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Macrophage/microglial modulation plays a critical role in the pathogenesis of multiple sclerosis (MS), which is an inflammatory disorder of the central nervous system. Dynamin-related protein 1 is a cytoplasmic molecule that regulates mitochondrial fission. It has been proven that mitochondrial fission inhibitor 1 (Mdivi-1), a small molecule inhibitor of Drp1, can relieve experimental autoimmune encephalomyelitis (EAE), a preclinical animal model of MS. Whether macrophages/microglia are involved in the pathological process of Mdivi-1-treated EAE remains to be determined. Here, we studied the anti-inflammatory effect of Mdivi-1 on mice with oligodendrocyte glycoprotein peptide35-55 (MOG35-55)-induced EAE. We found that Drp1 phosphorylation at serine 616 in macrophages/microglia was decreased with Mdivi-1 treatment, which was accompanied by decreased antigen presentation capacity of the macrophages/microglia in the EAE mouse spinal cord. The Mdivi-1 treatment caused macrophage/microglia to produce low levels of proinflammatory molecules, such as CD16/32, iNOS, and TNF-α, and high levels of anti-inflammatory molecules, such as CD206, IL-10, and Arginase-1, suggesting that Mdivi-1 promoted the macrophage/microglia shift from the inflammatory M1 phenotype to the anti-inflammatory M2 phenotype. Moreover, Mdivi-1 was able to downregulate the expression of TRL2, TRL4, GSK-3β, and phosphorylated NF-κB-p65 and prevent NF-κB-mediated IL-1β and IL-6 production. In conclusion, these results indicate that Mdivi-1 significantly alleviates inflammation in mice with EAE by promoting M2 polarization by inhibiting TLR2/4- and GSK3β-mediated NF-κB activation.

Mammalian cell-produced recombinant human erythropoietin (rhuEPO(M)) has been shown to be a multimodal neuroprotectant targeting an array of key pathological mechanisms in experimental stroke models. However, the rhuEPO(M) clinical trials were terminated due to increased risk of thrombosis, largely ascribed to its erythropoietic function. We recently took advantage of a plant-based expression system lacking sialylation capacity to produce asialo-rhuEPO(P), a rhuEPO derivative without sialic acid residues. In the present study, we proved that asialo-rhuEPO(P) is non-erythropoietic by repeated intravenous injection (44 μg/kg bw) in mice showing no increase in hemoglobin levels and red blood cell counts, and confirmed that it is non-immunogenic by measuring humoral response after immunizing the mice. We demonstrate that it is neuroprotective in a cerebral ischemia and reperfusion (I/R) mouse model, exhibiting ~ 50% reduction in cerebral infarct volume and edema, and significant improvement in neurological deficits and histopathological outcome. Our studies further revealed that asialo-rhuEPO(P), like rhuEPO(M), displays pleiotropic neuroprotective effects, including restoring I/R-interrupted mitochondrial fission and fusion proteins, preventing I/R injury-induced increase in mitophagy and autophagy markers, and inhibiting apoptosis to benefit nerve cell survival. Most importantly, asialo-rhuEPO(P) lacking erythropoietic activity and immunogenicity holds great translational potential as a multimodal neuroprotectant for stroke treatment.

There is an increasing body of evidence to show that impairment in mitochondrial dynamics including excessive fission and insufficient fusion has been observed in the pre-diabetic condition. In pre-diabetic rats with cardiac ischemia–reperfusion (I/R) injury, acute treatment with a mitochondria fission inhibitor (Mdivi-1) and a fusion promoter (M1) showed cardioprotection. However, the potential preventive effects of chronic Mdivi-1 and M1 treatment in a pre-diabetic model of cardiac I/R have never been elucidated. Male Wistar rats (n = 40) were fed with a high-fat diet (HFD) for 12 weeks to induce prediabetes. Then, all pre-diabetic rats received the following treatments daily via intraperitoneal injection for 2 weeks: (1) HFDV (Vehicle, 0.1% DMSO); (2) HFMdivi1 (Mdivi-1 1.2 mg/kg); (3) HFM1 (M1 2 mg/kg); and (4) HFCom (Mdivi-1 + M1). At the end of treatment protocols, all rats underwent 30 min of coronary artery ligation followed by reperfusion for 120 min. Chronic Mdivi-1, M1, and the combined treatment showed markedly improved cardiac mitochondrial function and dynamic control, leading to a decrease in cardiac arrhythmias, myocardial cell death, and infarct size (49%, 42%, and 51% reduction for HFMdivi1, HFM1, and HFCom, respectively vs HFDV). All of these treatments improved cardiac function following cardiac I/R injury in pre-diabetic rats. Chronic inhibition of mitochondrial fission and promotion of fusion exerted cardioprevention in prediabetes with cardiac I/R injury through the relief of cardiac mitochondrial dysfunction and dynamic alterations, and reduction in myocardial infarction, thus improving cardiac function.

Chemotherapy based on cisplatin (CDDP) has been established as the treatment of choice for head and neck squamous cell carcinoma (HNSCC). Malignant tumors respond to microenvironmental alterations through a dynamic balance between mitochondrial fission and fusion. HNSCCs are known to exhibit hypoxic conditions, yet the respective effects and underlying mechanisms of hypoxia on chemosensitivity and mitochondrial dynamics remain to be resolved. The effect of hypoxia on the chemosensitivity of HNCC cells was determined by flow cytometry. Mitochondrial fission factor (Mff) expression was assessed by RT-PCR and Western blotting in hypoxic HNSCC cells, and further verified in primary CDDP-sensitive and CDDP-resistant HSNCC samples. The biological function of Mff was evaluated by loss of function and gain of function analyses, both in vitro and in vivo. We found that hypoxia promoted mitochondrial fission and CDDP sensitivity in HNSCC cells. Importantly, Mff was found to be correlated with chemosensitivity in primary clinical samples under hypoxic conditions. Hypoxia-inducible factor 1α (HIF-1α) was found to markedly increase Mff transcription and to directly bind to Mff. Hypoxia enhanced the release of reactive oxygen species (ROS) and upregulated the expression of Mff via HIF-1α in HNSCC cells. ROS depletion in HNSCC cells attenuated HIF-1α expression, Mff expression and mitochondrial fission. Moreover, Mff knockdown led to suppression of hypoxia-induced mitochondrial fission and to decreased CDDP chemosensitivity in vivo and in vitro. Our findings indicate that hypoxia-induced release of ROS can promote mitochondrial fission and CDDP chemosensitivity via HIF1α/Mff regulation in HNSCC cells, indicating that Mff may serve as a biomarker to predict neoadjuvant chemosensitivity in HNSCC patients and as a target for overcoming chemoresistance.

Cardiomyocyte apoptosis is critical for the development of viral myocarditis (VMC), which is one of the leading causes of cardiac sudden death in young adults. Our previous studies have demonstrated that elevated calpain activity is involved in the pathogenesis of VMC. This study aimed to further explore the underlying mechanisms. Neonatal rat cardiomyocytes (NRCMs) and transgenic mice overexpressing calpastatin were infected with coxsackievirus B3 (CVB3) to establish a VMC model. Apoptosis was detected with flow cytometry, TUNEL staining, and western blotting. Cardiac function was measured using echocardiography. Mitochondrial function was measured using ATP assays, JC-1, and MitoSOX. Mitochondrial morphology was observed using MitoTracker staining and transmission electron microscopy. Colocalization of dynamin-related protein 1 (Drp-1) in mitochondria was examined using immunofluorescence. Phosphorylation levels of Drp-1 at Ser637 site were determined using western blotting analysis. We found that CVB3 infection impaired mitochondrial function as evidenced by increased mitochondrial ROS production, decreased ATP production and mitochondrial membrane potential, induced myocardial apoptosis and damage, and decreased myocardial function. These effects of CVB3 infection were attenuated by inhibition of calpain both by PD150606 treatment and calpastatin overexpression. Furthermore, CVB3-induced mitochondrial dysfunction was associated with the accumulation of Drp-1 in the outer membrane of mitochondria and subsequent increase in mitochondrial fission. Mechanistically, calpain cleaved and activated calcineurin A, which dephosphorylated Drp-1 at Ser637 site and promoted its accumulation in the mitochondria, leading to mitochondrial fission and dysfunction. In summary, calpain inhibition attenuated CVB3-induced myocarditis by reducing mitochondrial fission, thereby inhibiting cardiomyocyte apoptosis. Calpain is activated by CVB3 infection. Activated calpain cleaves calcineurin A and converts it to active form which could dephosphorylate Drp-1 at Ser637 site. Then, the active Drp-1 translocates from the cytoplasm to mitochondria and triggers excessive mitochondrial fission. Eventually, the balance of mitochondrial dynamics is broken, and apoptosis occurs.

