SUMOylation of MFF coordinates fission complexes to promote stress-induced mitochondrial fragmentation.

Mitochondria undergo fragmentation in response to bioenergetic stress, mediated by dynamin-related protein 1 (DRP1) recruitment to the mitochondria. The major pro-fission DRP1 receptor is mitochondrial fission factor (MFF), and mitochondrial dynamics proteins of 49 and 51 kilodaltons (MiD49/51), which can sequester inactive DRP1. Together, they form a trimeric DRP1-MiD-MFF complex. Adenosine monophosphate-activated protein kinase (AMPK)-mediated phosphorylation of MFF is necessary for mitochondrial fragmentation, but the molecular mechanisms are unclear. Here, we identify MFF as a target of small ubiquitin-like modifier (SUMO) at Lys151, MFF SUMOylation is enhanced following AMPK-mediated phosphorylation and that MFF SUMOylation regulates the level of MiD binding to MFF. The mitochondrial stressor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) promotes MFF SUMOylation and mitochondrial fragmentation. However, CCCP-induced fragmentation is impaired in MFF-knockout mouse embryonic fibroblasts expressing non-SUMOylatable MFF K151R. These data suggest that the AMPK-MFF SUMOylation axis dynamically controls stress-induced mitochondrial fragmentation by regulating the levels of MiD in trimeric fission complexes.

Real time precisely tracing the fluctuations of mitochondrial SO2 in cells during ferroptosis and tissues using a mitochondrial-immobilized ratiometric fluorescent probe.

Mitochondrial sulfur dioxide (SO2) plays important roles in physiological and pathological activities. Unfortunately, it is lack of a reliable tool to precisely visualize the mitochondrial SO2 and elaborate its complicated functions in various cytoactivities. Here we report a mitochondrial-immobilized fluorescent probe PM-Cl consisting of coumarin and benzyl chloride modified benzothiazole, which enables selective visualization of mitochondrial SO2via chemical immobilization. The spectral results demonstrated that probe PM-Cl could respond to SO2 with high selectivity and sensitivity. Co-localization and the fluorescence of cytolysis extraction verified the excellent mitochondrial targeting and anchoring abilities. Due to the chemical immobilization, probe PM-Cl could firmly retain into mitochondria after stimulation of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and H2O2. Significantly, a series of fluorescence images are indicative of capability for detecting the fluctuations of SO2 in mitochondria during ferroptosis. Furthermore, PM-Cl also could visualize SO2 in myocardium and muscle tissues after the stimulation of CCCP. Taken together, probe PM-Cl is a very potential molecular tool for precisely detecting mitochondrial SO2 to explore its complex functions in physiological and pathological activities.

Assessing the mechanism of facilitated proton transport across GUVs trapped in a microfluidic device.

Proton transport across lipid membranes is one of the most fundamental reactions that make up living organisms. In vitro, however, the study of proton transport reactions can be very challenging due to limitations imposed by proton concentrations, compartment size, and unstirred layers as well as buffer exchange and buffer capacity. In this study, we have developed a proton permeation assay based on the microfluidic trapping of giant vesicles enclosing the pH-sensitive dye pyranine to address some of these challenges. Time-resolved fluorescence imaging upon a rapid pH shift enabled us to investigate the facilitated H+ permeation mediated by either a channel or a carrier. Specifically, we compared the proton transport rates as a function of different proton gradients of the channel gramicidin D and the proton carrier carbonyl cyanide-m-chlorophenyl hydrazone. Our results demonstrate the efficacy of the assay in monitoring proton transport reactions and distinguishing between a channel-like and a carrier-like mechanism. This groundbreaking result enabled us to elucidate the enigmatic mode of the proton permeation mechanism of the recently discovered natural fibupeptide lugdunin.

Influence of carbonyl cyanide m-chlorophenyl hydrazone on biofilm dynamics, protease, and siderophore production by Burkholderia pseudomallei.

