The therapeutic effect of hesperetin on doxorubicin-induced testicular toxicity: Potential roles of the mechanistic target of rapamycin kinase (mTOR) and dynamin-related protein 1 (DRP1).

Clinical utilization of doxorubicin (DOX), which is a commonly used chemotherapeutic, is restricted due to toxic effects on various tissues. Using hesperetin (HST), an antioxidant used in Chinese traditional medicine protects testis against DOX-induced toxicity although the molecular mechanisms are not well-known. The study was aimed to examine the possible role of the mechanistic target of rapamycin kinase (mTOR) and dynamin 1-like dynamin-related protein 1 (DRP1) in the therapeutic effects of HST on the DOX-induced testicular toxicity. Rats were divided into Control, DOX, DOX + HST, and HST groups (n = 7). Single-dose DOX (15 mg/kg) was administered intraperitoneally and HST (50 mg/kg) was administered by oral gavage every other day for 28 days. Total antioxidant status (TAS), histopathological evaluations, immunohistochemistry, and gene expression level detection analyses were performed. Histopathologically, DOX-induced testicular damage was ameliorated by HST treatment. DOX reduced testicular TAS levels and increased oxidative stress markers, 8-Hydroxy-deoxyguanosine (8-OHdG), and 4-Hydroxynonenal (4-HNE). Also, upregulated mTOR and DRP1 expressions with DOX exposure were decreased after HST treatment in the testis (p < 0.05). On the other hand, DOX-administration downregulated miR-150-5p and miR-181b-2-3p miRNAs, targeting mTOR and mRNA levels of beclin 1 (BECN1) and autophagy-related 5 (ATG5), autophagic markers. Furthermore, these levels were nearly similar to control testis samples in the DOX + HST group (p < 0.05). The study demonstrated that HST may have a therapeutic effect on DOX-induced testicular toxicity by removing reactive oxygen species (ROS) and by modulating the mTOR and DRP1 expressions, which have a critical role in regulating the balance of generation/elimination of ROS.

A Novel Role of Claudin-5 in Prevention of Mitochondrial Fission Against Ischemic/Hypoxic Stress in Cardiomyocytes.

BACKGROUND: Downregulation of claudin-5 in the heart is associated with the end-stage heart failure. However, the underlying mechanism ofclaudin-5 is unclear. Here we investigated the molecular actions of claudin-5 in perspective of mitochondria in cardiomyocytes to better understand the role of claudin-5 in cardioprotection during ischemia. METHODS: Myocardial ischemia/reperfusion (I/R; 30 min/24 h) and hypoxia/reoxygenation (H/R; 24 h/4 h) were used in this study. Confocal microscopy and transmission electron microscope (TEM) were used to observe mitochondrial morphology. RESULTS: Claudin-5 was detected in murine heart tissue and neonatal rat cardiomyocytes (NRCM). Its protein level was severely decreased after myocardial I/R or H/R. Confocal microscopy showedclaudin-5 presented in the mitochondria of NRCM. H/R-induced claudin-5 downregulation was accompanied by mitochondrial fragmentation. The mitofusin 2 (Mfn2) expressionwas dramatically decreased while the dynamin-related protein (Drp) 1 expression was significantly increased after H/R. The TEM indicatedH/R-induced mitochondrial swelling and fission. Adenoviral claudin-5 overexpression reversed these structural disintegration of mitochondria. The mitochondria-centered intrinsic pathway of apoptosis triggered by H/R and indicated by the cytochrome c and cleaved caspase 3 in the cytoplasm of NRCMs was also reduced by overexpressing claudin-5. Claudin-5 overexpression in mouse heart also significantly decreased cleaved caspase 3 and the infarct size in ischemic heart with improved systolic function. CONCLUSION: We demonstrated for the first time the presence of claudin-5 in the mitochondria in cardiomyocytes and provided the firm evidence for the cardioprotective role of claudin-5 in the preservation of mitochondrial dynamics and cell fate against hypoxia- or ischemia-induced stress.

Mitochondrial dysfunction underlying sporadic inclusion body myositis is ameliorated by the mitochondrial homing drug MA-5.

