specifically in complex I and often arise at specific homopolymeric hotspots. In contrast, complex V is depleted of all non-synonymous mutations, suggesting that impairment of ATP synthesis and mitochondrial membrane potential dissipation are under negative selection. Common truncating mutations and rarer missense alleles are both associated with a pan-lineage transcriptional programme, even in cancer types where mtDNA mutations are comparatively rare. Pathogenic mutations of mtDNA are associated with substantial increases in overall survival of colorectal cancer patients, demonstrating a clear functional relationship between genotype and phenotype. The mitochondrial genome is therefore frequently and functionally disrupted across many cancers, with major implications for patient stratification, prognosis and therapeutic development.

Parkinson's disease (PD) is a neurodegenerative disease characterized by the selective loss of dopaminergic neurons in the substantia nigra (SN). In a previous study, the authors demonstrated that ferritin heavy chain 1 (FTH1) inhibited ferroptosis in a model of 6‑hydroxydopamine (6‑OHDA)‑induced PD. However, whether and how microRNAs (miRNAs/miRs) modulate FTH1 in PD ferroptosis is not yet well understood. In the present study, in vivo and in vitro models of PD induced by 6‑OHDA were established. The results in vivo and in vitro revealed that the levels of the ferroptosis marker protein, glutathione peroxidase 4 (GPX4), and the PD marker protein, tyrosine hydroxylase (TH), were decreased in the model group, associated with a decreased FTH1 expression and the upregulation of miR‑335. In both the in vivo and in vitro models, miR‑335 mimic led to a lower FTH1 expression, exacerbated ferroptosis and an enhanced PD pathology. The luciferase 3'‑untranslated region reporter results identified FTH1 as the direct target of miR‑335. The silencing of FTH1 in 6‑OHDA‑stimulated cells enhanced the effects of miR‑335 on ferroptosis and promoted PD pathology. Mechanistically, miR‑335 enhanced ferroptosis through the degradation of FTH1 to increase iron release, lipid peroxidation and reactive oxygen species (ROS) accumulation, and to decrease mitochondrial membrane potential (MMP). On the whole, the findings of the present study reveal that miR‑335 promotes ferroptosis by targeting FTH1 in in vitro and in vivo models of PD, providing a potential therapeutic target for the treatment of PD.

The present study aimed to explore the role and mechanisms of proprotein convertase subtilisin/kexin type 9 (PCSK9) in the oxidized low‑density lipoprotein (oxLDL)‑induced pyroptosis of vascular endothelial cells. For this purpose, human umbilical vein endothelial cells (HUVECs) were incubated with oxLDL (100 µg/ml) for 24 h to induce pyroptosis, which was detected using PI/hoechst33342 double staining. The expression of pyroptosis‑associated molecules was measured by western blot analysis and RT‑qPCR. Reactive oxygen species (ROS) and membrane potential were examined through ROS probe and JC‑1 staining, respectively. PCSK9 and mitochondrial ubiquinol‑cytochrome c reductase core protein 1 (UQCRC1) protein were knocked down by small interfering RNA (siRNA). PCSK9 was overexpressed by lentivirus. The results revealed that oxLDL induced HUVEC injury, pyroptosis and inflammatory factor release, and upregulated the expression of PCSK9 protein in the HUVECs in a concentration‑dependent manner. The silencing of PCSK9 expression with siRNA suppressed the oxLDL‑induced damage to HUVECs, the release of inflammatory substances and the occurrence of pyroptosis. In addition, oxLDL inhibited UQCRC1 expression, promoted mitochondrial membrane potential collapse and damaged mitochondrial function; however, these processes were reversed by the silencing of PCSK9. PCSK9 overexpression induced the pyroptosis of HUVECs, the generation of ROS and the disorder of mitochondrial function by inhibiting UQCRC1. Therefore, PCSK9 mediates the oxLDL‑induced pyroptosis of vascular endothelial cells via the UQCRC1/ROS pathway.

This study aimed to investigate the protective effect of melatonin on the corneal epithelium in dry eye disease(DED) and explore its underlying mechanism. Human corneal epithelial(HCE) cells was exposure to t-butylhydroperoxide(tBH), C57BL/6 mice were injected of subcutaneous scopolamine to imitate DED. Melatonin was used both in vivo and in vitro. Cell viability was detected by Cell Counting Kit-8 assay and Lactate Dehydrogenase Leakage. The change of cellular reactive oxygen species (ROS) levels, mitochondrial membrane potential (MMP), and apoptosis was analyzed by flow cytometry. Western blot assays and immunofluorescence were carried out to measure protein changes. mRNA expression was investigated by RNA sequencing (RNA-Seq) and quantitative real-time PCR. The change of autophagic flux were observed through mCherry-GFP-LC3 transfection and electron microscopy(TEM). Clinical parameters of corneal epithelium defects, conjunctival goblet cells, tear volume, and level of ocular surface inflammation was recorded. Melatonin was able to reduce excessive ROS production and maintain mitochondrial function. TEM assay found melatonin rescued impaired autophagic flux under tBH. Moreover, melatonin significantly preserved cell viability, abolished LDH release, and decreased apoptosis. RNA-Seq indicated that melatonin greatly activating hemeoxygenase-1 (HO-1) expression. Interestingly, HO-1 ablation largely attenuated its protective effects. Besides, in dry eye mouse model, intraperitoneal injection of melatonin showed greatly improved clinical parameters, inhibited activated NLRP3 inflammation cascade, and increased density of goblet cells and tear volume. Thus, melatonin protects corneal epithelial cells from oxidative damage, maintain normal level of autophagy, and reduce inflammation via trigging HO-1 expression in DED., (Copyright © 2021. Published by Elsevier Ltd.)