Inhibition of autophagy has been accepted as a promising therapeutic strategy in cancer, but its clinical application is hindered by lack of effective and specific autophagy inhibitors. We previously identified cepharanthine (CEP) as a novel autophagy inhibitor, which inhibited autophagy/mitophagy through blockage of autophagosome-lysosome fusion in human breast cancer cells. In this study we investigated whether and how inhibition of autophagy/mitophagy by cepharanthine affected the efficacy of chemotherapeutic agent epirubicin in triple negative breast cancer (TNBC) cells in vitro and in vivo. In human breast cancer MDA-MB-231 and BT549 cells, application of CEP (2 μM) greatly enhanced cepharanthine-induced inhibition on cell viability and colony formation. CEP interacted with epirubicin synergistically to induce apoptosis in TNBC cells via the mitochondrial pathway. We demonstrated that co-administration of CEP and epirubicin induced mitochondrial fission in MDA-MB-231 cells, and the production of mitochondrial superoxide was correlated with mitochondrial fission and apoptosis induced by the combination. Moreover, we revealed that co-administration of CEP and epirubicin markedly increased the generation of mitochondrial superoxide, resulting in oxidation of the actin-remodeling protein cofilin, which promoted formation of an intramolecular disulfide bridge between Cys39 and Cys80 as well as Ser3 dephosphorylation, leading to mitochondria translocation of cofilin, thus causing mitochondrial fission and apoptosis. Finally, in mice bearing MDA-MB-231 cell xenografts, co-administration of CEP (12 mg/kg, ip, once every other day for 36 days) greatly enhanced the therapeutic efficacy of epirubicin (2 mg/kg) as compared with administration of either drug alone. Taken together, our results implicate that a combination of cepharanthine with chemotherapeutic agents could represent a novel therapeutic strategy for the treatment of breast cancer.

Graphical abstract Previous work has shown an association between vitamin D3 deficiency and an increased risk for acquiring various inflammatory diseases. Vitamin D3 can reduce morbidity and mortality in these patients via different mechanisms. Lung inflammation is an important event in the initiation and development of respiratory disorders. However, the anti-inflammatory effects of vitamin D3 and the underlying mechanisms remained to be determined. The purpose of this study was to examine the effects and mechanisms of action of vitamin D3 (Vit. D) on the expression of intercellular adhesion molecule-1 (ICAM-1) in vitro and in vivo with or without tumor necrosis factor α (TNF-α) treatment. Pretreatment with Vit. D reduced the expression of ICAM-1 and leukocyte adhesion in TNF-α-treated A549 cells. TNF-α increased the accumulation of mitochondrial reactive oxygen species (mtROS), while Vit. D reduced this effect. Pretreatment with Vit. D attenuated TNF-α-induced mitochondrial fission, as shown by the increased expression of mitochondrial fission factor (Mff), phosphorylated dynamin-related protein 1 (p-DRP1), and mitophagy-related proteins (BCL2/adenovirus E1B 19 kDa protein-interacting protein 3, Bnip3) in A549 cells. Inhibition of DRP1 or Mff significantly decreased ICAM-1 expression. In addition, we found that Vit. D decreased TNF-α-induced ICAM-1 expression, mitochondrial fission, and mitophagy via the AKT and NF-κB pathways. Moreover, ICAM-1 expression, mitochondrial fission, and mitophagy were increased in the lung tissues of TNF-α-treated mice, while Vit. D supplementation reduced these effects. In this study, we elucidated the mechanisms by which Vit. D reduces the expression of adhesion molecules in models of airway inflammation. Vit. D might be served as a novel therapeutic agent for the targeting of epithelial activation in lung inflammation. Graphical Headlights: • The expression of DRP1 and Mff, mitochondrial fission-related proteins, was increased in TNF-α-treated A549 cells. • The expression of Bnip3 and LC3B, mitophagy-related proteins, was increased in TNF-α-treated A549 cells. • Vit. D pretreatment decreased TNF-α-induced inflammation through the reduction of mitochondrial fission and mitophagy in A549 cells. Supplementary Information The online version contains supplementary material available at 10.1007/s10565-021-09629-6.

Aims/hypothesis Mitochondria are highly dynamic organelles continuously undergoing fission and fusion, referred to as mitochondrial dynamics, to adapt to nutritional demands. Evidence suggests that impaired mitochondrial dynamics leads to metabolic abnormalities such as non-alcoholic steatohepatitis (NASH) phenotypes. However, how mitochondrial dynamics are involved in the development of NASH is poorly understood. This study aimed to elucidate the role of mitochondrial fission factor (MFF) in the development of NASH. Methods We created mice with hepatocyte-specific deletion of MFF (MffLiKO). MffLiKO mice fed normal chow diet (NCD) or high-fat diet (HFD) were evaluated for metabolic variables and their livers were examined by histological analysis. To elucidate the mechanism of development of NASH, we examined the expression of genes related to endoplasmic reticulum (ER) stress and lipid metabolism, and the secretion of triacylglycerol (TG) using the liver and primary hepatocytes isolated from MffLiKO and control mice. Results MffLiKO mice showed aberrant mitochondrial morphologies with no obvious NASH phenotypes during NCD, while they developed full-blown NASH phenotypes in response to HFD. Expression of genes related to ER stress was markedly upregulated in the liver from MffLiKO mice. In addition, expression of genes related to hepatic TG secretion was downregulated, with reduced hepatic TG secretion in MffLiKO mice in vivo and in primary cultures of MFF-deficient hepatocytes in vitro. Furthermore, thapsigargin-induced ER stress suppressed TG secretion in primary hepatocytes isolated from control mice. Conclusions/interpretation We demonstrated that ablation of MFF in liver provoked ER stress and reduced hepatic TG secretion in vivo and in vitro. Moreover, MffLiKO mice were more susceptible to HFD-induced NASH phenotype than control mice, partly because of ER stress-induced apoptosis of hepatocytes and suppression of TG secretion from hepatocytes. This study provides evidence for the role of mitochondrial fission in the development of NASH. Graphical abstract

Glutamine is essential for maintaining the TCA cycle in cancer cells yet they undergo glutamine starvation in the core of tumors. Cancer stem cells (CSCs), responsible for tumor recurrence are often found in the nutrient limiting cores. Our study uncovers the molecular basis and cellular links between glutamine deprivation and stemness in the cancer cells. We showed that glutamine is dispensable for the survival of ovarian and colon cancer cells while it is required for their proliferation. Glutamine starvation leads to the metabolic reprogramming in tumor cells with enhanced glycolysis and unaltered oxidative phosphorylation. Production of reactive oxygen species (ROS) in glutamine limiting condition induces MAPK-ERK1/2 signaling pathway to phosphorylate dynamin-related protein-1(DRP1) at Ser616. Moreover, p-DRP1 promotes mitochondrial fragmentation and enhances numbers of CD44 and CD117/CD45 positive CSCs. Besides the established features of cancer stem cells, glutamine deprivation induces perinuclear localization of fragmented mitochondria and reduction in proliferation rate which are usually observed in CSCs. Treatment with glutaminase inhibitor (L-DON) mimics the effects of glutamine starvation without altering cell survival in in vitro as well as in in vivo model. Interestingly, the combinatorial treatment of L-DON with DRP1 inhibitor (MDiVi-1) reduces the stem cell population in tumor tissue in mouse model. Collectively our data suggest that glutamine deficiency in the core of tumors can increase the cancer stem cell population and the combination therapy with MDiVi-1 and L-DON is a useful approach to reduce CSCs population in tumor.

Caffeine induces multiple vascular effects. In this study we investigated the angiogenic effect of physiological concentrations of caffeine with focus on endothelial cell behaviors (migration and proliferation) during angiogenesis and its mitochondrial and bioenergetic mechanisms. We showed that caffeine (10–50 μM) significantly enhanced angiogenesis in vitro, evidenced by concentration-dependent increases in tube formation, and migration of human umbilical vein endothelial cells (HUVECs) without affecting cell proliferation. Caffeine (50 μM) enhanced endothelial migration via activation of cAMP/PKA/AMPK signaling pathway, which was mimicked by cAMP analog 8-Br-cAMP, and blocked by PKA inhibitor H89, adenylate cyclase inhibitor SQ22536 or AMPK inhibitor compound C. Furthermore, caffeine (50 μM) induced significant mitochondrial shortening through the increased phosphorylation of mitochondrial fission protein dynamin-related protein 1 (Drp1) in HUVECs, which increased its activity to regulate mitochondrial fission. Pharmacological blockade of Drp1 by Mdivi-1 (10 μM) or disturbance of mitochondrial fission by Drp1 silencing markedly suppressed caffeine-induced lamellipodia formation and endothelial cell migration. Moreover, we showed that caffeine-induced mitochondrial fission led to accumulation of more mitochondria in lamellipodia regions and augmentation of mitochondrial energetics, both of which were necessary for cell migration. In a mouse model of hindlimb ischemia, administration of caffeine (0.05% in 200 mL drinking water daily, for 14 days) significantly promoted angiogenesis and perfusion as well as activation of endothelial AMPK signaling in the ischemic hindlimb. Taken together, caffeine induces mitochondrial fission through cAMP/PKA/AMPK signaling pathway. Mitochondrial fission is an integral process in caffeine-induced endothelial cell migration by altering mitochondrial distribution and energetics.