Sporadic inclusion body myositis (sIBM) is the most common idiopathic inflammatory myopathy, and several reports have suggested that mitochondrial abnormalities are involved in its etiology. We recruited 9 sIBM patients and found significant histological changes and an elevation of growth differential factor 15 (GDF15), a marker of mitochondrial disease, strongly suggesting the involvement of mitochondrial dysfunction. Bioenergetic analysis of sIBM patient myoblasts revealed impaired mitochondrial function. Decreased ATP production, reduced mitochondrial size and reduced mitochondrial dynamics were also observed in sIBM myoblasts. Cell vulnerability to oxidative stress also suggested the existence of mitochondrial dysfunction. Mitochonic acid-5 (MA-5) increased the cellular ATP level, reduced mitochondrial ROS, and provided protection against sIBM myoblast death. MA-5 also improved the survival of sIBM skin fibroblasts as well as mitochondrial morphology and dynamics in these cells. The reduction in the gene expression levels of Opa1 and Drp1 was also reversed by MA-5, suggesting the modification of the fusion/fission process. These data suggest that MA-5 may provide an alternative therapeutic strategy for treating not only mitochondrial diseases but also sIBM.

Luteolin enhances TRAIL sensitivity in non-small cell lung cancer cells through increasing DR5 expression and Drp1-mediated mitochondrial fission.

Cancer cells exhibit extreme sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) over normal cells, highlighting TRAIL's potential as a novel and effective cancer drug. However, the therapeutic effect of TRAIL is limited due to drug resistance. In the present study, we sought to investigate the potential effects of luteolin as a TRAIL sensitizer in non-small cell lung cancer (NSCLC) cells. A549 and H1975 cells had low sensitivity or were resistant to TRAIL. Luteolin alone or in combination with TRAIL decreased cell viability and increased apoptosis. Furthermore, luteolin alone or in combination with TRAIL enhanced death receptor 5 (DR5) expression and dynamin-related protein 1 (Drp1)-dependent mitochondrial fission. However, the synergistic effect of luteolin on cell viability and apoptosis was reversed by DR5 and Drp1 inhibition, suggesting that DR5 upregulation and mitochondrial dynamics may be essential for luteolin as a sensitizer of TRAIL-based therapy in NSCLC. Moreover, luteolin treatment alone or in combination with TRAIL increased the phosphorylation of c-Jun N-terminal kinase (JNK), while SP600125 (the JNK inhibitor) significantly abolished the synergistic effect on DR5 expression and Drp1 translocation, indicating that JNK signaling activation was greatly associated with the synergistic effect exerted by luteolin in NSCLC cells. Therefore, TRAIL combined with luteolin could be as an effective chemotherapeutic strategy for NSCLC.

Higher Levels of Dynamin-related Protein 1 are Associated with Reduced Radiation Sensitivity of Glioblastoma Cells.

BACKGROUND: Dynamin-related protein 1 (DRP1) is a GTPase involved in mitochondrial fission, mitochondrial protein import, and drug sensitivity, suggesting an association with cancer progression. This study was conducted to evaluate the prognostic significance of DRP1 in glioblastoma multiforme (GBM). METHODS: DRP1 expression was measured by immunohistochemistry and Western blotting. Correlations between DRP1 expression and clinicopathological parameters were determined by statistical analysis. Differences in survival were compared using the log-rank test. DRP1 expression was detected in 87.2% (41/47) of the investigated patients with GBM. RESULTS: The patients with higher DRP1 levels had worse survival (p = 0.0398). In vitro, the silencing of DRP1 reduced cell proliferation, invasive potential, and radiation resistance. The addition of shikonin inhibited DRP1 expression and increased drug uptake. Moreover, shikonin reduced the nuclear entry of DNA repair-associated enzymes and increased radiation sensitivity, suggesting that reducing DRP1 expression could inhibit DNA repair and increase the radiation sensitivity of GBM cells. CONCLUSION: Our results indicate that DRP1 overexpression is a prospective radio-resistant phenotype in GBM. Therefore, DRP1 could be a potential target for improving the effectiveness of radiation therapy.

The Expression of Dynamin 1, 2, and 3 in Human Hepatocellular Carcinoma and Patient Prognosis.