Inhibition of amyloid β (Aβ)-induced mitochondrial damage is considered crucial for reducing the pathological damage in Alzheimer's disease (AD). We evaluated the effect of neural stem cell-conditioned medium (NSC-CDM) on Aβ25-35-induced damage in SH-SY5Y cells. An in vitro model of AD was established by treating SH-SY5Y cells with 40 µM Aβ25-35 for 24 h. SH-SY5Y cells were divided into control, Aβ25-35 (40 µM), Aβ25-35 (40 µM) + NSC-CDM, and Aβ25-35 (40 µM) + neural stem cell-complete medium (NSC-CPM) groups. Cell viability was detected by CCK-8 assay. Apoptosis, reactive oxygen species (ROS) production, and mitochondrial membrane potential (MMP) were detected by flow cytometry. Malondialdehyde content was detected by ELISA assay. Western blot analysis was used to detect cytochrome c release and apoptosis-related proteins. Transmission electron microscopy was used to observe mitochondrial morphology. Cell viability significantly decreased and apoptosis significantly increased in SH-SY5Y cells treated with Aβ25-35, and both effects were rescued by NSC-CDM. In addition, NSC-CDM reduced ROS production and significantly inhibited the reduction of MMP caused by Aβ25-35. Furthermore, NSC-CDM ameliorated Aβ25-35-induced reduction in Bcl-2 expression levels and increased the expression levels of cytochrome c, caspase-9, caspase-3, and Bax. Moreover, Aβ25-35 induced the destruction of mitochondrial ultrastructure and this effect was reversed by NSC-CDM. Collectively, our findings demonstrated the protective effect of NCS-CDM against Aβ25-35-induced SH-SY5Y cell damage and clarified the mechanism of action of Aβ25-35 in terms of mitochondrial maintenance and mitochondria-associated apoptosis signaling pathways, thus providing a theoretical basis for the development of novel anti-AD treatments.

Mitochondrial apoptosis is one of the main mechanisms for cancer cells to overcome chemoresistance. Hexokinase 2 (HK2) can resist cancer cell apoptosis by expressing on mitochondria and binding to voltage-dependent anion channel 1 (VDAC1). We previously reported that peroxisome proliferator-activated receptor coactivator 1 α (PGC1α) is highly expressed in ovarian cancer cisplatin-resistant cells. However, the underlying mechanism remains unclear. Therefore, we evaluated the interaction between PGC1α and HK2 in ovarian cancer cisplatin-resistant cells. We found that the knockdown of PGC1α promotes the apoptosis of ovarian cancer cisplatin-resistant cells and increases their sensitivity to cisplatin. In addition, we found that the knockdown of PGC1α affects the mitochondrial membrane potential and the binding of HK2 and VDAC1. As the heat shock protein 70 (HSP70) family can help protein transport, we detected it and found that PGC1α can promote HSP70 gene transcription. Furthermore, HSP70 can promote an increase of HK2 expression on mitochondria and an increase of binding to VDAC1. Based on these results, PGC1α may reduce apoptosis through the HSP70/HK2/VDAC1 signaling pathway, thus promoting cisplatin resistance of ovarian cancer. These findings provide strong theoretical support for PGC1α as a potential therapeutic target of cisplatin resistance in ovarian cancer.

Remote ischemic postconditioning (RI-postC) is an effective measure to improve nerve function after cardiac arrest. However, the brain protective mechanism of RI-postC has not been fully elucidated, and whether it is related to mitophagy is unclear. In this study, we used the rat model of cardiac arrest to study the effect of RI-postC on mitophagy and explore its possible signaling pathways. Rats were randomly divided into Sham group, CA/CPR group, Mdivi-1 group and RI-postC group. The animal model of cardiac arrest was established by asphyxia. RI-postC was performed by clamping and loosening the left femoral artery. Mdivi-1 was treated with a single intravenous injection. Levels of TOMM20, TIM23, Mfn1, PINK1 and parkin were detected by western blots. Mitochondrial membrane potential was measured by flow cytometry. Real-time PCR was used to detect relative mitochondrial DNA levels. The apoptosis of hippocampal neurons was detected by flow and TUNEL. In addition, Histopathological tests were performed. The results showed that RI-postC was similar to the mitophagy inhibitor Mdivi-1, which could inhibit the decrease of mitophagy-related protein level, improve mitochondrial membrane potential and up-regulate the ratio of mt-Atp6/Rpl13 after cardiopulmonary resuscitation (CPR). Furthermore, RI-postC could also reduce the rate of hippocampal nerve apoptosis and the damage of hippocampal neurons after CPR. Moreover, RI-postC and Mdivi-1 could reduce the protein levels of PINK1 and parkin in mitochondria after CPR, while increasing PINK1 levels in the cytoplasm. These findings suggested that RI-postC could inhibit the overactivation mitophagy through the PINK1/parkin signaling pathway, thus providing neuroprotective effects.