3-Chloro-1, 2-propanediol (3-MCPD) is a food-borne toxic substance well-known for more than 40 years that is mainly associated with nephrotoxicity. A better understanding of 3-MCPD nephrotoxicity is required to devise efficacious strategies to counteract its toxicity. In the present work, the role of endoplasmic reticulum (ER) stress along with its underlying regulatory mechanism in 3-MCPD-mediated renal cytotoxicity was investigated in vivo and in vitro. Our data indicated that 3-MCPD-stimulated ER stress response evidenced by sustained activation of PERK-ATF4-p-CHOP and IRE1 branches in Sprague Dawley (SD) rats and human embryonic kidney (HEK293) cells. Moreover, ER stress-associated specific apoptotic initiator, caspase 12, was over-expressed. Blocking ER stress with its antagonist, 4-phenylbutyric acid (4-PBA), improved the morphology and function of kidney effectively. 4-PBA also increased cell viability, relieved mitochondrial vacuolation, and inhibited cell apoptosis through regulating caspase-dependent intrinsic apoptosis pathways. Furthermore, the enhanced expressions of two mitochondrial fission proteins, DRP1/p-DRP1 and FIS1, and the relocation of DRP1 on mitochondria subjected to 3-MPCD were reversed by 4-PBA, while the expression of the fusion protein, MFN2, was restored. Moreover, cellular Ca2+ overload, the over-expression of CaMKK2, and the loss of mitochondria-associated membranes (MAM) were also relieved after 4-PBA co-treatment. Collectively, our data emphasized that ER stress plays critical role in 3-MCPD-mediated mitochondrial dysfunction and subsequent apoptosis as well as blockage of ER stress ameliorated kidney injury through improving mitochondrial fission/fusion and Ca2+ homeostasis. These findings provide a novel insight into the regulatory role of ER stress in 3-MCPD-associated nephropathy and a potential therapeutic strategy. Graphical Headlights 1. 4-PBA inhibits ER stress mainly through regulating PERK-ATF4-CHOP and IRE1-XBP1s branches. 2. Inhibition of ER stress by 4-PBA mitigates ER associated and mitochondrial apoptosis 3. Inhibition of ER stress by 4-PBA helps maintaining calcium homeostasis and mitochondrial dynamic.

Mitochondria form dynamic networks in the cell that are balanced by the flux of iterative fusion and fission events of the organelles. It is now appreciated that mitochondrial fission also represents an end-point event in a signalling axis that allows cells to sense and respond to external cues. The fission process is orchestrated by membrane-associated adaptors, influenced by organellar and cytoskeletal interactions and ultimately executed by the dynamin-like GTPase DRP1. Here we invoke the framework of the 'mitochondrial divisome', which is conceptually and operationally similar to the bacterial cell-division machinery. We review the functional and regulatory aspects of the mitochondrial divisome and, within this framework, parse the core from the accessory machinery. In so doing, we transition from a phenomenological to a mechanistic understanding of the fission process.

The adenosine monophosphate (AMP)-activated protein kinase (Ampk) is a central regulator of metabolic pathways, and increasing Ampk activity has been considered to be an attractive therapeutic target. Here, we have identified an orphan ubiquitin E3 ligase subunit protein, Fbxo48, that targets the active, phosphorylated Ampkα (pAmpkα) for polyubiquitylation and proteasomal degradation. We have generated a novel Fbxo48 inhibitory compound, BC1618, whose potency in stimulating Ampk-dependent signaling greatly exceeds 5-aminoimidazole-4-carboxamide-1-β-ribofuranoside (AICAR) or metformin. This compound increases the biological activity of Ampk not by stimulating the activation of Ampk, but rather by preventing activated pAmpkα from Fbxo48-mediated degradation. We demonstrate that, consistent with augmenting Ampk activity, BC1618 promotes mitochondrial fission, facilitates autophagy and improves hepatic insulin sensitivity in high-fat-diet-induced obese mice. Hence, we provide a unique bioactive compound that inhibits pAmpkα disposal. Together, these results define a new pathway regulating Ampk biological activity and demonstrate the potential utility of modulating this pathway for therapeutic benefit.

Chloride intracellular channel 1 (CLIC1) is a sensor of oxidative stress in endothelial cells (EC). However, the mechanism by which CLIC1 mediate the regulation of endothelial dysfunction has not been established. In this study, overexpressed CLIC1 impaired the ability of the vascular cells to resist oxidative damage and promoted cellular senescence. Besides, suppressed CLIC1 protected against cellular senescence and dysfunction in Human Umbilical Vein Endothelial Cells (HUVECs) through the Nrf2/HO-1 pathway. We also found that ROS-activated CLIC1-induced oxidative stress in HUVECs. Nrf2 nuclear translocation was inhibited by CLIC1 overexpression, but was enhanced by IAA94 (CLICs inhibitor) treatment or knockdown of CLIC1. The Nrf2/HO-1 pathway plays a critical role in the anti-oxidative effect of suppressing CLIC1. And inhibition of CLIC1 decreases oxidative stress injury by downregulating the levels of ROS, MDA, and the expression of EC effectors (ICAM1 and VCAM1) protein expression and promotes the activity of superoxide dismutase (SOD). The AMPK-mediated signaling pathway activates Nrf2 through Nrf2 phosphorylation and nuclear translocation, which is also regulated by CLIC1. Moreover, the activation of CLIC1 contributes to H2O2-induced mitochondrial dysfunction and activation of mitochondrial fission. Therefore, elucidation of the mechanisms by which CLIC1 is involved in these pivotal pathways may uncover its therapeutic potential in alleviating ECs oxidative stress and age-related cardiovascular disease development.

This review describes the current knowledge about lipid metabolism in macrophages, its relation to mitochondrial dynamics, and the consequences in macrophage behavior. Lipid droplet (LD) expansion is necessary for optimal macrophage inflammatory response, as M1 (pro-inflammatory) macrophages accumulate LDs within the cytoplasm as a response to leptin signaling. The broken tricarboxylic acid (TCA) cycle, a hallmark of M1 macrophage metabolism alongside fissioned mitochondria, generates accumulated citrate that feeds fatty acid synthesis (FAS), which supplies LDs with fatty acids. All of these pathways are connected in order to induce and maintain the M1 phenotype. Macrophage profiles are closely related to their metabolism and mitochondrial network. Mitochondrial fission is linked with FAS in an essential mechanism for maintaining metabolic characteristics of M1 macrophages, in the context of LD expansion. While we do not know the full extent to which these factors interact, there are evident links between mitochondria dynamics and fatty acid metabolism in macrophage polarization which can be further explored.

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

Alzheimer's disease (AD) and diabetes mellitus (DM) share common pathophysiological findings, in particular, the mammalian target of rapamycin (mTOR) has been strongly implied to link to AD, while it also plays a key role in the insulin signaling pathway. However, the mechanism of how DM and AD is coupled remains elusive. In the present study, we found that streptozotocin (STZ)-induced DM mice significantly increased the levels P-mTOR Ser2448, P-p70S6K Thr389, P-tau Ser356 and Aβ levels (Aβ oligomer/monomer), as well as the levels of Drp1 and p-Drp1 S616 (mitochondrial fission proteins) are increased, whereas no change was found in the expression of Opa1, Mfn1 and Mfn2 (mitochondrial fusion proteins) compared with control mice. Moreover, the expression of 4-HNE and 8-OHdG showed an aberrant increase in the hippocampus of STZ-induced DM mice that is associated with a decreased capacity of spatial memory and a loss of synapses. Rapamycin, an inhibitor of mTOR, rescued the STZ-induced increases in mTOR/p70S6K activities, tau phosphorylation and Aβ levels, as well as mitochondria abnormality and cognitive impairment in mice. These findings imply that rapamycin prevents cognitive impairment and protects hippocampus neurons from AD-like pathology and mitochondrial abnormality, and also that rapamycin treatment could normalize these STZ-induced alterations by decreasing hippocampus mTOR/p70S6K hyperactivity.

Mitochondrial dysfunction is closely related to the occurrence of epilepsy. Homeostasis of mitochondrial fusion and division can alleviate mitochondrial dysfunction. The trafficking kinesin protein 1 (TRAK1) is a key regulator of mitochondrial movement and regulates mitochondrial fusion-fission balance. The pathogenic variants in TRAK1 result in the severe neurodevelopmental disorders. However, the role of TRAK1 in epilepsy remains unclear. In the present study, we report that TRAK1 has a crucial function in regulation of epileptogenesis in temporal lobe epilepsy (TLE). TRAK1 expression is decreased in the patient specimens and animal model of TLE. Knockdown of TRAK1 causes an increase in mitochondrial fission factor (MFF) in vitro and the susceptibility to seizures in vivo. Exogenous overexpression of TRAK1 can rescue the dysfunction caused by TRAK1 knockdown. These findings provide new insights into the fundamental mechanisms of TRAK1 in TLE and have important implications for understanding and treating TLE via targeting mitochondrion.

