Bacterial persister cells are phenotypic variants that exhibit transient antibiotic tolerance and play a leading role in chronic infections and the development of antibiotic resistance. Determining the mechanism that underlies persister formation and developing anti-persister strategies, therefore, are clinically important goals. Here, we report that many gram-negative and gram-positive bacteria become highly tolerant to typical bactericidal antibiotics when the carbon source for their antibiotic-sensitive exponential growth phase is shifted to fumarate, suggesting a role for fumarate in persister induction. Nutrient shift-induced Escherichia coli but not Staphylococcus aureus persister cells can be killed by aminoglycosides upon hypoionic shock (i.e., the absence of ions), which is achieved by suspending the persisters in aminoglycoside-containing pure water for only 1 or 2 min. Such potentiation can be abolished by inhibitors of the electron transport chain (e.g., NaN3) or proton motive force (e.g., CCCP). Additionally, we show that hypoionic shock facilitates the eradication of starvation-induced E. coli but not S. aureus persisters by aminoglycosides, and that such potentiation can be significantly suppressed by NaN3 or CCCP. Mechanistically, hypoionic shock dramatically enhances aminoglycoside uptake by both nutrient shift- and starvation-induced E. coli persisters, whereas CCCP can diminish this uptake. Results of our study illustrate the general role of fumarate in bacterial persistence and may open new avenues for persister eradication and aminoglycoside use.

Ocean acidification (OA) resulting from anthropogenic CO2 emissions is impairing the reproduction of marine organisms. While parental exposure to OA can protect offspring via carryover effects, this phenomenon is poorly understood in many marine invertebrate taxa. Here, we examined how parental exposure to acidified (pH 7.40) versus ambient (pH 7.72) seawater influenced reproduction and offspring performance across six gametogenic cycles (13 weeks) in the estuarine sea anemone Nematostella vectensis. Females exhibited reproductive plasticity under acidic conditions, releasing significantly fewer but larger eggs compared to ambient females after 4 weeks of exposure, and larger eggs in two of the four following spawning cycles despite recovering fecundity, indicating long-term acclimatization and greater investment in eggs. Males showed no changes in fecundity under acidic conditions but produced a greater percentage of sperm with high mitochondrial membrane potential (MMP; a proxy for elevated motility), which corresponded with higher fertilization rates relative to ambient males. Finally, parental exposure to acidic conditions did not significantly influence offspring development rates, respiration rates, or heat tolerance. Overall, this study demonstrates that parental exposure to acidic conditions impacts gamete production and physiology but not offspring performance in N. vectensis, suggesting that increased investment in individual gametes may promote fitness.

NLRX1 is a member of the of the Nod-like receptor (NLR) family, and it represents a unique pattern recognition molecule (PRM) as it localizes to the mitochondrial matrix in resting conditions. Over the past fifteen years, NLRX1 has been proposed to regulate multiple cellular processes, including antiviral immunity, apoptosis, reactive oxygen species (ROS) generation and mitochondrial metabolism. Similarly, in vivo models have shown that NLRX1 was associated with the control of a number of diseases, including multiple sclerosis, colorectal cancer and ischemia-reperfusion injury. This apparent versatility in function hinted that a common and general overarching role for NLRX1 may exist. Recent evidence has suggested that NLRX1 controls mitophagy through the detection of a specific "danger signal", namely the defective import of proteins into mitochondria, or mitochondrial protein import stress (MPIS). In this review article, we propose that mitophagy regulation may represent the overarching process detected by NLRX1, which could in turn impact on a number of diseases if dysfunctional.