Background: The lack of effective treatments for Alzheimer's disease (AD) reflects an incomplete understanding of disease mechanisms. Alterations in proteins involved in mitochondrial dynamics, an essential process for mitochondrial integrity and function, have been reported in AD brains. Impaired mitochondrial dynamics causes mitochondrial dysfunction and has been associated with cognitive impairment in AD. Here, we investigated a possible link between pro-inflammatory interleukin-1 (IL-1), mitochondrial dysfunction, and cognitive impairment in AD models., Methods: We exposed primary hippocampal cell cultures to amyloid-β oligomers (AβOs) and carried out AβO infusions into the lateral cerebral ventricle of cynomolgus macaques to assess the impact of AβOs on proteins that regulate mitochondrial dynamics. Where indicated, primary cultures were pre-treated with mitochondrial division inhibitor 1 (mdivi-1), or with anakinra, a recombinant interleukin-1 receptor (IL-1R) antagonist used in the treatment of rheumatoid arthritis. Cognitive impairment was investigated in C57BL/6 mice that received an intracerebroventricular (i.c.v.) infusion of AβOs in the presence or absence of mdivi-1. To assess the role of interleukin-1 beta (IL-1β) in AβO-induced alterations in mitochondrial proteins and memory impairment, interleukin receptor-1 knockout (Il1r1 -/- ) mice received an i.c.v. infusion of AβOs., Results: We report that anakinra prevented AβO-induced alteration in mitochondrial dynamics proteins in primary hippocampal cultures. Altered levels of proteins involved in mitochondrial fusion and fission were observed in the brains of cynomolgus macaques that received i.c.v. infusions of AβOs. The mitochondrial fission inhibitor, mdivi-1, alleviated synapse loss and cognitive impairment induced by AβOs in mice. In addition, AβOs failed to cause alterations in expression of mitochondrial dynamics proteins or memory impairment in Il1r1 -/- mice., Conclusion: These findings indicate that IL-1β mediates the impact of AβOs on proteins involved in mitochondrial dynamics and that strategies aimed to prevent pathological alterations in those proteins may counteract synapse loss and cognitive impairment in AD.

Little data exist about the physiological role of ion channels during the freeze-thaw process in mammalian sperm. Herein, we determined the relevance of potassium channels, including SLO1, and of voltage-gated proton channels (HVCN1) during mammalian sperm cryopreservation, using the pig as a model and through the addition of specific blockers (TEA: tetraethyl ammonium chloride, PAX: paxilline or 2-GBI: 2-guanidino benzimidazole) to the cryoprotective media at either 15 °C or 5 °C. Sperm quality of the control and blocked samples was performed at 30- and 240-min post-thaw, by assessing sperm motility and kinematics, plasma and acrosome membrane integrity, membrane lipid disorder, intracellular calcium levels, mitochondrial membrane potential, and intracellular O 2 - ⁻ and H 2 O 2 levels. General blockade of K + channels by TEA and specific blockade of SLO1 channels by PAX did not result in alterations in sperm quality after thawing as compared to control samples. In contrast, HVCN1-blocking with 2-GBI led to a significant decrease in post-thaw sperm quality as compared to the control, despite intracellular O 2 - ⁻ and H 2 O 2 levels in 2-GBI blocked samples being lower than in the control and in TEA- and PAX-blocked samples. We can thus conclude that HVCN1 channels are related to mammalian sperm cryotolerance and have an essential role during cryopreservation. In contrast, potassium channels do not seem to play such an instrumental role.

Downregulating miR-217-5p could protect cardiomyocytes against ischemia/reperfusion (I/R) injury, but its role in restoring mitochondrial function of I/R-injured cardiomyocytes remained unclear. H9C2 cardiomyocyte-derived cell line with I/R injury was established in vitro on the basis of hypoxia/reperfusion (H/R) model. Cell viability and apoptosis were respectively detected by MTT assay and flow cytometry. Contents of lactate dehydrogenase (LDH) and adenosine triphosphate (ATP) were determined. Flow cytometry was performed to measure the production of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP). Target gene and potential binding sites between miR-217-5p and Sirtuin1 (SIRT1) were predicted by TargetScan and confirmed by dual-luciferase reporter assay. Relative SIRT1 and expressions of autophagy-related and apoptosis-related genes were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. After I/R treatment, the viability of H9C2 cardiomyocyte-derived cell line and ATP contents were reduced, but LDH and ROS contents were increased, at the same time, cell apoptosis and the expressions of miR-217-5p, p62 and cleaved caspase-3 were increased, whereas the expressions of SIRT1, LC3 (light chain 3), PINK1 (PTEN-induced kinase 1), Parkin, Bcl-2, and c-IAP (inhibitor of apoptosis protein) were reduced. However, downregulating miR-217-5p expression reversed the effects of I/R. SIRT1 was predicted and verified to be the target of miR-217-5p, and silencing SIRT1 reversed the effects of downregulating miR-217-5p on I/R-injured cells. Downregulating miR-217-5p could help restore mitochondrial function via targeting SIRT1, so as to protect cardiomyocytes against I/R-induced injury.