Efflux pump inhibitors are a potential therapeutic strategy for managing antimicrobial resistance and biofilm formation. This article evaluated the effect of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) on the biofilm growth dynamics and the production of virulence factors by Burkholderia pseudomallei. The effects of CCCP on planktonic, growing, and mature biofilm, interaction with antibacterial drugs, and protease and siderophore production were assessed. CCCP MICs ranged between 128 and 256 µM. The CCCP (128 µM) had a synergic effect with all the antibiotics tested against biofilms. Additionally, CCCP reduced (p < .05) the biomass of biofilm growth and mature biofilms at 128 and 512 µM, respectively. CCCP also decreased (p < .05) protease production by growing (128 µM) and induced (p < .05) siderophore release by planktonic cells (128 µM) growing biofilms (12.8 and 128 µM) and mature biofilms (512 µM). CCCP demonstrates potential as a therapeutic adjuvant for disassembling B. pseudomallei biofilms and enhancing drug penetration.

Elevated Membrane Potential as a Tetracycline Resistance Mechanism in Escherichia coli.

The metabolic environment is responsible for antibiotic resistance, which highlights the way in which the antibiotic resistance mechanism works. Here, GC-MS-based metabolomics with iTRAQ-based proteomics was used to characterize a metabolic state in tetracycline-resistant Escherichia coli K12 (E. coli-RTET) compared with tetracycline-sensitive E. coli K12. The repressed pyruvate cycle against the elevation of the proton motive force (PMF) and ATP constructed the most characteristic feature as a consequence of tetracycline resistance. To understand the role of the elevated PMF in tetracycline resistance, PMF inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and the pH gradient were used to investigate how the elevation influences bacterial viability and intracellular antibiotic concentration. A strong synergy was detected between CCCP and tetracycline to the viability, which was consistent with increasing intracellular drug and decreasing external pH. Furthermore, E. coli-RTET and E. coli-RGEN with high and low PMF concentrations were susceptible to gentamicin and tetracycline, respectively. The elevated PMF in E. coli-RTET was attributed to the activation of other metabolic pathways, except for the pyruvate cycle, including a malate-oxaloacetate-phosphoenolpyruvate-pyruvate-malate cycle. These results not only revealed a PMF-dependent mechanism for tetracycline resistance but also provided a solution to tetracycline-resistant pathogens by aminoglycosides and aminoglycoside-resistant bacteria by tetracyclines.

Theoretical and Experimental Study of the Interaction of Protonophore Uncouplers and Decoupling Agents with Functionally Active Mitochondria.

The purpose of this work was to quantitatively characterize the effectiveness of oxidative phosphorylation uncouplers and decoupling agents in functionally active mitochondria, taking into account their content in the hydrophobic region of the inner membrane of these organelles. When conducting theoretical studies, it is accepted that uncouplers and decouplers occupy part of the volume of mitochondria to exhibit their activity, which is defined as the effective volume. The following quantities characterizing the action of these reagents are considered: (1) concentrations of reagents that cause double stimulation of mitochondrial respiration in state 4 ( C 200 ); (2) effective distribution coefficient ( E MW ) - the ratio of the amount of reagents in the effective volume of mitochondria and the water volume; (3) the relative amount of reagents associated with the effective volume of mitochondria ( U M / U T ); (4) specific activity of reagents localized in the effective volume of mitochondria ( A M ). We have developed methods for determining these values, based on an analysis of the dependence of the rate of mitochondrial respiration on the concentration of uncouplers and decoupling agents at two different concentrations of mitochondrial protein in the incubation medium. During experimental studies, we compared the effects of the classical protonophore uncouplers 2,4-dinitrophenol (DNP) and сarbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), the natural uncouplers lauric and palmitic acids, and the natural decouplers α,ω-tetradecanedioic (TDA) and α,ω-hexadecanedioic (HDA) acids that differ both in the structure of the molecule and in the degree of solubility in lipids. Using the developed methods, we have clarified the dependence of the degree of activity of these uncouplers and decoupling agents on the distribution of their molecules between the effective volume of mitochondria and the water volume.

Repurposing approved drugs as potential efflux pump inhibitors in multidrug-resistant Pseudomonas aeruginosa.