Pyrazinoic acid is the active form of pyrazinamide, a first-line antibiotic used to treat Mycobacterium tuberculosis infections. However, the mechanism of action of pyrazinoic acid remains a subject of debate, and alternatives to pyrazinamide in cases of resistance are not available. The work presented here demonstrates that pyrazinoic acid and known protonophores including salicylic acid, benzoic acid, and carbonyl cyanide m-chlorophenyl hydrazone all exhibit pH-dependent inhibition of mycobacterial growth activity over a physiologically relevant range of pH values. Other anti-tubercular drugs, including rifampin, isoniazid, bedaquiline, and p-aminosalicylic acid, do not exhibit similar pH-dependent growth-inhibitory activities. The growth inhibition curves of pyrazinoic, salicylic, benzoic, and picolinic acids, as well as carbonyl cyanide m-chlorophenyl hydrazone, all fit a quantitative structure-activity relationship (QSAR) derived from acid-base equilibria with R2 values > 0.95. The QSAR model indicates that growth inhibition relies solely on the concentration of the protonated forms of these weak acids (rather than the deprotonated forms). Moreover, pyrazinoic acid, salicylic acid, and carbonyl cyanide m-chlorophenyl hydrazone all caused acidification of the mycobacterial cytoplasm at concentrations that inhibit bacterial growth. Thus, it is concluded that pyrazinoic acid acts as an uncoupler of oxidative phosphorylation and that disruption of proton motive force is the primary mechanism of action of pyrazinoic acid rather than the inhibition of a classic enzyme activity.

The antibacterial activity of honeys derived from the endemic flora of the southwest corner of Western Australia, including the trees Jarrah (Eucalyptus marginata) and Marri (Corymbia calophylla), remains largely unexplored. Investigation of these honeys showed minimum inhibitory concentrations (MICs) of 6.7-28.0% (w/v) against Gram positive and negative bacteria. Honey solutions showed enhanced antibacterial activity after hydrogen peroxide was allowed to accumulate prior to testing, with a mean MIC after accumulation of 14.3% compared to 17.4% before accumulation. Antibacterial activity was reduced after treatment with catalase enzyme, with a mean MIC of 29.4% with catalase compared to 15.2% without catalase. Tests investigating the role of the Gram negative outer membrane in honey susceptibility revealed increases in activity after destabilisation of the outer membrane. Honeys reduced both the formation of biofilm and the production of bacterial pigments, which are both regulated by quorum sensing. However, these reductions were closely correlated with global growth inhibition. Honey applied to existing biofilms resulted in decreased metabolic activity and minor decreases in viability. These results enhance our understanding of the mechanisms of antibacterial action of Jarrah and Marri honeys, and provide further support for the use of honey in the treatment of infected wounds.

Bacterial persisters exhibit noninherited antibiotic tolerance and are linked to the recalcitrance of bacterial infections. It is very urgent but also challenging to develop antipersister strategies. Here, we report that 10-s freezing with liquid nitrogen dramatically enhances the bactericidal action of aminoglycoside antibiotics by 2 to 6 orders of magnitude against many Gram-negative pathogens, with weaker potentiation effects on Gram-positive bacteria. In particular, antibiotic-tolerant Escherichia coli and Pseudomonas aeruginosa persisters-which were prepared by treating exponential-phase cells with ampicillin, ofloxacin, the protonophore cyanide m-chlorophenyl hydrazone (CCCP), or bacteriostatic antibiotics-can be effectively killed. We demonstrated, as a proof of concept, that freezing potentiated the aminoglycosides' killing of P. aeruginosa persisters in a mouse acute skin wound model. Mechanistically, freezing dramatically increased the bacterial uptake of aminoglycosides regardless of the presence of CCCP, indicating that the effects are independent of the proton motive force (PMF). In line with these results, we found that the effects were linked to freezing-induced cell membrane damage and were attributable, at least partly, to the mechanosensitive ion channel MscL, which was able to directly mediate such freezing-enhanced aminoglycoside uptake. In view of these results, we propose that the freezing-induced aminoglycoside potentiation is achieved by freezing-induced cell membrane destabilization, which, in turn, activates the MscL channel, which is able to effectively take up aminoglycosides in a PMF-independent manner. Our work may pave the way for the development of antipersister strategies that utilize the same mechanism as freezing but do so without causing any injury to animal cells.IMPORTANCE Antibiotics have long been used to successfully kill bacterial pathogens, but antibiotic resistance/tolerance usually has led to the failure of antibiotic therapy, and it has become a severe threat to human health. How to improve the efficacy of existing antibiotics is of importance for combating antibiotic-resistant/tolerant pathogens. Here, we report that 10-s rapid freezing with liquid nitrogen dramatically enhanced the bactericidal action of aminoglycoside antibiotics by 2 to 6 orders of magnitude against many bacterial pathogens in vitro and also in a mouse skin wound model. In particular, such combined treatment was able to effectively kill persister cells of Escherichia coli and Pseudomonas aeruginosa, which are per se tolerant of conventional treatment with bactericidal antibiotics for several hours. We also demonstrated that freezing-induced aminoglycoside potentiation was apparently linked to freezing-induced cell membrane damage that may have activated the mechanosensitive ion channel MscL, which, in turn, was able to effectively uptake aminoglycoside antibiotics in a proton motive force-independent manner. Our report sheds light on the development of a new strategy against bacterial pathogens by combining existing antibiotics with a conventional physical treatment or with MscL agonists.