Oxidative stresss in the microenvironment surrounding lesions induces apoptosis of transplanted bone-marrow-derived mesenchymal stem cells (BMSCs). Hence, there is an urgent need for improving antioxidative-stress processes of transplanted BMSCs to further promote their survival. The present study reports the role and mechanism of Parkinson's disease protein 7 (PARK7) in enhancing antioxidative activity in BMSCs. We used a PARK7 lentivirus to transfect BMSCs to up- or downregulate PARK7, and then used H 2 O 2 to simulate oxidative stress in BMSCs in vitro. Overexpression of PARK7 effectively reduced reactive oxygen species and malondialdehyde, protected mitochondrial membrane potential, and resisted oxidative-stress-induced apoptosis of BMSCs, but the expression of PARK7 was downregulated, these results were reversed. At the same time, we also found that overexpression of PARK7 increased extracellular-regulated protein kinase 1/2 (ERK1/2) phosphorylation and nuclear translocation, as well as upregulated Elk1 phosphorylation and superoxide dismutase (SOD) expression. In contrast, when U0126 was used to block the ERK1/2 pathway, ERK1/2 and Elk1 phosphorylation levels were downregulated, ERK1/2 nuclear translocation and SOD content were significantly reduced, and PARK7-overexperssion-induced antioxidative activity was completely blocked. Collectively, our results suggest that PARK7 overexpression increased antioxidative-stress processes and survival of BMSCs subjected to H 2 O 2 via activating the ERK1/2 signaling pathway. Our findings may guide the development of a PARK7-specific strategy for improving the transplantation efficacy of BMSCs., (© 2020 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals LLC.)

We analyzed the state of intracellular compartments and production of cytokines in MSC depending on the culture density. MSC were growth-arrested with mitomycin C and seeded at a density of 300-7000 cell/cm2. MSC in low-density cultures had 2-fold higher levels of transmembrane mitochondrial potential (MitoTracker Red) and endogenous ROS (CMH 2 DCFDA), lysosomal compartments were less acidified (LysoTracker Green DND26), the production of immunoregulatory and angiogenic mediators VEGF, IL-6, IL-8, MCP-1, TGF-β was more intensive. It was assumed that culture density can be an effective tool for phenotypic polarization of MSC providing directional changes in their properties.

Mitochondria operate as a central hub for many metabolic processes by sensing and responding to the cellular environment. Developmental cues from the environment have been implicated in selective autophagy, or mitophagy, of mitochondria during cell differentiation and tissue development. Mitophagy occurring in this context, termed programmed mitophagy, responds to cell state rather than mitochondrial damage and is often accompanied by a metabolic transition. However, little is known about the mechanisms that engage and execute mitophagy under physiological or developmental conditions. As the mammary gland undergoes post-natal development and lactation challenges mitochondrial homeostasis, we investigated the contribution of mitochondria to differentiation of mammary epithelial cells (MECs). Using lactogenic differentiation of the HC11 mouse MEC line, we demonstrated that HC11 cells transition to a highly energetic state during differentiation by engaging both oxidative phosphorylation and glycolysis. Interestingly, this transition was lost when autophagy was inhibited with bafilomycin A 1 or knockdown of Atg7 ( autophagy related 7 ). To evaluate the specific targeting of mitochondria, we traced mitochondrial oxidation and turnover in vitro with the fluorescent probe, pMitoTimer . Indeed, we found that differentiation engaged mitophagy. To further evaluate the requirement of mitophagy during differentiation, we knocked down the expression of Prkn/parkin in HC11 cells. We found that MEC differentiation was impaired in shPrkn cells, implying that PRKN is required for MEC differentiation. These studies suggest a novel regulation of MEC differentiation through programmed mitophagy and provide a foundation for future studies of development and disease associated with mitochondrial function in the mammary gland. Abbreviations : AA: antimycin A; ATG5: autophagy related 5; BAF: bafilomycin A 1 ; BNIP3: BCL2 interacting protein 3; BNIP3L/NIX: BCL2 interacting protein 3 like; COX8A: cytochrome c oxidase subunit 8A; CQ: chloroquine; CSN2: casein beta; ECAR: extracellular acidification rate; FCCP: trifluoromethoxy carbonylcyanide phenylhydrazone; FUNDC1: FUN14 domain containing 1; HIF1A: hypoxia inducible factor 1 subunit alpha; L1: lactation day 1; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MEC: mammary epithelial cell; mitoQ: mitoquinol; mROS: mitochondrial reactive oxygen species; OCR: oxygen consumption rate; P: priming; P16: pregnancy day 16; PARP1: poly(ADP-ribose) polymerase 1; PINK1: PTEN induced kinase 1; PPARGC1A: PPARG coactivator 1 alpha; PRKN: parkin RBR E3 ubiquitin protein ligase; shNT : short hairpin non-targeting control; SQSTM1: sequestosome 1; STAT3: signal transducer and activator of transcription 3; TEM: transmission electron microscopy; TFAM: transcription factor A, mitochondrial; U: undifferentiated.