Aim: To evaluate the action of promethazine, fluoxetine and carbonyl cyanide 3-chlorophenylhydrazone as efflux pump inhibitors (EPIs) against multidrug-resistant Pseudomonas aeruginosa. Methods: The effect of the compounds was evaluated in planktonic cells and bacterial biofilms. Accumulation tests were performed with ethidium bromide to prove their action as EPIs. Then, they were associated with antimicrobials. Results: Effect on planktonic cells and biofilms was found. Assays with ethidium bromide indicate their action as EPIs. Significant reductions in the metabolic activity of biofilms were observed after the association with the antimicrobials, especially for meropenem. Conclusion: It is possible to prove the action of these compounds as EPIs for P. aeruginosa and demonstrate the relevance of efflux pumps in antimicrobial resistance.

[Box: see text].

Efflux pump effects on levofloxacin resistance in Mycobacterium abscessus.

Mycobacterium abscessus (M. abscessus) inherently displays resistance to most antibiotics, with the underlying drug resistance mechanisms remaining largely unexplored. Efflux pump is believed to play an important role in mediating drug resistance. The current study examined the potential of efflux pump inhibitors to reverse levofloxacin (LFX) resistance in M. abscessus. The reference strain of M. abscessus (ATCC19977) and 60 clinical isolates, including 41 M. abscessus subsp. abscessus and 19 M. abscessus subsp. massilense, were investigated. The drug sensitivity of M. abscessus against LFX alone or in conjunction with efflux pump inhibitors, including verapamil (VP), reserpine (RSP), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), or dicyclohexylcarbodiimide (DCC), were determined by AlarmarBlue microplate assay. Drug-resistant regions of the gyrA and gyrB genes from the drug-resistant strains were sequenced. The transcription level of the efflux pump genes was monitored using qRT-PCR. All the tested strains were resistant to LFX. The drug-resistant regions from the gyrA and gyrB genes showed no mutation associated with LFX resistance. CCCP, DCC, VP, and RSP increased the susceptibility of 93.3% (56/60), 91.7% (55/60), 85% (51/60), and 83.3% (50/60) isolates to LFX by 2 to 32-fold, respectively. Elevated transcription of seven efflux pump genes was observed in isolates with a high reduction in LFX MIC values in the presence of efflux pump inhibitors. Efflux pump inhibitors can improve the antibacterial activity of LFX against M. abscessus in vitro. The overexpression of efflux-related genes in LFX-resistant isolates suggests that efflux pumps are associated with the development of LFX resistance in M. abscessus.

Enhancing bactericidal activities of ciprofloxacin by targeting the trans-translation system that is involved in stress responses in Klebsiella pneumoniae.

Although the trans-translation system is a promising target for antcibiotic development, its antibacterial mechanism in Klebsiella pneumoniae (KP) is unclear. Considering that tmRNA was the core component of trans-translation, this study firstly investigated phenotypic changes caused by various environmental stresses in KP lacking trans-translation activities (tmRNA-deleted), and then aimed to evaluate antibacterial activities of the trans-translation-targeting antibiotic combination (tobramycin/ciprofloxacin) in clinical KP isolates based on inhibition activities of aminoglycosides against trans-translation. We found that the tmRNA-deleted strain P4325/ΔssrA was significantly more susceptible than the wild-type KP strain P4325 under environments with hypertonicity (0.5 and 1 M NaCl), hydrogen peroxide (40 mM), and UV irradiation. No significant differences in biofilm formation and survivals under human serum were observed between P4325/ΔssrA and P4325. tmRNA deletion caused twofold lower MIC values for aminoglycosides. As for the membrane permeability, tmRNA deletion increased ethidium bromide (EtBr) uptake of KP in the presence or absence of verapamil and carbonyl cyanide-m-chlorophenylhydrazone (CCCP), decreased EtBr uptake in presence of reserpine in P4325/ΔssrA, and reduced EtBr efflux in P4325/ΔssrA in the presence of CCCP. The time-kill curve and in vitro experiments revealed significant bactericidal activities of the tmRNA-targeting aminoglycoside-based antibiotic combination (tobramycin/ciprofloxacin). Thus, the corresponding tmRNA-targeting antibiotic combinations (aminoglycoside-based) might be effective and promising treatment options against multi-drug resistant KP.