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.

Health care associated infections (HAIs) are currently among the major challenges to the care of hematopoietic stem cell transplantation (HSCT) patients. The objective of the present study was to evaluate the impact of 2% chlorhexidine (CHG) bathing on the incidence of colonization and infection with vancomycin-resistant Enterococcus (VRE), multidrug-resistant (MDR) gram-negative pathogens, and to evaluate their CHG minimum inhibitory concentration (MIC) after the intervention.A quasi-experimental study with duration of 9 years was conducted. VRE colonization and infection, HAI rates, and MDR gram-negative infection were evaluated by interrupted time series analysis. The antibacterial susceptibility profile and mechanism of resistance to CHG were analyzed in both periods by the agar dilution method in the presence or absence of the efflux pump inhibitor carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) and presence of efflux pumps (qacA/E, qacA, qacE, cepA, AdeA, AdeB, and AdeC) by polymerase chain reaction (PCR).The VRE colonization and infection rates were significantly reduced in the postintervention period (P = 0.001). However, gram-negative MDR rates in the unit increased in the last years of the study. The CHG MICs for VRE increased during the period of exposure to the antiseptic. A higher MIC at baseline period was observed in MDR gram-negative strains. The emergence of a monoclonal Pseudomonas aeruginosa clone was observed in the second period.Concluding, CHG bathing was efficient regarding VRE colonization and infection, whereas no similar results were found with MDR gram-negative bacteria.

Lipin-1 has multiple functions that regulate lipid and energy metabolism according to its subcellular localization. The subcellular localization of Lipin-1 is determined by kinase-dependent phosphorylation; however, the phosphatase that dephosphorylates and inactivates Lipin-1 has remained elusive. Using an immunoprecipitation and LC-MS/MS approach we have identified phosphoglycerate mutase family member 5 (PGAM5), a serine/threonine specific protein phosphatase, as a regulator of Lipin-1 activity. Treatment of human hepatocellular carcinoma cells with carbonyl cyanide m-chlorophenyl hydrazone (CCCP), which activates endogenous PGAM5, promoted dephosphorylation and nuclear accumulation of Lipin-1. Our findings further elucidate the molecular mechanisms that regulate Lipin-1.

Lignin is the principal natural source of phenolics but its structural complexity and variability make it difficult to valorize through chemical depolymerization approaches. White rots are one of the rare groups of organisms that are able to degrade lignin in ecosystems. This biodegradation starts through extracellular enzymes producing oxidizing agents to depolymerize lignin and continue with the uptake of the generated oligomers by fungal cells for further degradation. Phanerochaete chrysosporium is one of the most studied species for the elucidation of these biodegradation mechanisms. Although the extracellular depolymerization step appears interesting for phenolics production from lignin, the uptake and intracellular degradation of oligomers occurring in the course of the depolymerization limits its potential. In this study, we aimed at inhibiting the phenolics uptake mechanism through metabolic inhibitors to favor extracellular oligomers accumulation without preventing the ligninases production that is necessary for extracellular depolymerization. The use of sodium azide confirmed that an active transportation phenomenon is involved in the phenolics uptake in P. chrysosporium. A protocol based on carbonyl cyanide m-chlorophenyl hydrazone enabled reaching 85% inhibition for vanillin uptake. This protocol was shown not to inhibit, but on the contrary, to stimulate the depolymerization of both dehydrogenation polymers (DHPs) and industrial purified lignins.

