Inhibition of Peroxynitrite-Induced Mitophagy Activation Attenuates Cerebral Ischemia-Reperfusion Injury

Activated autophagy/mitophagy has been intensively observed in ischemic brain, but its roles remain controversial. Peroxynitrite (ONOO^?), as a representative of reactive nitrogen species, is considered as a critical neurotoxic factor in mediating cerebral ischemia-reperfusion (I/R) injury, but its roles in autophagy/mitophagy activation remain unclear. Herein, we hypothesized that ONOO^? could induce PINK1/Parkin-mediated mitophagy activation via triggering dynamin-related protein 1 (Drp1) recruitment to damaged mitochondria, contributing to cerebral I/R injury. Firstly, we found PINK1/Parkin-mediated mitophagy activation was predominant among general autophagy, leading to rat brain injury at the reperfusion phase after cerebral ischemia. Subsequently, increased nitrotyrosine was found in the plasma of ischemic stroke patients and ischemia-reperfused rat brains, indicating the generation of ONOO^? in ischemic stroke. Moreover, in vivo animal experiments illustrated that ONOO^? was dramatically increased, accompanied with mitochondrial recruitment of Drp1, PINK1/Parkin-mediated mitophagy activation, and progressive infarct size in rat ischemic brains at the reperfusion phase. FeTMPyP, a peroxynitrite decomposition catalyst, remarkably reversed mitochondrial recruitment of Drp1, mitophagy activation, and brain injury. Intriguingly, further study revealed that ONOO^? induced tyrosine nitration of Drp1 peptide, which might contribute to mitochondrial recruitment of Drp1 for mitophagy activation. In vitro cell experiments yielded consistent results with in vivo animal experiments. Taken together, all above findings support the hypothesis that ONOO^?-induced mitophagy activation aggravates cerebral I/R injury via recruiting Drp1 to damaged mitochondria.     

Parkin‐independent mitophagy requires Drp1 and maintains the integrity of mammalian heart and brain

Mitochondrial dynamics and mitophagy have been linked to cardiovascular and neurodegenerative diseases. Here, we demonstrate that the mitochondrial division dynamin Drp1 and the Parkinson's disease‐associated E3 ubiquitin ligase parkin synergistically maintain the integrity of mitochondrial structure and function in mouse heart and brain. Mice lacking cardiac Drp1 exhibited lethal heart defects. In Drp1KO cardiomyocytes, mitochondria increased their connectivity, accumulated ubiquitinated proteins, and decreased their respiration. In contrast to the current views of the role of parkin in ubiquitination of mitochondrial proteins, mitochondrial ubiquitination was independent of parkin in Drp1KO hearts, and simultaneous loss of Drp1 and parkin worsened cardiac defects. Drp1 and parkin also play synergistic roles in neuronal mitochondrial homeostasis and survival. Mitochondrial degradation was further decreased by combination of Drp1 and parkin deficiency, compared with their single loss. Thus, the physiological importance of parkin in mitochondrial homeostasis is revealed in the absence of mitochondrial division in mammals.     

Nrf2对缺氧/复氧诱导的SH-SY5Y细胞线粒体分裂的影响

目的:通过体外模拟脑缺血再灌注损伤的细胞模型,探究核转录相关因子2(nuclear factor erythroid-2 related factor 2,Nrf2)与线粒体分裂是否存在调控关系。方法:通过三气培养箱和无糖培养基模拟氧糖剥夺/复氧(Oxygen and glucose deprivation/reperfusion,OGD/R)的条件,将OGD4 h、6h、8h和10h与R0h、6h、12h、18h、24h多个时间点组合,通过CCK8试剂盒(Cell Counting Kit-8,CCK8)检测细胞的存活率,最终以细胞凋亡明显但仍有半数存活的OGD4 h/恢复R18 h为条件建立细胞OGD/R模型;用Nrf2激动剂叔丁基对苯二酚(Tert-butylhydroquinone,t BHQ)和抑制剂鸦胆子苦醇(Brusatol,Bru)对细胞进行干预处理;蛋白免疫印迹(Western blot,WB)法检测Nrf2、动力相关蛋白1(Dynamin-related protein 1,Drp1)的表达量;制备细胞沉淀的切片,通过电镜观察细胞内线粒体的形态。结果:OGD/R+t BHQ组Nrf2蛋白的表达明显高于OGD/R组,且OGD/R+t BHQ组Drp1蛋白的表达则低于OGD/R组(P0.05),而OGD/R+Bru组Nrf2蛋白的表达低于OGD/R组,且OGD/R+Bru组Drp1蛋白的表达高于OGD/R组(P0.05);电镜观察结果显示OGD/R+t BHQ组线粒体分裂的程度和比例较OGD/R组有所减少,而在OGD/R+Bru组有增多(P0.05)。结论:Nrf2对线粒体分裂有负向调控作用,可能机制是经由Drp1蛋白发挥作用。     