BACKGROUND The classical dynamin family consists of dynamin 1, 2, and 3, which have different expression levels in different tissues to regulate cell membrane fission and endocytosis. Recent studies have reported increased expression of dynamins in human cancer, but their expression in hepatocellular carcinoma (HCC) remains to be determined. This study aimed to investigate the expression of dynamin 1, 2, and 3 in tissue sections of human HCC using quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry. MATERIAL AND METHODS The expression of dynamin 1, 2, and 3 were investigated in 192 cases of HCC and 14 paired samples of HCC and adjacent normal liver tissue by qRT-PCR and immunohistochemistry. The clinical significance of dynamin 1, 2, and 3 were determined by correlating their expression levels with patient clinicopathological factors and survival rates. Independent prognostic factors were determined using the Cox regression hazard model. RESULTS In tissue samples from 192 patients with HCC, the expression of dynamin 1, 2, and 3 were upregulated in 41.15%, 29.69%, and 8.33% of cases, respectively. Dynamin 1 had a significantly increased mRNA expression level in HCC compared with adjacent normal liver tissues and was significantly correlated with alpha fetoprotein (AFP) levels, T stage, and TNM stage. Only dynamin 1 expression was correlated with the reduced overall survival (OS), and was identified as an independent prognostic biomarker of human HCC. CONCLUSIONS Upregulation of dynamin 1 at the protein and mRNA level was an independent prognostic biomarker of reduced OS in patients with HCC.

Neuropeptide Y Induces Cardiomyocyte Hypertrophy via Attenuating miR-29a-3p in Neonatal Rat Cardiomyocytes.

BACKGROUND: Neuropeptide Y (NPY) has been well known to induce Cardiomyocyte Hypertrophy (CH), which is possibly caused by disruption of cardiac cell energy balance. As mitochondria is losely related to energy metabolism, in this study, we investigated the changes in mitochondrial Dynamics-related protein (Drp1) expression under the action of NPY. miRNA-29a, a endogenous noncoding small molecule RNA which is involved in many cardiac diseases, by using a bioinformatics tool, we found a potential binding site of miRNA-29a on the Drp1 mRNA, and suggesting that miRNA-29a might play a regulatory role. OBJECTIVE: To investigate the role of miR-29a-3p in the process of NPY-induced CH, and further explore it's predicted relationship with Drp1. METHODS: The expression levels of miR-29a-3p and Atrial Natriuretic Peptide (ANP) were performed by the method of fluorescence quantitative PCR, in addition, expression of Drp1 in treated and control groups were performed by western blot analysis.] Results: We found NPY leads to the CH and up-regulation of ANP expression levels. We also found significant up-regulation of Drp1 expression and down-regulation of miR-29a-3p expression in NPY-treated cells. The decrease in miR-29a-3p expression may lead the increase expression level of Drp1. We found that the expression of ANP increased after NPY treatment. When Drp1 protein was silenced, the high expression of ANP was inhibited. CONCLUSION: In this study, we found up-regulation of Drp1 in cells treated with NPY. Drp1 mRNA is a predicted target for miR-29a-3p, and the expression of Drp1 was attenuated by miR-29a-3p. Therefore, NPY leads to down-regulation of miR-29a-3p expression, up-regulation of Drp1 expression, and NPY leads to CH. Correspondingly, miR-29a-3p can counteract the effects of NPY. This may be a new way, which could be used in diagnosis and treatment plan for CH.

Androgen-induced expression of DRP1 regulates mitochondrial metabolic reprogramming in prostate cancer.