CCCP inhibits DPV infection in DEF cells by attenuating DPV manipulated ROS, apoptosis, and mitochondrial stability.

Duck plague virus (DPV) is extremely infectious and lethal, so antiviral drugs are urgently needed. Our previous study shows that DPV infection with duck embryo fibroblast (DEF) induces reactive oxygen species (ROS) changes and promotes apoptosis. In this study, we tested the antiviral effect of the carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a common mitochondrial autophagy inducer. Our results demonstrated a dose-dependent anti-DPV effect of CCCP, CCCP-treatment blocked the intercellular transmission of DPV after infection, and we also proved that CCCP could have an antiviral effect up to 48 hpi. The addition of CCCP reversed the DPV-induced ROS changes, CCCP can inhibit virus-induced apoptosis; meanwhile, CCCP can affect mitochondrial fusion and activate mitophagy to inhibit DPV. In conclusion, CCCP can be an effective antiviral candidate against DPV.

Knockout of adenylosuccinate synthase purA increases susceptibility to colistin in Escherichia coli.

Colistin is a cationic cyclic antimicrobial peptide used as a last resort against multidrug-resistant gram-negative bacteria. To understand the factors involved in colistin susceptibility, we screened colistin-sensitive mutants from an E. coli gene-knockout library (Keio collection). The knockout of purA, whose product catalyzes the synthesis of adenylosuccinate from IMP in the de novo purine synthesis pathway, resulted in increased sensitivity to colistin. Adenylosuccinate is subsequently converted to AMP, which is phosphorylated to produce ADP, a substrate for ATP synthesis. The amount of ATP was lower in the purA-knockout mutant than that in the wild-type strain. ATP synthesis is coupled with proton transfer, and it contributes to the membrane potential. Using the membrane potential probe, 3,3'-diethyloxacarbocyanine iodide [DiOC2(3)], we found that the membrane was hyperpolarized in the purA-knockout mutant compared to that in the wild-type strain. Treatment with the proton uncoupler, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), abolished the hyperpolarization and colistin sensitivity in the mutant. The purA-knockout mutant exhibited increased sensitivity to aminoglycosides, kanamycin, and gentamicin; their uptake requires a membrane potential. Therefore, the knockout of purA, an adenylosuccinate synthase, decreases ATP synthesis concurrently with membrane hyperpolarization, resulting in increased sensitivity to colistin.

Detection of Aminoglycoside Modifying Enzyme (AME) genes in Acinetobacter baumannii isolates and the inhibitory effect of efflux pump activity on drug susceptibility pattern.

INTRODUCTION: The development of aminoglycoside modifying enzymes (AMEs) and increased efflux activity are considered important aminoglycosides resistance mechanisms. AIM: This study is focused on the detection of the AMEs gene and assessing the effect of efflux pump inhibitor on the reversal of A. baumannii drug susceptibility. METHODOLOGY: Bacterial DNA was amplified using AMEs gene-specific primers. Isolates were also investigated for efflux pump activity using efflux pump inhibitor (EPI) i.e. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and the impact of both mechanisms was analyzed. RESULTS: Among A. baumannii isolates, 55% isolates (n â€‹= â€‹22/40) were identified to have aminoglycoside modifying enzymes genes; ant(3')-I gene (50%, 11/22), aac(6')-Ib gene (45.4%, 10/22), aph(3')-I gene (18.1%, 4/22) and aac(3)-I (9.1%, 2/22). Total 70% isolates have shown MIC alteration in different classes of drugs in response to EPI-CCCP. Such alteration was found in 100% amikacin sensitive and 58.6% amikacin resistant, 93.7% and 57.1% gentamicin sensitive and resistant isolates respectively. CONCLUSION: The presence of aminoglycosides modifying enzymes was frequent among aminoglycosides resistant A. baumannii isolates and the coexistence of efflux pumps activity also plays an important role to increase drug resistance. REPOSITORIES: Genbank and their accession numbers are MT903331[aac(3)-I], MT903332 MT903333 [ant(3')-I], MT903334, MT903335 [aph(3')-I)] and MT903336, MT940242 [ aac(6')-Ib].