The redox states of NAD and NADP are linked to each other in the mitochondria thanks to the enzyme nicotinamide nucleotide transhydrogenase (NNT) which, by utilizing the mitochondrial membrane potential (mΔΨ), catalyzes the transfer of redox potential between these two coenzymes, reducing one at the expense of the oxidation of the other. In order to define NNT reaction direction in CF cells, NNT activity under different redox states of cell has been investigated. Using spectrophotometric and western blotting techniques, the presence, abundance and activity level of NNT were determined. In parallel, the levels of NADPH and NADH as well as of mitochondrial and cellular ROS were also quantified. CF cells showed a 70% increase in protein expression compared to the Wt sample; however, regarding NNT activity, it was surprisingly lower in CF cells than healthy cells (about 30%). The cellular redox state, together with the low mΔΨ, pushes to drive NNT reverse reaction, at the expense of its antioxidant potential, thus consuming NADPH to support NADH production. At the same time, the reduced NNT activity prevents the NADH, produced by the reaction, from causing an explosion of ROS by the damaged respiratory chain, in accordance with the reduced level of mitochondrial ROS in NNT-loss cells. This new information on cellular bioenergetics represents an important building block for further understanding the molecular mechanisms responsible for cellular dysfunction in cystic fibrosis.

Human body temperature limits below 40°C during heat stroke or fever. The implications of prolonged exposure to the physiologically relevant temperature (40°C) on cellular mechanobiology is poorly understood. Here, we have examined the effects of heat stress (40°C for 72 h incubation) in human lung adenocarcinoma (A549), mouse melanoma (B16F10), and non-cancerous mouse origin adipose tissue cells (L929). Hyperthermia increased the level of ROS, γ-H2AX and HSP70 and decreased mitochondrial membrane potential in the cells. Heat stress impaired cell division, caused G1 arrest, induced cellular senescence, and apoptosis in all the tested cell lines. The cells incubated at 40°C for 72 h displayed a significant decrease in the f-actin level and cellular traction as compared with cells incubated at 37°C. Also, the cells showed a larger focal adhesion area and stronger adhesion at 40°C than at 37°C. The mitotic cells at 40°C were unable to round up properly and displayed retracting actin stress fibers. Hyperthermia down-regulated HDAC6, increased the acetylation level of microtubules, and perturbed the chromosome alignment in the mitotic cells at 40°C. Overexpression of HDAC6 rescued the cells from the G1 arrest and reduced the delay in cell rounding at 40°C suggesting a crucial role of HDAC6 in hyperthermia mediated responses. This study elucidates the significant role of cellular traction, focal adhesions, and cytoskeletal networks in mitotic cell rounding and chromosomal misalignment. It also highlights the significance of HDAC6 in heat-evoked senile cellular responses., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Paraquat (methyl viologen), is a non-selective contact herbicide and well known mitochondrial toxicant. Mitochondria are the center of cellular metabolism, and involved in the development, lifespan, and reproduction of an organism. Mitochondria are dynamic organelles that are inherited maternally through the germline and carry multiple copies of their own genome (mtDNA). It is important to understand the effects of acute and chronic stress caused by mitochondrial toxicants over multiple generations at the cellular and organism levels. Using the model nematode C. elegans, we show that acute and chronic exposure to paraquat affects reproduction, longevity, gene expression, and mitochondrial physiology. Acute exposure to paraquat in N2 (wild type) causes induction of mitochondrial unfolded protein response (mtUPR), increased expression of mitochondrial superoxide dismutase, decreased mitochondrial membrane potential (Δψm), a dose-dependent progression from linear to fragmented mitochondria, and dose-dependent changes in longevity. Chronic exposure to a low dose of paraquat (0.035 mM) over multiple generations in N2 causes a progressive decline of fertility, leading to complete loss of fertile embryo production by the third generation. The mutation in CEP-1 [cep-1(gk138)], a key regulator of stress-induced apoptosis in the germline, causes increased sensitivity to chronic paraquat relative to N2 with no fertile embryo production beyond the second generation. Whereas, mitochondrial electron transport chain (complex III) mutant [isp-1(qm150)], which display constitutive activation of mtUPR showed increased tolerance and produced fertile embryo out to the fourth generation. The N2, cep-1(gk138), and isp-1(qm150) strain's lifespan over multiple generations exposed to chronic paraquat were measured. Fertility and lifespan data together indicate a trade-off between reproduction and somatic maintenance during chronic paraquat exposure. We have proposed that mitochondrial signaling, dynamics, and CEP-1 mediated germline apoptosis is involved in this trade-off., (Copyright © 2020. Published by Elsevier B.V.)