Androgen receptor (AR) signaling plays a central role in metabolic reprogramming for prostate cancer (PCa) growth and progression. Mitochondria are metabolic powerhouses of the cell and support several hallmarks of cancer. However, the molecular links between AR signaling and the mitochondria that support the metabolic demands of PCa cells are poorly understood. Here, we demonstrate increased levels of dynamin-related protein 1 (DRP1), a mitochondrial fission mediator, in androgen-sensitive and castration-resistant AR-driven PCa. AR signaling upregulates DRP1 to form the VDAC-MPC2 complex, increases pyruvate transport into mitochondria, and supports mitochondrial metabolism, including oxidative phosphorylation and lipogenesis. DRP1 inhibition activates the cellular metabolic stress response, which involves AMPK phosphorylation, induction of autophagy, and the ER unfolded protein response, and attenuates androgen-induced proliferation. Additionally, DRP1 expression facilitates PCa cell survival under diverse metabolic stress conditions, including hypoxia and oxidative stress. Moreover, we found that increased DRP1 expression was indicative of poor prognosis in patients with castration-resistant PCa. Collectively, our findings link androgen signaling-mediated mitochondrial dynamics to metabolic reprogramming; moreover, they have important implications for understanding PCa progression.

Inhibitor 1 of Protein Phosphatase 1 Regulates Ca2+/Calmodulin-Dependent Protein Kinase II to Alleviate Oxidative Stress in Hypoxia-Reoxygenation Injury of Cardiomyocytes.

Ca2+/calmodulin-dependent protein kinase II (CaMKII), regulated by inhibitor 1 of protein phosphatase 1 (I1PP1), is vital for maintaining cardiovascular homeostasis. However, the role and mechanism of I1PP1 against hypoxia-reoxygenation (H/R) injury in cardiomyocytes remain a question. In our study, after I1PP1 overexpression by adenovirus infection in the neonatal cardiomyocytes followed by hypoxia for 4 h and reoxygenation for 12 h, the CaMKIIδ alternative splicing subtype, ATP content, and lactate dehydrogenase (LDH) release were determined. CaMKII activity was evaluated by phosphoprotein phosphorylation at Thr17 (p-PLB Thr17), CaMKII phosphorylation (p-CaMKII), and CaMKII oxidation (ox-CaMKII). Reactive oxygen species (ROS), mitochondrial membrane potential, dynamin-related protein 1 (DRP1), and optic atrophy 1 (OPA1) expressions were assessed. Our study verified that I1PP1 overexpression attenuated the CaMKIIδ alternative splicing disorder; suppressed PLB phosphorylation at Thr17, p-CaMKII, and ox-CaMKII; decreased cell LDH release; increased ATP content; attenuated ROS production; increased mitochondrial membrane potential; and decreased DRP1 expression but increased OPA1 expression in the cardiomyocytes after H/R. Contrarily, CaMKIIδ alternative splicing disorder, LDH release, ATP reduction, and ROS accumulation were aggravated after H/R injury with the I1PP1 knockdown. Collectively, I1PP1 overexpression corrected disorders of CaMKIIδ alternative splicing, inhibited CaMKII phosphorylation, repressed CaMKII oxidation, suppressed ROS production, and attenuated cardiomyocyte H/R injury.

Zinc accumulation in mitochondria promotes ischemia-induced BBB disruption through Drp1-dependent mitochondria fission.

High concentration of zinc has been reported to act as a critical mediator of neuronal death in the ischemic brain. Our previous studies showed that labile zinc accumulates in cerebromicrovessels and contributes to blood-brain barrier (BBB) permeability increase after cerebral ischemia. However, the role of mitochondrial zinc in ischemia-induced BBB permeability alteration is still unclear. In this study, we showed that ischemia/reperfusion induced free zinc accumulation in endothelial cells (ECs), resulting in increased generation of reactive oxygen species (ROS) in both cultured ECs and in microvessels isolated from the brain of ischemic rats. Furthermore, we found that zinc was highly accumulated in mitochondria, leading to mitochondrial ROS generation under the ischemic condition. Moreover, zinc overload in mitochondria resulted in the collapse of the network of mitochondria, which was mediated through Dynamin-related protein-1 (Drp-1) dependent mitochondrial fission pathway. Finally, the zinc overload in mitochondria activated matrix metalloproteinase-2 and led to ischemia-induced BBB permeability increase. This study demonstrated that zinc-ROS pathway in mitochondria contributes to the ischemia-induced BBB disruption via Drp-1 dependent mitochondrial fission pathway.

Acute spinal cord injury is associated with mitochondrial dysfunction in mouse urothelium.