Septa of filamentous ascomycetes are perforated by septal pores that allow communication between individual hyphal compartments. Upon injury, septal pores are plugged rapidly by Woronin bodies (WBs), thereby preventing extensive cytoplasmic bleeding. The mechanism by which WBs translocate into the pore is not known, but it has been suggested that wound-induced cytoplasmic bleeding "flushes" WBs into the septal opening. Alternatively, contraction of septum-associated tethering proteins may pull WBs into the septal pore. Here, we investigate WB dynamics in the wheat pathogen Zymoseptoria tritici. Ultrastructural studies showed that 3.4 ± 0.2 WBs reside on each side of a septum and that single WBs of 128.5 ± 3.6 nm in diameter seal the septal pore (41 ± 1.5 nm). Live cell imaging of green fluorescent ZtHex1, a major protein in WBs, and the integral plasma membrane protein ZtSso1 confirms WB translocation into the septal pore. This was associated with the occasional formation of a plasma membrane "balloon," extruding into the dead cell, suggesting that the plasma membrane rapidly seals the wounded septal pore wound. Minor amounts of fluorescent ZtHex1-enhanced green fluorescent protein (eGFP) appeared associated with the "ballooning" plasma membrane, indicating that cytoplasmic ZtHex1-eGFP is recruited to the extending plasma membrane. Surprisingly, in ~15% of all cases, WBs moved from the ruptured cell into the septal pore. This translocation against the cytoplasmic flow suggests that an active mechanism drives WB plugging. Indeed, treatment of unwounded and intact cells with the respiration inhibitor carbonyl cyanide m-chlorophenyl hydrazone induced WB translocation into the pores. Moreover, carbonyl cyanide m-chlorophenyl hydrazone treatment recruited cytoplasmic ZtHex1-eGFP to the lateral plasma membrane of the cells. Thus, keeping the WBs out of the septal pores, in Z. tritici, is an ATP-dependent process.

Acetate, together with other short chain fatty acids has been implicated in colorectal cancer (CRC) prevention/therapy. Acetate was shown to induce apoptosis in CRC cells. The precise mechanism underlying acetate transport across CRC cells membrane, that may be implicated in its selectivity towards CRC cells, is not fully understood and was addressed here. We also assessed the effect of acetate in CRC glycolytic metabolism and explored its use in combination with the glycolytic inhibitor 3-bromopyruvate (3BP). We provide evidence that acetate enters CRC cells by the secondary active transporters MCT1 and/or MCT2 and SMCT1 as well as by facilitated diffusion via aquaporins. CRC cell exposure to acetate upregulates the expression of MCT1, MCT4 and CD147, while promoting MCT1 plasma membrane localization. We also observed that acetate increases CRC cell glycolytic phenotype and that acetate-induced apoptosis and anti-proliferative effect was potentiated by 3BP. Our data suggest that acetate selectivity towards CRC cells might be explained by the fact that aquaporins and MCTs are found overexpressed in CRC clinical cases. Our work highlights the importance that acetate transport regulation has in the use of drugs such as 3BP as a new therapeutic strategy for CRC.

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.