Hepatic ischemic reperfusion injury (IRI) is a common complication of liver surgery. Although an imbalance between mitochondrial fission and fusion has been identified as the cause of IRI, the detailed mechanism remains unclear. Augmenter of liver regeneration (ALR) was reported to prevent mitochondrial fission by inhibiting dynamin-related protein 1 (Drp1) phosphorylation, contributing partially to its liver protection. Apart from phosphorylation, Drp1 activity is also regulated by small ubiquitin-like modification (SUMOylation), which accelerates mitochondrial fission. This study aimed to investigate whether ALR-mediated protection from hepatic IRI might be associated with an effect on Drp1 SUMOylation. Liver tissues were harvested from both humans and from heterozygous ALR knockout mice, which underwent IRI. The SUMOylation and phosphorylation of Drp1 and their modulation by ALR were investigated. Hepatic Drp1 SUMOylation was significantly increased in human transplanted livers and IRI-livers of mice. ALR-transfection significantly decreased Drp1 SUMOylation, attenuated the IRI-induced mitochondrial fission and preserved mitochondrial stability and function. This study showed that the binding of transcription factor Yin Yang-1 (YY1) to its downstream target gene UBA2, a subunit of SUMO-E1 enzyme heterodimer, was critical to control Drp1 SUMOylation. By interacting with YY1, ALR inhibits its nuclear import and dramatically decreases the transcriptional level of UBA2. Consequently, mitochondrial fission was significantly reduced, and mitochondrial function was maintained. This study showed that the regulation of Drp1 SUMOylation by ALR protects mitochondria from fission, rescuing hepatocytes from IRI-induced apoptosis. These new findings provide a potential target for clinical intervention to reduce the effects of IRI during hepatic surgery.

This review focuses on the central role of mitochondrial fission-fusion imbalance in heart failure. We also discuss the development of pharmacological strategies capable of re-establishing mitochondrial dynamics in heart failure. Heart failure is a degenerative disease and a major cause of morbidity and mortality worldwide. Loss of mitochondrial fission-fusion balance and consequent impaired cardiac bioenergetics are hallmarks of heart failure. Therefore, the identification of maladaptive molecular signatures that contribute to impaired mitochondrial dynamics and bioenergetics, such as activation of protein kinase C βII, becomes a valuable platform and strategy to generate novel, and more effective, pharmacological tools to treat heart failure. Here, we discuss critical post-translational modifications (phosphorylation) of mitochondrial dynamics-related proteins in failing hearts. We also highlight some druggable protein-protein interactions that control mitochondrial dynamics as potential targets to treat heart failure.

Phase separation concentrates mitochondrial RNA granules. Here Rey et al., show that mitochondrial RNA granules (MRGs) behaviour is consistent with liquid-liquid phase separation (LLPS) and their fusion coincides with mitochondrial remodelling. Mitochondria contain the genetic information and expression machinery to produce essential respiratory chain proteins. Within the mitochondrial matrix, newly synthesized RNA, RNA processing proteins and mitoribosome assembly factors form punctate sub-compartments referred to as mitochondrial RNA granules (MRGs)(1-3). Despite their proposed importance in regulating gene expression, the structural and dynamic properties of MRGs remain largely unknown. We investigated the internal architecture of MRGs using fluorescence super-resolution localization microscopy and correlative electron microscopy, and found that the MRG ultrastructure consists of compacted RNA embedded within a protein cloud. Using live-cell super-resolution structured illumination microscopy and fluorescence recovery after photobleaching, we reveal that MRGs rapidly exchange components and can undergo fusion, characteristic properties of fluid condensates(4). Furthermore, MRGs associate with the inner mitochondrial membrane and their fusion coincides with mitochondrial remodelling. Inhibition of mitochondrial fission or fusion leads to an aberrant accumulation of MRGs into concentrated pockets, where they remain as distinct individual units despite their close apposition. Together, our findings reveal that MRGs are nanoscale fluid compartments, which are dispersed along mitochondria via membrane dynamics.

Heat shock protein B8 (HSPB8) impacts on tumor proliferation and migration of malignancy. However, the role of HSPB8 in lung adenocarcinoma (LUAC) remains unclear. The aim of this study, therefore, was to clarify whether HSPB8 could bring benefits to proliferation and migration of LUAC and its underlying mechanisms. The expression of HSPB8 was first evaluated by immunohistochemistry in 35 LUAC samples. Then, A549 lung adenocarcinoma cells were transfected with pcDNA-HSPB8 or si-HSPB8 to induce HSPB8 overexpression and silence. Cellular activity was evaluated with a Cell Counting Kit-8 (CCK-8) assay. Cell proliferation and migration were observed by EdU assay and scratch assay. Mitochondria-specific reactive oxygen species (mtROS) and membrane potential were measured using MitoSOX Red probe and JC-1 staining. Superoxide dismutase (SOD) activities and malondialdehyde (MDA) level were measured using commercial kits, respectively. HSPB8 protein, mitochondrial fusion protein MFN2 and mitochondrial fission protein p-Drp1/Drp1 were measured using western blot. Compared with the normal tissues, the expression of HSPB8 protein was higher in LUAC tissues and upregulation of HSPB8 protein was related to tumor size and tumor location. Furthermore, HSPB8 overexpression aggravated cell proliferation and migration of A549 cells. Mechanistically, HSPB8 suppressed mitochondrial impairment, leading to promoting the progress of A549 lung adenocarcinoma cells. These data demonstrate that HSPB8 plays an important role in progression of LUAC and may be a new target to treat LUAC.

Amyotrophic lateral sclerosis (ALS) is a multifactorial and progressive neurodegenerative disease of unknown etiology. Due to ALS’s unpredictable onset and progression rate, the search for biomarkers that allow the detection and tracking of its development and therapeutic efficacy would be of significant medical value. Considering that alterations of energy supply are one of ALS’s main hallmarks and that a correlation has been established between gene expression in human brain tissue and peripheral blood mononuclear cells (PBMCs), the present work investigates whether changes in mitochondrial function could be used to monitor ALS. To achieve this goal, PBMCs from ALS patients and control subjects were used; blood sampling is a quite non-invasive method and is cost-effective. Different parameters were evaluated, namely cytosolic calcium levels, mitochondrial membrane potential, oxidative stress, and metabolic compounds levels, as well as mitochondrial dynamics and degradation. Altogether, we observed lower mitochondrial calcium uptake/retention, mitochondria depolarization, and redox homeostasis deregulation, in addition to a decrease in critical metabolic genes, a diminishment in mitochondrial biogenesis, and an augmentation in mitochondrial fission and autophagy-related gene expression. All of these changes can contribute to the decreased ATP and pyruvate levels observed in ALS PBMCs. Our data indicate that PBMCs from ALS patients show a significant mitochondrial dysfunction, resembling several findings from ALS’ neural cells/models, which could be exploited as a powerful tool in ALS research. Our findings can also guide future studies on new pharmacological interventions for ALS since assessments of brain samples are challenging and represent a relevant limited strategy. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s12035-020-02059-1) contains supplementary material, which is available to authorized users.

The neonatal heart possesses the ability to proliferate and the capacity to regenerate after injury; however, the mechanisms underlying these processes are not fully understood. Melatonin has been shown to protect the heart against myocardial injury through mitigating oxidative stress, reducing apoptosis, inhibiting mitochondrial fission, etc. In this study, we investigated whether melatonin regulated cardiomyocyte proliferation and promoted cardiac repair in mice with myocardial infarction (MI), which was induced by ligation of the left anterior descending coronary artery. We showed that melatonin administration significantly improved the cardiac functions accompanied by markedly enhanced cardiomyocyte proliferation in MI mice. In neonatal mouse cardiomyocytes, treatment with melatonin (1 μM) greatly suppressed miR-143-3p levels. Silencing of miR-143-3p stimulated cardiomyocytes to re-enter the cell cycle. On the contrary, overexpression of miR-143-3p inhibited the mitosis of cardiomyocytes and abrogated cardiomyocyte mitosis induced by exposure to melatonin. Moreover, Yap and Ctnnd1 were identified as the target genes of miR-143-3p. In cardiomyocytes, inhibition of miR-143-3p increased the protein expression of Yap and Ctnnd1. Melatonin treatment also enhanced Yap and Ctnnd1 protein levels. Furthermore, Yap siRNA and Ctnnd1 siRNA attenuated melatonin-induced cell cycle re-entry of cardiomyocytes. We showed that the effect of melatonin on cardiomyocyte proliferation and cardiac regeneration was impeded by the melatonin receptor inhibitor luzindole. Silencing miR-143-3p abrogated the inhibition of luzindole on cardiomyocyte proliferation. In addition, both MT1 and MT2 siRNA could cancel the beneficial effects of melatonin on cardiomyocyte proliferation. Collectively, the results suggest that melatonin induces cardiomyocyte proliferation and heart regeneration after MI by regulating the miR-143-3p/Yap/Ctnnd1 signaling pathway, providing a new therapeutic strategy for cardiac regeneration.

Transplantation of autologous mesenchymal stem cells (MSCs) is an effective therapy for several diseases. Mitochondria modulate several important aspects of MSC function, but might be damaged by comorbidities and cardiovascular risk factors. We hypothesized that metabolic syndrome (MetS) compromises 3D mitochondrial structure, dynamics, and function in swine adipose tissue-derived MSCs. Domestic pigs were fed a Lean or MetS diet (n = 6 each) for 16 weeks. MSCs were collected from subcutaneous abdominal fat and their mitochondria analyzed using state-of-the-art Serial Block Face Electron Microscopy and 3D reconstruction. Mitochondrial dynamics (fusion/fission) were assessed by mRNA sequencing and Western blotting, and bioenergetics by membrane potential (TMRE), cytochrome-c oxidase (COX)-IV activity, and Seahorse Analyzer. Expression of mitochondria-associated microRNAs (mitomiRs) was measured by quantitative polymerase chain reaction (qPCR). MetS pigs developed obesity, hypertension, insulin resistance, and hyperlipidemia. Mitochondrial density was similar between the groups, but 3D mitochondrial and matrix volumes were lower in MetS-MSCs versus Lean-MSCs. Mitochondrial fission was higher, but fusion lower in MetS-MSCs versus Lean-MSCs, as were membrane potential, COX-IV activity, and ATP production. Contrarily, expression of the mitomiRs miR15a, miR-137, and miR-181c, which target mitochondrial genes that support mitochondrial structure, energy pathways, and dynamics, was higher in MetS-MSCs compared to Lean-MSCs, suggesting a potential to modulate their expression. MetS damages MSC 3D mitochondrial structure, dynamics, and function, and may modulate genes encoding for mitochondrial proteins. These observations support development of mitoprotective strategies to preserve the regenerative potency of MSCs and their suitability for autologous transplantation in patients with MetS.