Aims: Traumatic brain injury (TBI) is a major cause of disability and death, and a better understanding of the underlying mechanisms of mitochondrial dysfunction will provide important targets for preventing damage from neuronal insults. Phosphoglycerate mutase 5 (PGAM5) is localized to the mitochondrial outer-inner membrane contact sites, and the PGAM5-Drp1 pathway is involved in mitochondrial dysfunction and cell death. The purpose of this project was to evaluate the effects of PGAM5 on neuronal injury and mitochondrial dysfunction. Results: PGAM5 was overexpressed in mice subjected to TBI and in primary cortical neurons injured by mechanical equiaxial stretching. PGAM5 deficiency alleviated neuroinflammation, blocked Parkin, PINK1, and Drp1 translocation to mitochondria and abnormal phosphorylation of Drp1, mitochondrial ultrastructural changes, and nerve malfunction in TBI mouse model. PGAM5-shRNA (short hairpin RNA) reduced Drp1 translocation and activation, including dephosphorylation of p-Drp1 on Ser622 (human Drp1 Ser616) and phosphorylation of Drp1 on Ser643 (human Drp1 Ser637). The levels of inflammatory cytokines, the degree of mitochondrial impairment (mitochondrial membrane potential, ADP/ATP, AMP/ADP, antioxidant capacity), and neuronal injury in stretch-induced primary cortical neurons were reduced by blocking expression of PGAM5. The inhibition of PGAM5 is neuroprotective via attenuation of Drp1 activation, similar to that achieved by mitochondrial division inhibitor-1 (Mdivi1)-mediated Drp1 inhibition. Innovation and Conclusion: Our findings demonstrate the critical role of PGAM5 in progression of neuronal injury from TBI via Drp1 activation (dephosphorylation of p-Drp1 on Ser622 and phosphorylation of Drp1 on Ser643)-mediated mitochondrial dysfunction. The data may open a window for developing new drugs to prevent the neuropathology of TBI.

Reductions in the activities of mitochondrial electron transport chain (ETC) enzymes have been implicated in the pathogenesis of numerous chronic neurodegenerative disorders. Maintenance of the mitochondrial membrane potential (Δψ m ) is a primary function of these enzyme complexes, and is essential for ATP production and neuronal survival. We examined the effects of inhibition of mitochondrial ETC complexes I, II/III, III and IV activities by titrations of respective inhibitors on Δψ m in synaptosomal mitochondria. Small perturbations in the activity of complex I, brought about by low concentrations of rotenone (1-50 nM), caused depolarisation of Δψ m . Small decreases in complex I activity caused an immediate and partial Δψ m depolarisation, whereas inhibition of complex II/III activity by more than 70% with antimycin A was required to affect Δψ m . A similarly high threshold of inhibition was found when complex III was inhibited with myxothiazol, and inhibition of complex IV by more than 90% with KCN was required. The plasma membrane potential (Δψ p ) had a complex I inhibition threshold of 40% whereas complex III and IV had to be inhibited by more than 90% before changes in Δψ p were registered. These data indicate that in synaptosomes, both Δψ m and Δψ p are more susceptible to reductions in complex I activity than reductions in the other ETC complexes. These findings may be of relevance to the mechanism of neuronal cell death in Parkinson's disease in particular, where such reductions in complex I activity are present.

Peptide toxins of arthropods are one of the potential sources of bioactive substances. Toxins are able to bind to calcium channels and block them. Ca 2+ ions play an important role in many cell processes, in particular, in apoptosis. In this work, we study the effect of some arthropod toxins on intracellular processes associated with the induction of apoptosis. Synthetic analogs of U 5 -scytotoxin-Sth1a, ω-hexatoxin-Hv1a, ω-theraphotoxin-Hhn2a, and μ-agatoxin-Aa1a toxins-inhibitors of calcium L, P, and Q channels and sodium channels were used in the study. Apoptosis was induced by AC-1001 H3 peptide. We study the effect of toxins on the level of apoptosis, ROS, mitochondrial potential, GSH, and ATP in CHO-K1 cells. We show that all the tested toxins are able to dose dependently block the induction of apoptosis triggered by AC-1001 H3 and reduce the level of natural apoptosis in CHO-K1 cells. Cell incubation with apoptosis inducer AC-1001 H3 in the presence and absence of toxins causes an increase in the intracellular concentrations of ROS, ATP, and mitochondrial potential and decreases the GSH concentration. The present study reveals the antiapoptotic effect of a number of arthropod peptide toxins. The toxins studied can represent a novel approach used in the treatment of pathologies associated with the activation of apoptotic mechanisms., (© 2020 European Peptide Society and John Wiley & Sons, Ltd.)