AIM: To characterize the effects of acute spinal cord injury (SCI) on mitochondrial morphology and function in bladder urothelium and to test the therapeutic efficacy of early treatment with the mitochondrially targeted antioxidant, MitoTempo. METHODS: We used a mouse model of acute SCI by spinal cord transection between the T8-T9 vertebrae with or without MitoTempo delivery at the time of injury followed by tissue processing at 3 days after SCI. Control, SCI, and SCI-MitoTempo-treated mice were compared in all experimental conditions. Assessments included analysis of markers of mitochondrial health including accumulation of reactive oxygen species (ROS), morphological changes in the ultrastructure of mitochondria by transmission electron microscopy, and Western blot analysis to quantify protein levels of markers for autophagy and altered mitochondrial dynamics. RESULTS: SCI resulted in an increase in oxidative stress markers and ROS production, confirming mitochondrial dysfunction. Mitochondria from SCI mice developed large electron-dense inclusions and these aberrant mitochondria accumulated throughout the cytoplasm suggesting an inability to clear dysfunctional mitochondria by mitophagy. SCI mice also exhibited elevated levels of dynamin-related protein 1 (DRP1), consistent with a disruption of mitochondrial dynamics. Remarkably, treatment with MitoTempo reversed many of the SCI-induced abnormalities that we observed. CONCLUSIONS: Acute SCI negatively and severely affects mitochondrial health of bladder urothelium. Early treatment of SCI with MitoTempo may be a viable therapeutic agent to mitigate these deleterious effects.

Atorvastatin exerts inhibitory effect on endothelial senescence in hyperlipidemic rats through a mechanism involving down-regulation of miR-21-5p/203a-3p.

Statins are reported to exert benefits on endothelial function through a mechanism involving in prevention of endothelial senescence. This study aims to explore whether atorvastatin exerts inhibitory effect on endothelial senescence in hyperlipidemic rats or ox-LDL-treated HUVECs through a mechanism involving suppress of miR-21-5p/203a-3p expression and their downstream pathway. The rats were fed with high-fat diet to establish a hyperlipidemic model, which showed an increase in plasma lipids and endothelial senescence, accompanied by the elevation in plasma levels of miR-21-5p/203a-3p, down-regulation of Drp1 and up-regulation of p53 in the aorta of hyperlipidemic rats; these phenomena were reversed by atorvastatin. Next, HUVECs were incubated with ox-LDL to establish a senescent model in vitro. Consistent with the finding in vivo, atorvastatin treatment decreased the level of miR-21-5p and miR-203a-3p in the ox-LDL-treated HUVECs, restored Drp1 expression and mitochondrial function, as well as suppressed p53 and p16 expression and endothelial senescence. Based on these observations, we conclude that atorvastatin exerts inhibitory effect on endothelial senescence in hyperlipidemic rats through a mechanism involving down-regulation of miR-21-5p/203a-3p, which leads to the restoration of Drp1 level and recovery of mitochondrial function. Our findings highlight a novel non-lipid effect for atorvastatin besides its function in modulation of lipids.

miR-21-5p/203a-3p promote ox-LDL-induced endothelial cell senescence through down-regulation of mitochondrial fission protein Drp1.

This study aims to identify both endothelia-specific/enriched and senescence-associated miRNAs as well as their functions. The rats were fed on high-fat diet to establish a hyperlipidemic model, which showed an increase in plasma lipids and acceleration in endothelial senescence and endothelial dysfunction, accompanied by alterations in 7 endothelia-specific/enriched and senescence-associated miRNAs. Among the 7 selected miRNAs, miR-21-5p and miR-203a-3p were significantly up-regulated in a human umbilical vein endothelial cells (HUVECs) senescent model induced by ox-LDL, consistent with their changes in the hyperlipidemic rats. After performing the bioinformatic analysis, dynamin-related protein 1 (Drp1) was predicted to be a potential target for both miR-21-5p and miR-203a-3p. In ox-LDL-induced senescent HUVECs, Drp1 was significantly down-regulated, concomitant with mitochondrial dysfunctions and the activation of AMPK-p53/p16 pathway, while these phenomena were attenuated by miR-21-5p or miR-203a-3p inhibitor. Luciferase reporter gene assay confirmed a direct interaction between miR-21-5p and Drp1 but not between miR-203a-3p and Drp1. Based on these observations, we conclude that miR-21-5p/203a-3p promote ox-LDL-induced endothelial senescence through down-regulation of Drp1 in a direct or indirect way. Our findings highlight the plasma levels of miR-21-5p/203a-3p may serve as novel biomarkers to evaluate the degree of endothelial senescence in hyperlipidemia.