Excessive mitochondrial fission has been implicated in the etiology of neuronal cell death in traumatic brain injury (TBI). In the present study, we examined the efficacy of melatonin (Mel) as a neuroprotective agent against TBI-induced oxidative damage and mitochondrial dysfunction. We assessed the impact of Mel post-treatment (10 mg/kg b.wt., i.p.) at different time intervals in TBI-subjected Wistar rats. We found that the Mel treatment significantly attenuated brain edema, oxidative damage, mitochondrial fission, and promoted mitochondrial fusion. Additionally, Mel-treated rats showed restoration of mitochondrial membrane potential and oxidative phosphorylation with a concomitant reduction in cytochrome-c release. Further, Mel treatment significantly inhibited the translocation of Bax and Drp1 proteins to mitochondria in TBI-subjected rats. The restorative role of Mel treatment in TBI rats was supported by the mitochondrial ultra-structural analysis, which showed activation of mitochondrial fusion mechanism. Mel enhanced mitochondrial biogenesis by upregulation of PGC-1α protein. Our results demonstrated the remedial role of Mel in ameliorating mitochondrial dysfunctions that are modulated in TBI-subjected rats and provided support for mitochondrial-mediated neuroprotection as a putative therapeutic agent in the brain trauma.

Selenium has been shown to possess antioxidant and neuroprotective effects by modulating mitochondrial function and activating mitochondrial biogenesis. Our previous study has also suggested that selenium protected neurons against glutamate toxicity and hyperglycemia-induced damage by regulating mitochondrial fission and fusion. However, it is still not known whether the mitochondrial biogenesis is involved in selenium alleviating hyperglycemia-aggravated cerebral ischemia reperfusion (I/R) injury. The object of this study is to define whether selenium protects neurons against hyperglycemia-aggravated cerebral I/R injury by promoting mitochondrial biogenesis. In vitro oxygen deprivation plus high glucose model decreased cell viability, enhanced reactive oxygen species production, and meanwhile stimulated mitochondrial biogenesis signaling. Pretreated with selenium significantly decreased cell death and further activated the mitochondrial biogenesis signaling. In vivo 30 min of middle cerebral artery occlusion in the rats under hyperglycemic condition enhanced neurological deficits, enlarged infarct volume, exacerbated neuronal damage and oxidative stress compared with normoglycemic ischemic rats after 24 h reperfusion. Consistent to the in vitro results, selenium treatment alleviated ischemic damage in hyperglycemic ischemic animals. Furthermore, selenium reduced the structural changes of mitochondria caused by hyperglycemic ischemia and further promoted the mitochondrial biogenesis signaling. Selenium activates mitochondrial biogenesis signaling, protects mitochondrial structure integrity and ameliorates cerebral I/R injury in hyperglycemic rats.

Clinical advances in the treatment of intracranial hemorrhage (ICH) are restricted by the incomplete understanding of the molecular mechanisms contributing to secondary brain injury. Acrolein is a highly active unsaturated aldehyde which has been implicated in many nervous system diseases. Our results indicated a significant increase in the level of acrolein after ICH in mouse brain. In primary neurons, acrolein induced an increase in mitochondrial fragmentation, loss of mitochondrial membrane potential, generation of reactive oxidative species, and release of mitochondrial cytochrome c. Mechanistically, acrolein facilitated the translocation of dynamin-related protein1 (Drp1) from the cytoplasm onto the mitochondrial membrane and led to excessive mitochondrial fission. Further studies found that treatment with hydralazine (an acrolein scavenger) significantly reversed Drp1 translocation and the morphological damage of mitochondria after ICH. In parallel, the neural apoptosis, brain edema, and neurological functional deficits induced by ICH were also remarkably alleviated. In conclusion, our results identify acrolein as an important contributor to the secondary brain injury following ICH. Meanwhile, we uncovered a novel mechanism by which Drp1-mediated mitochondrial oxidative damage is involved in acrolein-induced brain injury. Electronic supplementary material The online version of this article (10.1007/s12264-020-00505-7) contains supplementary material, which is available to authorized users.

Heat stress-induced reductions in milk yield and the dysfunction of mammary glands are economically important challenges that face the dairy industry, especially during summer. The aim of the present study is to investigate the effects of heat stress on mitochondrial function by using dairy cow mammary epithelial cells (DCMECs) as an in vitro model. Live cell imaging shows that the mitochondria continually change shape through fission and fusion. However, heat stress induces the fragmentation of mitochondria, as well as the decreased of ATP level, membrane potential, and anti-oxidant enzyme activity and the increased of respiratory chain complex I activity. In addition, the cytosolic Ca2+ concentration and cytochrome c expression (Cyto-c) were increased after heat stress treatment. Both qRT-PCR and western blot analysis indicate that mitofusin1/2 (Mfn1/2) and optic atrophy protein-1 (Opa-1) are downregulated after heat stress, whereas dynamin-related protein 1 (Drp1) and fission 1 (Fis-1) are upregulated, which explains the observed defect of mitochondrial network dynamics. Accordingly, the present study indicated that heat stress induced the dysfunction of DCMEC through disruption of the normal balance of mitochondrial fission and fusion.

Cells experiencing delays in mitotic progression are prone to undergo apoptosis unless they can exit mitosis before proapoptotic factors reach a critical threshold. Microtubule targeting agents (MTAs) arrest cells in mitosis and induce apoptotic cell death engaging the BCL2 network. Degradation of the antiapoptotic BCL2 family member MCL-1 is considered to set the time until onset of apoptosis upon MTA treatment. MCL1 degradation involves its interaction with one of its key binding partners, the proapoptotic BH3-only protein NOXA. Here, we report that the mitochondria-associated E3-ligase MARCH5, best known for its role in mitochondrial quality control and regulation of components of the mitochondrial fission machinery, controls the levels of MCL1/NOXA protein complexes in steady state as well as during mitotic arrest. Inhibition of MARCH5 function sensitizes cancer cells to the proapoptotic effects of MTAs by the accumulation of NOXA and primes cancer cells that may undergo slippage to escape death in mitosis to cell death in the next G1 phase. We propose that inhibition of MARCH5 may be a suitable strategy to sensitize cancer cells to antimitotic drug treatment.

Arterial media calcification is related to mitochondrial dysfunction. Protective mitophagy delays the progression of vascular calcification. We previously reported that lactate accelerates osteoblastic phenotype transition of VSMC through BNIP3-mediated mitophagy suppression. In this study, we investigated the specific links between lactate, mitochondrial homeostasis, and vascular calcification. Ex vivo, alizarin S red and von Kossa staining in addition to measurement of calcium content, RUNX2, and BMP-2 protein levels revealed that lactate accelerated arterial media calcification. We demonstrated that lactate induced mitochondrial fission and apoptosis in aortas, whereas mitophagy was suppressed. In VSMCs, lactate increased NR4A1 expression, leading to activation of DNA-PKcs and p53. Lactate induced Drp1 migration to the mitochondria and enhanced mitochondrial fission through NR4A1. Western blot analysis of LC3-II and p62 and mRFP-GFP-LC3 adenovirus detection showed that NR4A1 knockdown was involved in enhanced autophagy flux. Furthermore, NR4A1 inhibited BNIP3-related mitophagy, which was confirmed by TOMM20 and BNIP3 protein levels, and LC3-II co-localization with TOMM20. The excessive fission and deficient mitophagy damaged mitochondrial structure and impaired respiratory function, determined by mPTP opening rate, mitochondrial membrane potential, mitochondrial morphology under TEM, ATP production, and OCR, which was reversed by NR4A1 silencing. Mechanistically, lactate enhanced fission but halted mitophagy via activation of the NR4A1/DNA-PKcs/p53 pathway, evoking apoptosis, finally accelerating osteoblastic phenotype transition of VSMC and calcium deposition. This study suggests that the NR4A1/DNA-PKcs/p53 pathway is involved in the mechanism by which lactate accelerates vascular calcification, partly through excessive Drp-mediated mitochondrial fission and BNIP3-related mitophagy deficiency.