Mitochondrial membrane potential (Ψ m ) is a critical parameter that can be used to determine cellular well-being. As it is a direct measure of the cell's ATP generating capability, in recent years, this key component in cell biology has been the subject of thousands of biochemical and biophysical investigations. Membrane-permeant fluorescent dyes, like tetramethylrhodamine ethyl ester (TMRE), have been predominantly employed to monitor ΔΨ m in cells. These dyes are typically lipophilic cationic compounds that equilibrate across membranes in a Nernstian fashion, thus accumulating into the mitochondrial membrane matrix space in inverse proportion to Ψ m . However, the bath loading method practiced for labelling tissue slices with these cationic dyes poses limitations in the form of non-specificity and low signal to noise ratio, which compromises the precision of the results. Therefore, we introduce an alternative way for TMRE loading to image the ΔΨ m in tissue slices by utilizing a low resistance glass pipette attached to a pressure injector. This method shows highly precise fluorescent dye labelling of the mitochondria and offers maximum output intensity, in turn enhancing signal to noise ratio., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Mitochondria play a key role in various modes of cell death. Analysis of mitochondrial dysfunction and the release of proteins from the intermembrane space of mitochondria represent essential tools in cell death investigation. Here we describe how to evaluate release of intermembrane space proteins during apoptosis, alterations in the mitochondrial membrane potential, and oxygen consumption in apoptotic cells.

Brain is one of the most energy-demanding organs. Energy in the form of ATP is produced in brain cells predominantly in oxidative phosphorylation coupled to mitochondrial respiration. Any alteration of the mitochondrial metabolism or prolonged ischemic or anoxic conditions can lead to serious neurological conditions, including neurodegenerative disorders. Assessment of mitochondrial metabolism is important for understanding physiological and pathological processes in the brain. Bioenergetics in central nervous system is dependent on multiple parameters including neuron-glia interactions and considering this, in vivo or ex vivo, the measurements of mitochondrial metabolism should also be complimenting the experiments on isolated mitochondria or cell cultures. To assess the mitochondrial function, there are several key bioenergetic parameters which indicate mitochondrial health. One of the major characteristics of mitochondria is the mitochondrial membrane potential (ΔΨm) which is used as a proton motive force for ATP production and generated by activity of the electron transport chain. Major donor of electrons for the mitochondrial respiratory chain is NADH. Here we demonstrate how to measure mitochondrial NADH/NAD(P)H autofluorescence and ΔΨm in acute brain slices in a time-dependent manner and provide information for the identification of NADH redox index, mitochondrial NADH pool, and the rate of NADH production in the Krebs cycle. Additionally, non-mitochondrial NADH/NADPH autofluorescence can signify the level of activity of the pentose phosphate pathway.

Apoptin can specifically kill cancer cells but has no toxicity to normal cells. Human telomerase reverse transcriptase (hTERT) can act as a tumour-specific promoter by triggering the expression of certain genes in tumour cells. This study aims to investigate the inhibitory effects and to explore the inhibitory pathway of a dual cancer-specific recombinant adenovirus (Ad-apoptin-hTERTp-E1a, Ad-VT) on breast cancer stem cells. Breast cancer cell spheres were obtained from MCF-7 cells through serum-free suspension culture. The cell spheres were detected by flow cytometry for CD44 + CD24 - cell subsets. The stemness of MCF-7-CSC cells was confirmed by in vivo tumorigenesis experiments. The inhibitory effect of the recombinant adenoviruses on MCF-7-CSC cells was evaluated by CCK-8 assay. In addition, the stemness of adenovirus-infected MCF-7-CSC cells was analysed by testing the presence of CD44 + CD24 - cell subsets. The ability of the recombinant adenovirus to induce MCF-7-CSC cell apoptosis was detected by staining JC-1, TMRM and Annexin V. Our results showed that a significantly higher proportion of the CD44 + CD24 - cell subsets was present in MCF-7-CSC cells with a significantly increased expression of stem cell marker proteins. The MCF-7-CSC cells, whlist exhibited a strong tumorigenic ability with a certain degree of stemness in mice, were shown to be strongly inhibited by recombinant adenovirus Ad-VT through cell apoptosis. In addition, Ad-VT was shown to exert a killing effect on BCSCs. These results provide a new theoretical basis for the future treatment of breast cancer., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Cell-penetrating peptides are a promising therapeutic strategy for a wide variety of degenerative diseases, ageing, and cancer. Among the multitude of cell-penetrating peptides, PepFect14 has been preferentially used in our laboratory for oligonucleotide delivery into cells and in vivo mouse models. However, this activity has mainly been reported towards cytoplasm and nuclei, while the mentioned disorders have been linked to mitochondrial defects. Here, we report a library generated from a combinatorial covalent fusion of a mitochondrial-penetrating peptide, mtCPP1, and PepFect14 in order to deliver therapeutic biomolecules to influence mitochondrial protein expression. The non-covalent complexation of these peptides with oligonucleotides resulted in nano-complexes affecting biological functions in the cytoplasm and on mitochondria. This delivery system proved to efficiently target mitochondrial genes, providing a framework for the development of mitochondrial peptide-based oligonucleotide technologies with the potential to be used as a treatment for patients with mitochondrial disorders.