AMP-activated protein kinase (AMPK) suppresses Ibaraki virus propagation.

The Ibaraki virus (IBAV) causes Ibaraki disease in cattle. Our previous studies have shown that IBAV uses macropinocytosis to enter the host cell and exit from the endosome to the cytosol in response to endosomal acidification. To further explore the mechanism of IBAV infection and replication, we examined the effect of inhibitors of mitochondrial oxidative phosphorylation, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and antimycin A, on IBAV propagation. These inhibitors significantly suppressed IBAV propagation, with reduced cellular ATP levels resulting from suppression of ATP synthesis. Furthermore, we identified AMP-activated protein kinase (AMPK), which is activated by CCCP or antimycin A, as a key signaling molecule in IBAV suppression. We also observed that IBAV infection induces ATP depletion and increases AMPK activity. Our findings suggest that AMPK is a potential target in Ibaraki disease.

Protocol for evaluating mitochondrial morphology changes in response to CCCP-induced stress through open-source image processing software.

Mitochondrial morphology is an indicator of cellular health and function; however, its quantification and categorization into different subclasses is a complicated process. Here, we present a protocol for mitochondrial morphology quantification in the presence and absence of carbonyl cyanide m-chlorophenyl hydrazone stress. We describe steps for the preparation of cells for immunofluorescence microscopy, staining, and morphology quantification. The quantification protocol generates an aspect ratio that helps to categorize mitochondria into two clear subclasses. For complete details on the use and execution of this protocol, please refer to Nag et al.1.

Trichostatin A enhances the titanium rods osseointegration in osteoporotic rats by the inhibition of oxidative stress through activating the AKT/Nrf2 pathway.

The use of titanium implants as fixed supports following fractures in patients with OP can often result in sterile loosening and poor osseointegration. Oxidative stress has been shown to play a particularly important role in this process. While TSA has been reported to facilitate in vivo osteogenesis, the underlying mechanisms remain to be clarified. It also remains unclear whether TSA can improve the osseointegration of titanium implants. This study investigated whether TSA could enhance the osseointegration of titanium rods by activating AKT/Nrf2 pathway signaling, thereby suppressing oxidative stress. MC3T3-E1 cells treated with CCCP to induce oxidative stress served as an in vitro model, while an OVX-induced OP rat model was employed for in vivo analysis of titanium rod implantation. In vitro, TSA treatment of CCCP-treated MC3T3-E1 cells resulted in the upregulation of osteogenic proteins together with increased AKT, total Nrf2, nuclear Nrf2, HO-1, and NQO1 expression, enhanced mitochondrial functionality, and decreased oxidative damage. Notably, the PI3K/AKT inhibitor LY294002 reversed these effects. In vivo, TSA effectively enhanced the microstructural characteristics of distal femur trabecular bone, increased BMSCs mineralization capacity, promoted bone formation, and improved the binding of titanium implants to the surrounding tissue. Finally, our results showed that TSA could reverse oxidative stress-induced cell damage while promoting bone healing and improving titanium rods' osseointegration through AKT/Nrf2 pathway activation.

Synergistic action of indole-3-carbinol with membrane-active agents against multidrug-resistant Gram-negative bacteria.

The purpose of this study was to evaluate the antimicrobial activity of indole-3-carbinol (I3C) with membrane-active agents, namely carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and ethylenediaminetetraacetic acid (EDTA) against multidrug-resistant (MDR) Gram-negative bacteria and bacterial persisters. The determination of minimal inhibitory concentration (MIC) showed that I3C was effective against Acinetobacter baumannii (3.13‒6.25 × 10-3 mol l-1), Klebsiella pneumoniae (8 × 10-3 mol l-1), Pseudomonas aeruginosa (6.25‒12.5 × 10-3 mol l-1), and Escherichia coli (6.25‒12.5 × 10-3 mol l-1). Our study demonstrated that EDTA synergistically enhanced the bactericidal activity of I3C against most MDR Gram-negative bacteria isolates and contributed to an 8- to 64-fold MIC reduction compared with that of I3C alone, yet CCCP only displayed synergy with I3C against P. aeruginosa and A. baumannii. The EDTA-I3C combination also significantly reduced the viable number of testing bacteria (P = 7.2E-05), effectively reduced bacterial persisters, and repressed bacterial growth compared with that the use of I3C alone. Our data demonstrate that use of EDTA as adjuvant molecules can effectively improve the antibacterial activity of I3C and may help to reduce the development of antimicrobial resistance.