Current evidence indicates that coronary microcirculation is a key target for protecting against cardiac ischemia–reperfusion (I/R) injury. Mitochondrial calcium uniporter (MCU) complex activation and mitochondrial calcium ([Ca2+]m) overload are underlying mechanisms involved in cardiovascular disease. Histidine triad nucleotide-binding 2 (HINT2) has been reported to modulate [Ca2+]m via the MCU complex, and our previous work demonstrated that HINT2 improved cardiomyocyte survival and preserved heart function in mice with cardiac ischemia. This study aimed to explore the benefits of HINT2 on cardiac microcirculation in I/R injury with a focus on mitochondria, the MCU complex, and [Ca2+]m overload in endothelial cells. The present work demonstrated that HINT2 overexpression significantly reduced the no-reflow area and improved microvascular perfusion in I/R-injured mouse hearts, potentially by promoting endothelial nitric oxide synthase (eNOS) expression and phosphorylation. Microvascular barrier function was compromised by reperfusion injury, but was repaired by HINT2 overexpression via inhibiting VE-Cadherin phosphorylation at Tyr731 and enhancing the VE-Cadherin/β-Catenin interaction. In addition, HINT2 overexpression inhibited the inflammatory response by suppressing vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). Mitochondrial fission occurred in cardiac microvascular endothelial cells (CMECs) subjected to oxygen–glucose deprivation/reoxygenation (OGD/R) injury and resulted in mitochondrial dysfunction and mitochondrion-dependent apoptosis, the effects of which were largely relieved by HINT2 overexpression. Additional experiments confirmed that [Ca2+]m overload was an initiating factor for mitochondrial fission and that HINT2 suppressed [Ca2+]m overload via modulation of the MCU complex through directly interacting with MCU in CMECs. Regaining [Ca2+]m overload by spermine, an MCU agonist, abolished all the protective effects of HINT2 on OGD/R-injured CMECs and I/R-injured cardiac microcirculation. In conclusion, the present report demonstrated that HINT2 overexpression inhibited MCU complex-mitochondrial calcium overload-mitochondrial fission and apoptosis pathway, and thereby attenuated cardiac microvascular ischemia–reperfusion injury.

O-GlcNAcylation is a prevalent form of glycosylation that regulates proteins within the cytosol, nucleus, and mitochondria. The O-GlcNAc modification can affect protein cellular localization, function, and signaling interactions. The specific impact of O-GlcNAcylation on mitochondrial morphology and function has been elusive. In this manuscript, the role of O-GlcNAcylation on mitochondrial fission, oxidative phosphorylation (Oxphos), and the activity of electron transport chain (ETC) complexes were evaluated. In a cellular environment with hyper O-GlcNAcylation due to the deletion of O-GlcNAcase (OGA), mitochondria showed a dramatic reduction in size and a corresponding increase in number and total mitochondrial mass. Because of the increased mitochondrial content, OGA knockout cells exhibited comparable coupled mitochondrial Oxphos and ATP levels when compared to WT cells. However, we observed reduced protein levels for complex I and II when comparing normalized mitochondrial content and reduced linked activity for complexes I and III when examining individual ETC complex activities. In assessing mitochondrial fission, we observed increased amounts of O-GlcNAcylated dynamin-related protein 1 (Drp1) in cells genetically null for OGA and in glioblastoma cells. Individual regions of Drp1 were evaluated for O-GlcNAc modifications, and we found that this post-translational modification (PTM) was not limited to the previously characterized residues in the variable domain (VD). Additional modification sites are predicted in the GTPase domain, which may influence enzyme activity. Collectively, these results highlight the impact of O-GlcNAcylation on mitochondrial dynamics and ETC function and mimic the changes that may occur during glucose toxicity from hyperglycemia.

Mitochondrial mass imbalance is one of the key causes of cardiovascular dysfunction after hypoxia. The activation of dynamin-related protein 1 (Drp1), as well as its mitochondrial translocation, play important roles in the changes of both mitochondrial morphology and mitochondrial functions after hypoxia. However, in addition to mediating mitochondrial fission, whether Drp1 has other regulatory roles in mitochondrial homeostasis after mitochondrial translocation is unknown. In this study, we performed a series of interaction and colocalization assays and found that, after mitochondrial translocation, Drp1 may promote the excessive opening of the mitochondrial permeability transition pore (mPTP) after hypoxia. Firstly, mitochondrial Drp1 maximumly recognizes mPTP channels by binding Bcl-2-associated X protein (BAX) and a phosphate carrier protein (PiC) in the mPTP. Then, leucine-rich repeat serine/threonine-protein kinase 2 (LRRK2) is recruited, whose kinase activity is inhibited by direct binding with mitochondrial Drp1 after hypoxia. Subsequently, the mPTP-related protein hexokinase 2 (HK2) is inactivated at Thr-473 and dissociates from the mitochondrial membrane, ultimately causing structural disruption and overopening of mPTP, which aggravates mitochondrial and cellular dysfunction after hypoxia. Thus, our study interprets the dual direct regulation of mitochondrial Drp1 on mitochondrial morphology and functions after hypoxia and proposes a new mitochondrial fission-independent mechanism for the role of Drp1 after its translocation in hypoxic injury.

The endoplasmic reticulum (ER) is the organelle responsible for the folding of secretory/membrane proteins and acts as a dynamic calcium ion (Ca2+) store involved in various cellular signalling pathways. Previously, we reported that the ER-resident disulfide reductase ERdj5 is involved in the ER-associated degradation (ERAD) of misfolded proteins in the ER and the activation of SERCA2b, a Ca2+ pump on the ER membrane. These results highlighted the importance of the regulation of redox activity in both Ca2+ and protein homeostasis in the ER. Here, we show that the deletion of ERdj5 causes an imbalance in intracellular Ca2+ homeostasis, the activation of Drp1, a cytosolic GTPase involved in mitochondrial fission, and finally the aberrant fragmentation of mitochondria, which affects cell viability as well as phenotype with features of cellular senescence. Thus, ERdj5-mediated regulation of intracellular Ca2+ is essential for the maintenance of mitochondrial homeostasis involved in cellular senescence.

Background Mitochondrial dynamics is the result of a dynamic balance between fusion and fission events, which are driven via a set of mitochondria-shaping proteins. These proteins are generally considered to be binary components of either the fission or fusion machinery, but potential crosstalk between the fission and fusion machineries remains less explored. In the present work, we analyzed the roles of mitochondrial elongation factors 1 and 2 (MIEF1/2), core components of the fission machinery in mammals. Results We show that MIEFs (MIEF1/2), besides their action in the fission machinery, regulate mitochondrial fusion through direct interaction with the fusion proteins Mfn1 and Mfn2, suggesting that MIEFs participate in not only fission but also fusion. Elevated levels of MIEFs enhance mitochondrial fusion in an Mfn1/2- and OPA1-dependent but Drp1-independent manner. Moreover, mitochondrial localization and self-association of MIEFs are crucial for their fusion-promoting ability. In addition, we show that MIEF1/2 can competitively decrease the interaction of hFis1 with Mfn1 and Mfn2, alleviating hFis1-induced mitochondrial fragmentation and contributing to mitochondrial fusion. Conclusions Our study suggests that MIEFs serve as a central hub that interacts with and regulates both the fission and fusion machineries, which uncovers a novel mechanism for balancing these opposing forces of mitochondrial dynamics in mammals.

With global warming, heat stress has become an important challenge for the global dairy industry. Sirtuin 3 (SIRT3), an important mitochondrial NAD+dependent decarboxylase and a major regulator of cellular energy metabolism and antioxidant defense, is integral to maintaining normal mitochondrial function. The aim of this study was to assess the protective effect of SIRT3 on damage to bovine mammary epithelial cells (BMECs) induced by heat stress and to explore its potential mechanism. Our results indicate that SIRT3 is significantly downregulated in heat-stressed mammary tissue and high-temperature-treated BMECs. SIRT3 knockdown significantly increased the expression of HSP70, Bax, and cleaved-caspase 3 and inhibited the production of antioxidases, thus promoting ROS production and cell apoptosis in BMECs. In addition, SIRT3 knockdown can aggravate mitochondrial damage by mediating the expression of genes related to mitochondrial fission and fusion, including dynamin-related protein 1, mitochondrial fission 1 protein, and mitochondrial fusion proteins 1and 2. In addition, SIRT3 knockdown substantially decreased AMPK phosphorylation in BMECs. In contrast, SIRT3 overexpression in high-temperature treatment had the opposite effect to SIRT3 knockdown in BMECs. SIRT3 overexpression reduced mitochondrial damage and weakened the oxidative stress response of BMECs induced by heat stress and promoted the phosphorylation of AMPK. Taken together, our results indicate that SIRT3 can protect BMECs from heat stress damage through the AMPK signaling pathway. Therefore, the reduction of oxidative stress by SIRT3 may be the primary molecular mechanism underlying resistance to heat stress in summer cows.

Background Our previous study proved that Shen Qi Li Xin formula (SQLXF) improved the heart function of chronic heart failure (CHF) patients, while the action mechanism remains unclear. Methods H&E staining and TUNEL staining were performed to measure myocardial damages. Western blot was used to examine the expression of proteins. Moreover, CCK-8 assay and flow cytometry were used to measure cell viability and cell apoptosis, respectively. Concentrations of ATP and ROS in cells, and mitochondrial membrane potential (MMP) were detected to estimate oxidative stress. Results In vivo, we found that SQLXF improved cardiac hemodynamic parameters, reduced LDH, CK-MB and BNP production, and attenuated myocardial damages in CHF rats. Besides, SQLXF promoted mitochondrial fusion-related proteins expression and inhibited fission-related proteins expression in CHF rats and oxygen glucose deprivation/reoxygenation (OGD/R)-induced cardiac myocytes (CMs). In vitro, our data show that certain dose of SQLXF inhibited OGD/R-induced CMs apoptosis, cell viability decreasing and oxidative stress. Conclusion Overall, certain dose of SQLXF could effectively improve the cardiac function of CHF rats through inhibition of CMs apoptosis via balancing mitochondrial fission and fusion. Our data proved a novel action mechanism of SQLXF in CHF improvement, and provided a reference for clinical.