Mitochondrial phosphatase PGAM5 regulates Keap1-mediated Bcl-xL degradation and controls cardiomyocyte apoptosis driven by myocardial ischemia/reperfusion injury.

Phosphoglycerate mutase 5 (PGAM5) is a mitochondrial membrane protein that plays crucial roles in necroptosis and apoptosis. Though PGAM5 is known to be required for inducing intrinsic apoptosis through interacting with BCL2 associated X protein (Bax) and dynamin-related protein 1 (Drp1), the expression and role of PGAM5 in cardiomyocyte apoptosis driven by myocardial ischemia/reperfusion injury(MIRI) has not been studied. The present study shows that PGAM5 expression decreased after MIRI in vivo, positively correlated with Bcl-xL expression, negatively correlated with Kelch-ECH associating protein 1 (Keap1) expression. Furthermore, PGAM5 expression also decreased in cardiomyocytes after hypoxia/reoxygenation (H/R) treatment in vitro. PGAM5 silence promoted cardiomyocyte apoptosis and inhibited Bcl-xL expression, but with no effect on Keap1 expression. Accordingly, Keap1 overexpression further inhibited Bcl-xL and PGAM5 expression. Additionally, PGAM5-Bcl-xL-Keap1 interaction was identified, suggesting that PGAM5 might participate in the degradation of Bcl-xL mediated by Keap1. In summary, PGAM5 controls cardiomyocyte apoptosis induced by MIRI through regulating Keap1-mediated Bcl-xL degradation, which may supply a novel molecular target for acute myocardial infarction (AMI) therapy. Graphical abstract ᅟ.

Curcumin prevents mitochondrial dynamics disturbances in early 5/6 nephrectomy: Relation to oxidative stress and mitochondrial bioenergetics.

Five-sixths nephrectomy (5/6NX) is a widely used model to study the mechanisms leading to renal damage in chronic kidney disease (CKD). However, early alterations on renal function, mitochondrial dynamics, and oxidative stress have not been explored yet. Curcumin is an antioxidant that has shown nephroprotection in 5/6NX-induced renal damage. The aim of this study was to explore the effect of curcumin on early mitochondrial alterations induced by 5/6NX in rats. In isolated mitochondria, 5/6NX-induced hydrogen peroxide production was associated with decreased activity of complexes I and V, decreased activity of antioxidant enzymes, alterations in oxygen consumption and increased MDA-protein adducts. In addition, it was found that 5/6NX shifted mitochondrial dynamics to fusion, which was evidenced by increased optic atrophy 1 and mitofusin 1 (Mfn1) and decreased fission 1 and dynamin-related protein 1 expressions. These data were confirmed by morphological analysis and immunoelectron microscopy of Mfn-1. All the above-described mechanisms were prevented by curcumin. Also, it was found that curcumin prevented renal dysfunction by improving renal blood flow and the total antioxidant capacity induced by 5/6NX. Moreover, in glomeruli and proximal tubules 5/6NX-induced superoxide anion production by uncoupled nitric oxide synthase (NOS) and nicotinamide adenine dinucleotide phosphate oxidase (NOX) dependent way, this latter was associated with increased phosphorylation of serine 304 of p47phox subunit of NOX. In conclusion, this study shows that curcumin pretreatment decreases early 5/6NX-induced altered mitochondrial dynamics, bioenergetics, and oxidative stress, which may be associated with the preservation of renal function. © 2016 BioFactors, 43(2):293-310, 2017.

Peroxisome proliferator-activated receptor-gamma dependent pathway reduces the phosphorylation of dynamin-related protein 1 and ameliorates hippocampal injury induced by global ischemia in rats.