Sodium butyrate enhances titanium nail osseointegration in ovariectomized rats by inhibiting the PKCα/NOX4/ROS/NF-κB pathways.

BACKGROUND: Elevated levels of oxidative stress as a consequence of estrogen deficiency serve as a key driver of the onset of osteoporosis (OP). In addition to increasing the risk of bone fractures, OP can reduce the bone volume proximal to titanium nails implanted to treat these osteoporotic fractures, thereby contributing to titanium nail loosening. Sodium butyrate (NaB) is a short-chain fatty acid produced by members of the gut microbiota that exhibits robust antioxidant and anti-inflammatory properties. METHODS: OP fracture model rats parameters including bone mineral density (BMD), new bone formation, and the number of bonelets around the implanted nail were analyzed via micro-CT scans, H&E staining, and Masson's staining. The protective effects of NaB on such osseointegration and the underlying mechanisms were further studied in vitro using MC3T3-E1 cells treated with carbonyl cyanide m-chlorophenylhydrazone (CCCP) to induce oxidative stress. Techniques including Western immunoblotting, electron microscopy, flow cytometry, alkaline phosphatase (ALP) staining, and osteoblast mineralization assays were employed to probe behaviors such as reactive oxygen species production, mineralization activity, ALP activity, protein expression, and the ability of cells to attach to and survive on titanium plates. RESULTS: NaB treatment was found to enhance ALP activity, mineralization capacity, and Coll-I, BMP2, and OCN expression levels in CCCP-treated MC3T3-E1 cells, while also suppressing PKC and NF-κB expression and enhancing Nrf2 and HO-1 expression in these cells. NaB further suppressed intracellular ROS production and malondialdehyde levels within the cytosol while enhancing superoxide dismutase activity and lowering the apoptotic death rate. In line with these results, in vivo work revealed an increase in BMD in NaB-treated rats that was associated with enhanced bone formation surrounding titanium nails. CONCLUSION: These findings indicate that NaB may represent a valuable compound that can be postoperatively administered to aid in treating OP fractures through the enhancement of titanium nail osseointegration.

The Highs and Lows of Memantine-An Autophagy and Mitophagy Inducing Agent That Protects Mitochondria.

Memantine is an FDA-approved, non-competitive NMDA-receptor antagonist that has been shown to have mitochondrial protective effects, improve cell viability and enhance clearance of Aß42 peptide. Currently, there are uncertainties regarding the precise molecular targets as well as the most favourable treatment concentrations of memantine. Here, we made use of an imaging-based approach to investigate the concentration-dependent effects of memantine on mitochondrial fission and fusion dynamics, autophagy and mitochondrial quality control using a neuronal model of CCCP-induced mitochondrial injury so as to better unpack how memantine aids in promoting neuronal health. GT1-7 murine hypothalamic cells were cultured under standard conditions, treated with a relatively high and low concentration (100 µM and 50 µM) of memantine for 48 h. Images were acquired using a Zeiss 780 PS1 platform. Utilising the mitochondrial event localiser (MEL), we demonstrated clear concentration-dependent effects of memantine causing a protective response to mitochondrial injury. Both concentrations maintained the mitochondrial network volume whilst the low concentration caused an increase in mitochondrial number as well as increased fission and fusion events following CCCP-induced injury. Additionally, we made use of a customised Python-based image processing and analysis pipeline to quantitatively assess memantine-dependent changes in the autophagosomal and lysosomal compartments. Our results revealed that memantine elicits a differential, concentration-dependent effect on autophagy pathway intermediates. Intriguingly, low but not high concentrations of memantine lead to the induction of mitophagy. Taken together, our findings have shown that memantine is able to protect the mitochondrial network by preserving its volume upon mitochondrial injury with high concentrations of memantine inducing macroautophagy, whereas low concentrations lead to the induction of mitophagy.