Equine spermatozoa highly rely on oxidative phosphorylation for their energy management. The present work aimed to characterize the role of mitochondria on horse sperm motility and ROS production by incubating spermatozoa with specific inhibitors of the different mitochondrial complexes. Equine spermatozoa were incubated 1 h and 3 h at 37 °C with: complex I inhibitor rotenone (5 μM, ROT), complex II inhibitor dimethyl-malonate (10 mM, DMM), complex III inhibitor antimycin A (1.8 μM, ANTI), the uncoupling agent carbonyl cyanide m-chlorophenyl hydrazine (5 μM, CCCP), ATP synthase inhibitor oligomycin (5 μM, OLIGO), and 2 μL vehicle DMSO (control, CTL). Samples were analyzed for sperm motility and for mitochondrial membrane potential (MMP), mitochondrial integrity, mitochondrial O2•- production, and cytoplasmic H2O2. A multivariate analysis was performed on the data. CCCP caused a pronounced MMP reduction at both time points while ROT and ANTI showed the same effect at 3 h. All treatments at 3 h incubation significantly reduced the percentage of sperm with early changes in membrane permeability with active mitochondria. The H2O2 production of live cells was low at 1 h incubation in all treatments; after 3 h a slight decrease in the percentage of low-H2O2 producing cells was recorded. All treatments, except DMM, induced a significant decline in sperm motility and kinematics and modified the pattern of sperm subpopulations. The effect of DMM was evident only after 3 h, increasing the percentage of slow sperm subpopulation. In conclusion, the disruption of mitochondrial integrity induces an increase of mitochondrial ROS production that could be detrimental for cell function and survivior.

Endothelial dysfunction is a critical mediator in the pathogenesis of vascular complications of diabetes. Herein, this study was conducted to investigate the therapeutic effects of Salvianolic acid B (Sal B) on diabetes-induced endothelial dysfunction and the underlying mechanisms. Diabetic models were established both in db/db mice and high glucose (HG)-induced human umbilical vein endothelial cells (HUVECs). Moreover, HUVECs were exposed to carbonyl cyanide m-chlorophenyl hydrazone (CCCP) to induce endothelial cell damage. Following Sal B treatment, pathological changes of thoracic aorta were investigated by hematoxylin and eosin, alcian blue (AB), elastic fiber, Masson, and reticular fiber staining. BCL2-associated X (BAX), B-cell lymphoma-2 (Bcl-2), Beclin1, Parkin and PTEN Induced Kinase 1 (Pink1) expression was detected by Western blot, immunohistochemistry, and immunofluorescence in thoracic aorta, HG- and CCCP-induced HUVECs. Cell scratch test, MitoTracker Red CMXRos staining and Flou-4 AM staining were separately presented to detect migration, mitochondrial activity and intracellular Ca2+ in HUVECs. Our results showed that Sal B significantly ameliorated hyperlipidemia, hyperglycemia, hyperinsulinemia, and insulin resistance in db/db mice. Furthermore, it significantly alleviated diabetes-induced vascular endothelial dysfunction according to histopathology analysis. In diabetic thoracic aorta, HG- and CCCP-induced HUVECs, Sal B distinctly increased Bcl-2 expression and reduced BAX, Beclin1, Parkin and Pink1 expression, thereby protecting endothelial cells from apoptosis and mitophagy. Moreover, Sal B markedly enhanced migration, mitochondrial activity and intracellular Ca2+ levels both in HG- and CCCP-induced HUVECs. Collectively, Sal B exhibited a potential to improve diabetes-induced endothelial and mitochondrial dysfunction through down-regulating apoptosis and mitophagy of endothelial cells.Abbreviations: DM: diabetes mellitus; T2DM: type 2 diabetes mellitus; Sal B: Salvianolic acid B; HG: high glucose; FBG: fasting blood glucose; TC: total cholesterol; TG: triglycerides; LDL-C: low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol; FINS: fasting insulin; HOMA-IR: homeostasis model assessment insulin resistance; QUICKI: quantitative insulin-sensitivity check index; H&E: hematoxylin and eosin; HUVECs: human umbilical vein endothelial cells; IHC: immunohistochemistry; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; FCM: flow cytometry; CCK-8: cell counting kit-8.

Peroxisomes serve as important centers for cellular redox metabolism and communication. However, fundamental gaps remain in our understanding of how the peroxisomal redox equilibrium is maintained. In particular, very little is known about the function of the nonenzymatic antioxidant glutathione in the peroxisome interior and how the glutathione antioxidant system balances with peroxisomal protein thiols. So far, only one human peroxisomal glutathione-consuming enzyme has been identified: glutathione S-transferase 1 kappa (GSTK1). To study the role of this enzyme in peroxisomal glutathione regulation and function, a GSTK1-deficient HEK-293 cell line was generated and fluorescent redox sensors were used to monitor the intraperoxisomal GSSG/GSH and NAD+/NADH redox couples and NADPH levels. We provide evidence that ablation of GSTK1 does not change the basal intraperoxisomal redox state but significantly extends the recovery period of the peroxisomal glutathione redox sensor po-roGFP2 upon treatment of the cells with thiol-specific oxidants. Given that this delay (i) can be rescued by reintroduction of GSTK1, but not its S16A active site mutant, and (ii) is not observed with a glutaredoxin-tagged version of po-roGFP2, our findings demonstrate that GSTK1 contains GSH-dependent disulfide bond oxidoreductase activity.