BACKGROUND: Dynamin-related protein 1 (Drp1) is a mitochondrial fission protein that, upon phosphorylation at serine 616 (p-Drp1(Ser616)), plays a pivotal role in neuronal death after ischemia. In the present study, we hypothesized that peroxisome proliferator-activated receptor-gamma (PPARγ)-dependent pathway can reduce the expression of p-Drp1(Ser616) and ameliorate hippocampal injury induced by global ischemia in rats. RESULTS: We found that pretreatment of the rats with Mdivi-1, a selective Drp1 inhibitor, decreased the level of transient global ischemia (TGI)-induced p-Drp1(Ser616) and reduced cellular contents of oxidized proteins, activated caspase-3 expression as well as the extent of DNA fragmentation. Delivery of siRNA against Drp1 attenuated the expression of p-Drp1(Ser616) that was accompanied by alleviation of the TGI-induced protein oxidation, activated caspase-3 expression and DNA fragmentation in hippocampal proteins. Exogenous application of pioglitazone, a PPARγ agonist, reduced the p-Drp1(Ser616) expression, decreased TGI-induced oxidative stress and activated caspase-3 expression, lessened the extents of DNA fragmentation, and diminished the numbers of TUNEL-positive neuronal cells; all of these effects were reversed by GW9662, a PPARγ antagonist. CONCLUSIONS: Our findings thus indicated that inhibition of TGI-induced p-Drp1(Ser616) expression by Drp1 inhibitor and Drp1-siRNA can decrease protein oxidation, activated caspase-3 expression and neuronal damage in the hippocampal CA1 subfield. PPARγ agonist, through PPARγ-dependent mechanism and via decreasing p-Drp1(Ser616) expression, can exert anti-oxidative and anti-apoptotic effects against ischemic neuronal injury.

Murine Mesenchymal Stem Cell Commitment to Differentiation Is Regulated by Mitochondrial Dynamics.

Mouse skin mesenchymal stem cells (msMSCs) are dermis CD105(+) CD90(+) CD73(+) CD29(+) CD34(-) mesodermal precursors which, after in vitro induction, undergo chondro, adipo, and osteogenesis. Extensive metabolic reconfiguration has been found to occur during differentiation, and the bioenergetic status of a cell is known to be dependent on the quality and abundance of the mitochondrial population, which may be regulated by fusion and fission. However, little is known regarding the impact of mitochondrial dynamics on the differentiation process. We addressed this knowledge gap by isolating MSCs from Swiss female mice, inducing these cells to differentiate into osteo, chondro, and adipocytes and measuring changes in mass, morphology, dynamics, and bioenergetics. Mitochondrial biogenesis was increased in adipogenesis, as evaluated through confocal microscopy, citrate synthase activity, and mtDNA content. The early steps of adipo and osteogenesis involved mitochondrial elongation, as well as increased expression of mitochondrial fusion proteins Mfn1 and 2. Chondrogenesis involved a fragmented mitochondrial phenotype, increased expression of fission proteins Drp1, Fis1, and 2, and enhanced mitophagy. These events were accompanied by profound bioenergetic alterations during the commitment period. Moreover, knockdown of Mfn2 in adipo and osteogenesis and the overexpression of a dominant negative form of Drp1 during chondrogenesis resulted in a loss of differentiation ability. Overall, we find that mitochondrial morphology and its regulating processes of fission/fusion are modulated early on during commitment, leading to alterations in the bioenergetic profile that are important for differentiation. We thus propose a central role for mitochondrial dynamics in the maintenance/commitment of mesenchymal stem cells.

Synaptic dysfunction, memory deficits and hippocampal atrophy due to ablation of mitochondrial fission in adult forebrain neurons.

Well-balanced mitochondrial fission and fusion processes are essential for nervous system development. Loss of function of the main mitochondrial fission mediator, dynamin-related protein 1 (Drp1), is lethal early during embryonic development or around birth, but the role of mitochondrial fission in adult neurons remains unclear. Here we show that inducible Drp1 ablation in neurons of the adult mouse forebrain results in progressive, neuronal subtype-specific alterations of mitochondrial morphology in the hippocampus that are marginally responsive to antioxidant treatment. Furthermore, DRP1 loss affects synaptic transmission and memory function. Although these changes culminate in hippocampal atrophy, they are not sufficient to cause neuronal cell death within 10 weeks of genetic Drp1 ablation. Collectively, our in vivo observations clarify the role of mitochondrial fission in neurons, demonstrating that Drp1 ablation in adult forebrain neurons compromises critical neuronal functions without causing overt neurodegeneration.