Vertebrate photoreceptors contain large numbers of closely-packed mitochondria which sustain the high metabolic demands of these cells. These mitochondria populations are dynamic and undergo fusion and fission events. This activity serves to maintain the population in a healthy state. In the event of mitochondrial damage, sub-domains, or indeed whole mitochondria, can be degraded and population homeostasis achieved. If this process is overwhelmed cell death may result. Death of photoreceptors contributes to loss of vision in aging individuals and is associated with many eye diseases. In this study we used serial block face scanning electron microscopy of adult (Macaca fascicularis) retinae to examine the 3D structure of mitochondria in rod and cone photoreceptors. Healthy-looking photoreceptors contain mitochondria with a range of shapes which are associated with different regions of the cell. In some photoreceptors we observe mitochondrial swelling and other changes we associate with stress or degeneration of the cell. In both rods and cones we identify elongated domains of mitochondria with densely-packed normal cristae associated with photoreceptor ciliary rootlet bundles. We observe mitochondrial fission and mitochondrion fragments localised to these domains. Swollen mitochondria with few intact cristae are located towards the periphery of the photoreceptor inner-segment in rods, whilst they are found throughout the cell in cones. Swollen mitochondria exhibit sites on the mitochondrial inner membrane which have undergone complex invagination resulting in membranous, electron-dense aggregates. Membrane contact occurs between the mitochondrion and the photoreceptor plasma membrane in the vicinity of these aggregates, and a series of subsequent membrane fusions results in expulsion of the mitochondrial aggregate from the photoreceptor. These events are primarily associated with rods, likely reflecting the ageing mechanism in primates where many rods die but cones do not. Possible consequences of this atypical mitochondrial degradation are discussed.

Megaconial Congenital Muscular Dystrophy (CMD) is a rare autosomal recessive disorder characterized by enlarged mitochondria located mainly at the periphery of muscle fibers and caused by mutations in the Choline Kinase Beta (CHKB) gene. Although the pathogenesis of this disease is not well understood, there is accumulating evidence for the presence of mitochondrial dysfunction. In this study, we aimed to investigate whether imbalanced mitochondrial dynamics affects mitochondrial function and bioenergetic efficiency in skeletal muscle cells of Megaconial CMD. Immunofluorescence, confocal and transmission electron microscopy studies revealed impaired mitochondrial network, morphology, and localization in primary skeletal muscle cells of Megaconial CMD. The organelle disruption was specific only to skeletal muscle cells grown in culture. The expression levels of mitochondrial fission proteins (DRP1, MFF, FIS1) were found to be decreased significantly in both primary skeletal muscle cells and tissue sections of Megaconial CMD by Western blotting and/or immunofluorescence analysis. The metabolomic and fluxomic analysis, which were performed in Megaconial CMD for the first time, revealed decreased levels of phosphonucleotides, Krebs cycle intermediates, ATP, and altered energy metabolism pathways. Our results indicate that reduced mitochondrial fission and altered mitochondrial energy metabolism contribute to mitochondrial dysmorphology and dysfunction in the pathogenesis of Megaconial CMD.

Porphyromonas gingivalis (P. gingivalis), a key pathogen in periodontitis, has been shown to accelerate the progression of atherosclerosis (AS). However, the definite mechanisms remain elusive. Emerging evidence supports an association between mitochondrial dysfunction and AS. In our study, the impact of P. gingivalis on mitochondrial dysfunction and the potential mechanism were investigated. The mitochondrial morphology of EA.hy926 cells infected with P. gingivalis was assessed by transmission electron microscopy, mitochondrial staining, and quantitative analysis of the mitochondrial network. Fluorescence staining and flow cytometry analysis were performed to determine mitochondrial reactive oxygen species (mtROS) and mitochondrial membrane potential (MMP) levels. Cellular ATP production was examined by a luminescence assay kit. The expression of key fusion and fission proteins was evaluated by western blot and immunofluorescence. Mdivi-1, a specific Drp1 inhibitor, was used to elucidate the role of Drp1 in mitochondrial dysfunction. Our findings showed that P. gingivalis infection induced mitochondrial fragmentation, increased the mtROS levels, and decreased the MMP and ATP concentration in vascular endothelial cells. We observed upregulation of Drp1 (Ser616) phosphorylation and translocation of Drp1 to mitochondria. Mdivi-1 blocked the mitochondrial fragmentation and dysfunction induced by P. gingivalis. Collectively, these results revealed that P. gingivalis infection promoted mitochondrial fragmentation and dysfunction, which was dependent on Drp1. Mitochondrial dysfunction may represent the mechanism by which P. gingivalis exacerbates atherosclerotic lesions.

Neuronal necrosis induced by excessive glutamate release is well known to contribute morbidity and mortality in ischemic stroke. Over the past decades, strategies on targeting glutamate receptor did not achieve desirable clinical outcomes. Finding the downstream mechanism of the glutamate receptor activation may provide new targets to suppress the cell death. Previously, our study demonstrated that the increase of H3K4 trimethylation (H3K4me3) played a key detrimental role on neuronal necrosis; however, the mechanism of this histone modification is unclear. Through a genome-wide small RNA sequencing, we identified several tRNA-derived fragments (tRFs) and piwi-interacting RNA (piRNAs) species were enriched in glutamate-induced neuronal necrosis in rat primary neuron cultures, and this enrichment was dependent on the H3K4me3 increase. Strikingly, when we transfected several synthesized tRFs and piRNA species into neurons, the tRFs but not the piRNAs induced neuron swelling and death. The cell death morphology recapitulated neuronal necrosis induced by glutamate. For the cytotoxic effect of tRFs, our data suggested that protein synthesis was inhibited likely through induction of ribosomal stalling. By proteomic analysis of tRFs effect, the most affected pathway was enriched in the mitochondrial metabolism. Consistently, mitochondrial fragmentation was increased in neuronal necrosis, and suppression of mitochondrial fission by genetic manipulation or drug rescued neuronal necrosis. Using our previously established Drosophila model of neuronal necrosis, we found that inhibition of small RNA transcription, blocking RNA transport from nucleus to cytosol, or knocking down Ago1/2 to suppress the RNA interference effect, all rescued the fly death, suggesting transcription and processing of small RNAs contribute to neuronal necrosis. Together, these results indicate that the abnormal transcription of tRFs may play a key role downstream of the H3K4me3 increase. This provides a potential new strategy to suppress neuronal necrosis.

Background Mitochondrial dynamics plays an important role in tumour progression. However, how these dynamics integrate tumour metabolism in hepatocellular carcinoma (HCC) metastasis is still unclear. Methods The mitochondrial fusion protein mitofusin-1 (MFN1) expression and its prognostic value are detected in HCC. The effects and underlying mechanisms of MFN1 on HCC metastasis and metabolic reprogramming are analysed both in vitro and in vivo. Results Mitochondrial dynamics, represented by constant fission and fusion, are found to be associated with HCC metastasis. High metastatic HCC displays excessive mitochondrial fission. Among genes involved in mitochondrial dynamics, MFN1 is identified as a leading downregulated candidate that is closely associated with HCC metastasis and poor prognosis. While promoting mitochondrial fusion, MFN1 inhibits cell proliferation, invasion and migration capacity both in vitro and in vivo. Mechanistically, disruption of mitochondrial dynamics by depletion of MFN1 triggers the epithelial-to-mesenchymal transition (EMT) of HCC. Moreover, MFN1 modulates HCC metastasis by metabolic shift from aerobic glycolysis to oxidative phosphorylation. Treatment with glycolytic inhibitor 2-Deoxy-d-glucose (2-DG) significantly suppresses the effects induced by depletion of MFN1. Conclusions Our results reveal a critical involvement of mitochondrial dynamics in HCC metastasis via modulating glucose metabolic reprogramming. MFN1 may serve as a novel potential therapeutic target for HCC.

The mammalian target of rapamycin (mTOR) was reported to regulate cell autophagy and outcomes of several neurological diseases. Mitochondria, which serve as critical organelles in neurons. are also involved in the pathology of neurological diseases. However, the role of mTOR in mitochondrial morphology has not been clarified especially in subarachnoid hemorrhage (SAH). In this study, we established SAH models both in vivo and in vitro. Rapamycin and 3-methyl adenine (3-MA) were then administered to alter mTOR activity. Post-SAH assessment included SAH grading, neurological evaluation, blood-brain barrier (BBB) permeability, brain water content, mitochondrial membrane potential (MMP), mitochondrial morphology, ATP content, cell viability, cytotoxicity, and expression of proteins related to apoptosis and mitochondrial fission. The results showed that (1) neurological deficits, BBB permeability, and brain edema were increased after SAH and that cell viability was exacerbated in brain tissue. (2) Excessive mitochondrial fission was evident based on changes in mitochondrial morphology, while MMP and ATP content were decreased in neurons after SAH. (3) Administration of rapamycin improved the excessive mitochondrial fission and restored mitochondrial function, which subsequently reduced apoptosis. (4) 3-MA showed an adverse effect on mitochondria and aggravated excessive mitochondrial fission and dysfunction in SAH. Neurological deficits and neuronal viability were also exacerbated following the administration of 3-MA. Therefore, our study suggests that mTOR inhibition has neuroprotective effects against neuronal injury after SAH via alleviating excessive mitochondrial fission.