Invasive growth of yeast cells into nutrient agar is induced by different stresses and contributes to the survival of yeast cells under several adverse conditions. The mechanism of invasive growth of Saccharomyces cerevisiae has been extensively investigated. However, there is very little information about the mechanism of invasive growth of another human pathogen yeast Cryptococcus neoformans. Here, we report that deletion of a small and secreted cysteine-rich protein Cpl1 in C. neoformans JEC21 leads to increased adhesive and invasive growth into nutrient agar. The increased adhesive and invasive growth does not depend on the only known adhesion protein Cfl1 and its main controller Znf2. Cpl1Δ accumulates significantly higher level of intracellular labile zinc ion, leading to increased glucose uptake, higher level of mitochondrial membrane potential, ATP and Reactive Oxygen Species(ROS) production. Higher level of ROS activates Snf1, leading to invasive growth of Cpl1Δ. Three cysteine residues at the N-terminals of the cysteine-rich domain controls the increased invasive growth under nutrient sufficient conditions. This is the first report that a small and secreted cysteine-rich protein negatively regulates invasive growth of C. neoformans through regulating the intracellular labile zinc ion level. The function of this cysteine-rich domain was systematically investigated by site-directed mutagenensis in C. neoformans. The work contributes to understanding the function of this protein family and the invasive growth mechanism in C. neoformans., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Neuronal mitochondrial fragmentation is a phenotype exhibited in models of neurodegeneration such as Parkinson's disease. Delineating the dysfunction in mitochondrial dynamics found in diseased states can aid our understanding of underlying mechanisms of disease progression and possibly identify novel therapeutic approaches. Advances in microscopy and the availability of intuitive open-access software have accelerated the rate of image acquisition and analysis, respectively. These developments allow routine biology researchers to rapidly turn hypotheses into results. In this protocol, we describe the utilization of cell culture techniques, high-content imaging (HCI), and the subsequent open-source image analysis pipeline for the quantification of mitochondrial fragmentation in the context of a rotenone-based in vitro Parkinson's disease model. © 2020 The Authors. Basic Protocol 1: SN4741 neuron culture and treatment in a rotenone-based model of Parkinson's disease Basic Protocol 2: Identification of cell nuclei, measurement of mitochondrial membrane potential, and measurement of mitochondrial fragmentation in mouse-derived midbrain dopaminergic neurons., (© 2020 The Authors.)

Glutamate N-methyl-D-aspartate (NMDA) receptor (NMDAR) is critical for neurotransmission as a Ca 2+ channel. Nonetheless, flux-independent signaling has also been demonstrated. Astrocytes express NMDAR distinct from its neuronal counterpart, but cultured astrocytes have no electrophysiological response to NMDA. We recently demonstrated that in cultured astrocytes, NMDA at pH6 (NMDA/pH6) acting through the NMDAR elicits flux-independent Ca 2+ release from the Endoplasmic Reticulum (ER) and depletes mitochondrial membrane potential (mΔΨ). Here we show that Ca 2+ release is due to pH6 sensing by NMDAR, whereas mΔΨ depletion requires both: pH6 and flux-dependent NMDAR signaling. Plasma membrane (PM) NMDAR guard a non-random distribution relative to the ER and mitochondria. Also, NMDA/pH6 induces ER stress, endocytosis, PM electrical capacitance reduction, mitochondria-ER, and -nuclear contacts. Strikingly, it also produces the formation of PM invaginations near mitochondria along with structures referred to here as PM-mitochondrial bridges (PM-m-br). These and earlier data strongly suggest PM-mitochondria communication. As proof of the concept of mass transfer, we found that NMDA/pH6 provoked mitochondria labeling by the PM dye FM-4-64FX. NMDA/pH6 caused PM depolarization, cell acidification, and Ca 2+ release from most mitochondria. Finally, the MCU and microtubules were not involved in mΔΨ depletion, while actin cytoskeleton was partially involved. These findings demonstrate that NMDAR has concomitant flux-independent and flux-dependent actions in cultured astrocytes., (© 2020 Federation of American Societies for Experimental Biology.)

This work analyzes the effects of red LED light on mammalian sperm mitochondrial function, using the pig as an animal model. Liquid-stored pig semen was stimulated with red-light for 1, 5 and 10 min in the presence or absence of oligomycin A, a specific inhibitor of mitochondrial ATP synthase, or carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), a specific disruptor of mitochondrial electron chain. Whereas exposure for 1 and 5 min significantly ( p < 0.05) decreased total motility and intracellular ATP levels, irradiation for 10 min induced the opposite effect. Oligomycin A abolished the light-effects on intracellular ATP levels, O 2 consumption and mitochondrial membrane potential, whereas compared to non-irradiated samples, FCCP significantly ( p < 0.05) increased O 2 consumption when sperm were irradiated for 1 min. Both oligomycin A and FCCP significantly ( p < 0.05) decreased total motility. Red-light increased cytochrome c oxidase activity with a maximal effect after 5 min of irradiation, which was abolished by both oligomycin A and FCCP. In conclusion, red-light modulates sperm mitochondrial function via electron chain activity in an exposition, time-dependent manner.