Defective mitochondrial fission augments NLRP3 inflammasome activation.

Despite the fact that deregulated NLRP3 inflammasome activation contributes to the pathogenesis of chronic inflammatory or metabolic disorders, the underlying mechanism by which NLRP3 inflammasome signaling is initiated or potentiated remains poorly understood. Much attention is being paid to mitochondria as a regulator of NLRP3 inflammasome activation, but little is known about the role of mitochondrial dynamics for the inflammasome pathway. Here, we present evidence that aberrant mitochondrial elongation caused by the knockdown of dynamin-related protein 1 (Drp1) lead to a marked increase in NLRP3-dependent caspase-1 activation and interleukin-1-beta secretion in mouse bone marrow-derived macrophages. Conversely, carbonyl cyanide m-chlorophenyl hydrazone, a chemical inducer of mitochondrial fission, clearly attenuated NLRP3 inflammasome assembly and activation. Augmented activation of NLRP3 inflammasome by mitochondrial elongation is not resulted from the increased mitochondrial damages of Drp1-knockdown cells. Notably, enhanced extracellular signal-regulated kinase (ERK) signaling in Drp1-knockdown macrophages is implicated in the potentiation of NLRP3 inflammasome activation, possibly via mediating mitochondrial localization of NLRP3 to facilitate the assembly of NLRP3 inflammasome. Taken together, our results provide a molecular insight into the importance of mitochondrial dynamics in potentiating NLRP3 inflammasome activation, leading to aberrant inflammation.

Pharmacological activation of AMPK prevents Drp1-mediated mitochondrial fission and alleviates endoplasmic reticulum stress-associated endothelial dysfunction.

BACKGROUND AND PURPOSE: This study aims to investigate whether and how pharmacological activation of AMP-activated protein kinase (AMPK) improves endothelial function by suppressing mitochondrial ROS-associated endoplasmic reticulum stress (ER stress) in the endothelium. Experimental approach Palmitate stimulation induced mitochondrial fission and ER stress-associated endothelial dysfunction. The effects of AMPK activators salicylate and AICA riboside (AICAR) on mitochondrial ROS production, Drp1 phosphorylation, mitochondrial fission, ER stress, thioredoxin-interacting protein (TXNIP)/NLRP3 inflammasome activation, inflammation, cell apoptosis and endothelium-dependent vasodilation were observed. Key results "Silencing" of TXNIP by RNA interference inhibited NLRP3 inflammasome activation in response to ER stress, indicating that TXNIP was a key link between ER stress and NLRP3 inflammasome activation. AMPK activators salicylate and AICAR prevented ROS-induced mitochondrial fission by enhancing dynamin-related protein 1 (Drp1) phosphorylation (Ser 637) and thereby attenuated IRE-1α and PERK phosphorylation, but their actions were blocked by knockdown of AMPK. Salicylate and AICAR reduced TXNIP induction and inhibited NLRP3 inflammasome activation by reducing NLRP3 and caspase-1 expression, leading to a reduction in IL-1ß secretion. As a result, salicylate and AICAR inhibited inflammation and reduced cell apoptosis. Meanwhile, salicylate and AICAR enhanced eNOS phosphorylation and restored the loss of endothelium-dependent vasodilation in the rat aorta. Immunohistochemistry staining showed that AMPK activation inhibited ER stress and NLRP3 inflammasome activation in the vascular endothelium. CONCLUSION AND IMPLICATIONS: Pharmacological activation of AMPK regulated mitochondrial morphology and ameliorated endothelial dysfunction by suppression of mitochondrial ROS-associated ER stress and subsequent TXNIP/NLRP3 inflammasome activation. These findings suggested that regulation of Drp1 phosphorylation by AMPK activation contributed to suppression of ER stress and thus presented a potential therapeutic strategy for AMPK activation in the regulation of endothelium homeostasis.