Background/Objectives: Impaired insulin-mediated glucose partitioning is an intrinsic metabolic defect in skeletal muscle from severely obese humans (BMI ≥ 40 kg/m2). Roux-en-Y gastric bypass (RYGB) surgery has been shown to improve glucose metabolism in severely obese humans. The purpose of the study was to determine the effects of RYGB surgery on glucose partitioning, mitochondrial network morphology, and markers of mitochondrial dynamics skeletal muscle from severely obese humans. Subject/Methods: Human skeletal muscle cells were isolated from muscle biopsies obtained from RYGB patients (BMI = 48.0 ± 2.1, n=7) prior to, 1-month and 7-months following surgery and lean control subjects (BMI = 22.4 ± 1.1, n=7). Complete glucose oxidation, non-oxidized glycolysis rates, mitochondrial respiratory capacity, mitochondrial network morphology and regulatory proteins of mitochondrial dynamics were determined in differentiated human myotubes. Results: Myotubes derived from severely obese humans exhibited enhanced glucose oxidation (13.5%; 95%CI [7.6, 19.4], P = 0.043) and reduced non-oxidized glycolysis (−1.3%; 95%CI [−11.1, 8.6]) in response to insulin stimulation at 7-months after RYGB when compared to the pre-surgery state (−0.6%; 95%CI [−5.2, 4.0] and 19.5%; 95%CI [4.0, 35.0], P =0.006), and were not different from the lean controls (16.7%; 95%CI [11.8, 21.5] and 1.9%; 95%CI [−1.6, 5.4], respectively). Further, number of fragmented mitochondria and Drp1(Ser616) phosphorylation and were trended to reduced/reduced (0.0104, 95%CI [0.0085, 0.0126], P = 0.091 and 0.0085, 95%CI [0.0068, 0.0102], P = 0.05) in myotubes derived from severely obese humans at 7-months after RYGB surgery in comparison to the pre-surgery state. Finally, Drp1(Ser616) phosphorylation was negatively correlated with insulin-stimulated glucose oxidation (r = −0.49, P = 0.037). Conclusion/Interpretation: These data indicate that an intrinsic metabolic defect of glucose partitioning in skeletal muscle from severely obese humans is restored by RYGB surgery. The restoration of glucose partitioning may be regulated through reduced mitochondrial fission protein Drp1 phosphorylation.

Dopamine deficiency is mainly caused by apoptosis of dopaminergic nerve cells in the substantia nigra of the midbrain and the striatum and is an important pathologic basis of Parkinson’s disease (PD). Recent research has shown that dynamin-related protein 1 (Drp1)-mediated aberrant mitochondrial fission plays a crucial role in dopaminergic nerve cell apoptosis. However, the upstream regulatory mechanism remains unclear. Our study showed that Drp1 knockdown inhibited aberrant mitochondrial fission and apoptosis. Importantly, we found that ROCK1 was activated in an MPP+-induced PD cell model and that ROCK1 knockdown and the specific ROCK1 activation inhibitor Y-27632 blocked Drp1-mediated aberrant mitochondrial fission and apoptosis of dopaminergic nerve cells by suppressing Drp1 dephosphorylation/activation. Our in vivo study confirmed that Y-27632 significantly improved symptoms in a PD mouse model by inhibiting Drp1-mediated aberrant mitochondrial fission and apoptosis. Collectively, our findings suggest an important molecular mechanism of PD pathogenesis involving ROCK1-regulated dopaminergic nerve cell apoptosis via the activation of Drp1-induced aberrant mitochondrial fission., Parkinson’s disease: Looking back identifies a new target Researchers in China have revealed how a protein molecule plays an early part in the molecular steps that can lead to Parkinson’s disease, which is caused by the death of nerve cells that make the neurotransmitter dopamine. Disruption of mitochondria, the energy-generating bodies inside cells, was already known to lead to the death of dopamine-producing cells. Rong Zhang, Guobing Li and colleagues at The Second Affiliated Hospital of Army Medical University in Chongqing, China traced the chain of cause and effect back to a protein called ROCK-1. Using a mouse model of Parkinson’s disease, they found that ROCK-1 activates another protein previously shown to trigger the disruption of mitochondria. ROCK-1’s early role in the sequence might make it a suitable target for treatment using drugs that inhibit its activity.

We investigated the contribution of mitochondrial dysfunction to the senescence of human endothelial progenitor cells (EPCs) expanded in vitro and the underlying molecular mechanism. Serial passage increased cell doubling time and those cells reaching the doubling time for more than 100% were defined as senescent EPCs, of which the activity of therapeutic angiogenesis was attenuated in mouse ischemic hindlimbs. The senescent cells, in medium free of glucose and bicarbonate, showed impaired activity in migration and tube formation. Flow cytometry indicated increased content of reactive oxygen species, mitochondria, and calcium, while bioenergetic analysis showed increased oxygen consumption and reduced ATP content. Examination of mitochondrial network showed that senescence increased the length of the network and ultrastructure analysis exhibited elongated mitochondria. Immunoblotting of the senescent EPCs demonstrated decreased expression level of fission protein1 (Fis1). In rat EPCs, the Fis1 level was decreased in the animals aged 24 months or older, compared to those of 3 months. Silencing of Fis1 in the young EPCs using Fis1-specific siRNA leads to appearance of phenotype resembling those of senescent cells, including elevated oxidative stress, disturbed mitochondrial network, reduced mitochondria membrane potential, decreasing ATP content, lower proliferation activity, and loss of therapeutic potential in ischemic hindlimbs. Fis1 over-expression in senescent EPCs reduced the oxidative stress, increased the proliferation, and restored the cobble stone-like morphology, senescence, bioenergetics, angiogenic potential, and therapeutic activity. In human EPCs, down-regulation of Fis1 is involved in mitochondrial dysfunction and contributes to the impaired activity of EPCs during the senescence process. Enhanced expression of Fis1 in senescent EPCs restores the youthful phenotype.

Irisin plays a protective effect in acute and chronic myocardial damage, but its role in septic cardiomyopathy is unclear. The aim of our study was to explore the in vivo and in vitro effects of irisin using an LPS-induced septic cardiomyopathy model. Our results demonstrated that irisin treatment attenuated LPS-mediated cardiomyocyte death and myocardial dysfunction. At the molecular level, LPS application was associated with mitochondrial oxidative injury, cardiomyocyte ATP depletion and caspase-related apoptosis activation. In contrast, the irisin treatment sustained mitochondrial function by inhibiting DRP1-related mitochondrial fission and the reactivation of mitochondrial fission impaired the protective action of irisin on inflammation-attacked mitochondria and cardiomyocytes. Additionally, we found that irisin modulated DRP1-related mitochondrial fission through the JNK-LATS2 signaling pathway. JNK activation and/or LATS2 overexpression abolished the beneficial effects of irisin on LPS-mediated mitochondrial stress and cardiomyocyte death. Altogether, our results illustrate that LPS-mediated activation of DRP1-related mitochondrial fission through the JNK-LATS2 pathway participates in the pathogenesis of septic cardiomyopathy. Irisin could be used in the future as an effective therapy for sepsis-induced myocardial depression because it corrects DRP1-related mitochondrial fission and normalizes the JNK-LATS2 signaling pathway.

The pathological form of amyloid beta (Aβ) peptide is shown to be toxic to the mitochondria and implicates this organelle in the progression and pathogenesis of Alzheimer's disease (AD). Mitochondria are dynamic structures constantly undergoing fission and fusion, and altering their shape and size while traveling through neurons. Mitochondrial fission (Drp1, Fis1) and fusion (OPA1, Mfn1, and Mfn2) proteins are balanced in healthy neuronal cells. Glia maturation factor (GMF), a neuroinflammatory protein isolated and cloned in our laboratory plays an important role in the pathogenesis of AD. We hypothesized that GMF, a brain-localized inflammatory protein, promotes oxidative stress-mediated disruption of mitochondrial dynamics by alterations in mitochondrial fission and fusion proteins which eventually leads to apoptosis in the Aβ (1-42)-treated human neuroblastoma (SH-SY5Y) cells. The SH-SY5Y cells were incubated with GMF and Aβ (1-42) peptide, and mitochondrial fission and fusion proteins were analyzed by immunofluorescence, western blotting, and co-immunoprecipitation. We report that SH-SY5Y cells incubated with GMF and Aβ (1-42) promote mitochondrial fragmentation, by potentiating oxidative stress, mitophagy and shifts in the Bax/Bcl2 expression and release of cytochrome-c, and eventual apoptosis. In the present study, we show that GMF and Aβ treatments significantly upregulate fission proteins and downregulate fusion proteins. The study shows that extracellular GMF is an important inflammatory mediator that mediates mitochondrial dynamics by altering the balance in fission and fusion proteins and amplifies similar effects promoted by Aβ. Upregulated GMF in the presence of Aβ could be an additional risk factor for AD, and their synergistic actions need to be explored as a potential therapeutic target to suppress the progression of AD.