The dynamin-related protein Opa1 is localized to the mitochondrial intermembrane space, where it facilitates fusion between mitochondria. Apoptosis causes Opa1 release into the cytosol and causes mitochondria to fragment. Loss of mitochondrial membrane potential also causes mitochondrial fragmentation but not Opa1 release into the cytosol. Both conditions induce the proteolytic cleavage of Opa1, suggesting that mitochondrial fragmentation is triggered by Opa1 inactivation. The opposite effect was observed with knockdown of the mitochondrial intermembrane space protease Yme1. Knockdown of Yme1 prevents the constitutive cleavage of a subset of Opa1 splice variants but does not affect carbonyl cyanide m-chlorophenyl hydrazone or apoptosis-induced cleavage. Knockdown of Yme1 also increases mitochondrial connectivity, but this effect is independent of Opa1 because it also occurs in Opa1 knockdown cells. We conclude that Yme1 constitutively regulates a subset of Opa1 isoforms and an unknown mitochondrial morphology protein, whereas the loss of membrane potential induces the further proteolysis of Opa1.

Inhibitory PAS domain protein (IPAS), a repressor of hypoxia-inducible factor-dependent transcription under hypoxia, was found to exert pro-apoptotic activity in oxidative stress-induced cell death. However, physiological and pathological processes associated with this activity are not known. Here we show that IPAS is a key molecule involved in neuronal cell death in Parkinson's disease (PD). IPAS was ubiquitinated by Parkin for proteasomal degradation following carbonyl cyanide m-chlorophenyl hydrazone treatment. Phosphorylation of IPAS at Thr12 by PTEN-induced putative kinase 1 (PINK1) was required for ubiquitination to occur. Activation of the PINK1-Parkin pathway attenuated IPAS-dependent apoptosis. IPAS was markedly induced in the midbrain following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration, and IPAS-deficient mice showed resistance to MPTP-induced degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc). A significant increase in IPAS expression was found in SNpc neurons in patients with sporadic PD. These results indicate a mechanism of neurodegeneration in PD.

Oxidative damage to retinal pigment epithelial (RPE) cells contributes to the development of age-related macular degeneration, which is among the leading causes of visual loss in elderly people. In the present study, we evaluated the protective role of triphenylphosphonium (TPP)-Niacin against hydrogen peroxide (H2O2)-induced oxidative stress in RPE cells.

Accumulation of damaged mitochondria is implicated in a number of neurodegenerative disorders, including Parkinson's disease. Therefore, the machinery for mitochondrial quality control is important for the prevention of such diseases. It has been reported that Parkin- and p62/sequestosome 1 (SQSTM1)-mediated clustering and subsequent elimination of damaged mitochondria (termed mitophagy) are critical for maintaining the quality of mitochondria under stress induced by uncoupling agents such as carbonyl cyanide m-chlorophenyl hydrazone. However, the molecular mechanisms underlying mitochondrial translocation to the perinuclear region during mitophagy have not been adequately addressed to date. In this study, we found that BCL2-associated athanogene 6 (BAG6; also known as BAT3 or Scythe) is required for this process. Indeed, RNA interference-mediated depletion of endogenous BAG6 prevented Parkin-dependent relocalization of mitochondrial clusters to the perinuclear cytoplasmic region, whereas BAG6 knockdown did not affect the translocation of Parkin and p62/SQSTM1 to the depolarized mitochondria and subsequent aggregation. These results suggest that BAG6 is essential for cytoplasmic redistribution, but not for clustering, of damaged mitochondria.