Study of mitochondrial function in thawed bull spermatozoa using selective electron transfer chain inhibitors.

Bull spermatozoa depend equally on glycolysis and oxidative phosphorylation for the maintenance of the energy necessary for their proper functioning. The aim of the present work was to delineate the mitochondrial activity of bull spermatozoa after incubation with specific inhibitors of the different mitochondrial complexes and evaluate their ROS production. Thawed bull sperm cells (30 × 106 mL-1 in Tyrode's extender) were incubated 1 and 3h at 37 °C with rotenone 5 µM (ROT), complex I inhibitor; dimethyl-malonate 10 mM (DMM), complex II inhibitor; carbonyl cyanide m-chlorophenyl hydrazine 5 µM (CCCP), uncoupling agent; antimycin A 1 µg/mL (ANTI), complex III inhibitor; oligomycin 5 µM (OLIGO), ATP synthase inhibitor, and 0.5% DMSO, vehicle (CTR). Sperm motility and kinematics were assessed by Hamilton Thorn IVOS 12.0. Mitochondrial membrane potential, mitochondrial O2•- production and H2O2 intracellular content were evaluated by BD FACSCalibur flow cytometer, and sperm viability (SYBR-14/PI) and mitochondrial activity (JC-1/SYBR-14/PI) were assessed by epifluorescence microscopy. A multivariate analysis was performed on the results. In addition, sperm kinematic features, registered for each motile spermatozoon, were studied by cluster analysis. The incubation during 1 or 3 h in presence of the inhibitors of mitochondrial functionality only had a minor effect on motility parameters, decreasing the proportion of the SP1 (fast progressive) subpopulation after 3 h of incubation with ROT, ANTI or OLIGO. The percentage of live spermatozoa with active mitochondria was reduced under the effect of ANTI and CCCP both at 1 and 3 h. In conclusion, mitochondrial function is somehow impaired in frozen thawed bull sperm as not all live cells showed active mitochondria. These results support the findings that bull spermatozoa can alternatively rely on oxidative phosphorylation or glycolysis for energy obtainment and that their mitochondria are less affected by ETC inhibitors.

The inner mitochondrial membrane fission protein MTP18 serves as a mitophagy receptor to prevent apoptosis in oral cancer.

MTP18 (also known as MTFP1), an inner mitochondrial membrane protein, plays a vital role in maintaining mitochondrial morphology by regulating mitochondrial fission. Here, we found that MTP18 functions as a mitophagy receptor that targets dysfunctional mitochondria into autophagosomes for elimination. Interestingly, MTP18 interacts with members of the LC3 (also known as MAP1LC3) family through its LC3-interacting region (LIR) to induce mitochondrial autophagy. Mutation in the LIR motif (mLIR) inhibited that interaction, thus suppressing mitophagy. Moreover, Parkin or PINK1 deficiency abrogated mitophagy in MTP18-overexpressing human oral cancer-derived FaDu cells. Upon exposure to the mitochondrial oxidative phosphorylation uncoupler CCCP, MTP18[mLIR]-FaDu cells showed decreased TOM20 levels without affecting COX IV levels. Conversely, loss of Parkin or PINK1 resulted in inhibition of TOM20 and COX IV degradation in MTP18[mLIR]-FaDu cells exposed to CCCP, establishing Parkin-mediated proteasomal degradation of outer mitochondrial membrane as essential for effective mitophagy. We also found that MTP18 provides a survival advantage to oral cancer cells exposed to cellular stress and that inhibition of MTP18-dependent mitophagy induced cell death in oral cancer cells. These findings demonstrate that MTP18 is a novel mitophagy receptor and that MTP18-dependent mitophagy has pathophysiologic implications for oral cancer progression, indicating inhibition of MTP18-mitophagy could thus be a promising cancer therapy strategy.