Defects in Mitochondrial Fission Protein Dynamin-Related Protein 1 Are Linked to Apoptotic Resistance and Autophagy in a Lung Cancer Model

Evasion of apoptosis is implicated in almost all aspects of cancer progression, as well as treatment resistance. In this study, resistance to apoptosis was identified in tumorigenic lung epithelial (A549) cells as a consequence of defects in mitochondrial and autophagic function. Mitochondrial function is determined in part by mitochondrial morphology, a process regulated by mitochondrial dynamics whereby the joining of two mitochondria, fusion, inhibits apoptosis while fission, the division of a mitochondrion, initiates apoptosis. Mitochondrial morphology of A549 cells displayed an elongated phenotype–mimicking cells deficient in mitochondrial fission protein, Dynamin-related protein 1 (Drp1). A549 cells had impaired Drp1 mitochondrial recruitment and decreased Drp1-dependent fission. Cytochrome c release and caspase-3 and PARP cleavage were impaired both basally and with apoptotic stimuli in A549 cells. Increased mitochondrial mass was observed in A549 cells, suggesting defects in mitophagy (mitochondrial selective autophagy). A549 cells had decreased LC3-II lipidation and lysosomal inhibition suggesting defects in autophagy occur upstream of lysosomal degradation. Immunostaining indicated mitochondrial localized LC3 punctae in A549 cells increased after mitochondrial uncoupling or with a combination of mitochondrial depolarization and ectopic Drp1 expression. Increased inhibition of apoptosis in A549 cells is correlated with impeded mitochondrial fission and mitophagy. We suggest mitochondrial fission defects contribute to apoptotic resistance in A549 cells.     

Cdk1, PKCδ and calcineurin-mediated Drp1 pathway contributes to mitochondrial fission-induced cardiomyocyte death

Myocardial ischemia–reperfusion (I/R) injury is one of the leading causes of death and disability worldwide. Mitochondrial fission has been shown to be involved in cardiomyocyte death. However, molecular machinery involved in mitochondrial fission during I/R injury has not yet been completely understood. In this study we aimed to investigate molecular mechanisms of controlling activation of dynamin-related protein 1 (Drp1, a key protein in mitochondrial fission) during anoxia-reoxygenation (A/R) injury of HL1 cardiomyocytes.     

Growth Factor erv1-like Modulates Drp1 to Preserve Mitochondrial Dynamics and Function in Mouse Embryonic Stem Cells

The relationship of mitochondrial dynamics and function to pluripotency are rather poorly understood aspects of stem cell biology. Here we show that growth factor erv1-like (Gfer) is involved in preserving mouse embryonic stem cell (ESC) mitochondrial morphology and function. Knockdown (KD) of Gfer in ESCs leads to decreased pluripotency marker expression, embryoid body (EB) formation, cell survival, and loss of mitochondrial function. Mitochondria in Gfer-KD ESCs undergo excessive fragmentation and mitophagy, whereas those in ESCs overexpressing Gfer appear elongated. Levels of the mitochondrial fission GTPase dynamin-related protein 1 (Drp1) are highly elevated in Gfer-KD ESCs and decreased in Gfer-overexpressing cells. Treatment with a specific inhibitor of Drp1 rescues mitochondrial function and apoptosis, whereas expression of Drp1-dominant negative resulted in the restoration of pluripotency marker expression in Gfer-KD ESCs. Altogether, our data reveal a novel prosurvival role for Gfer in maintaining mitochondrial fission–fusion dynamics in pluripotent ESCs.     

Ablation of dynamin-related protein 1 promotes diabetes-induced synaptic injury in the hippocampus

Dynamin-related protein 1 (Drp1)-mediated mitochondrial dysfunction is associated with synaptic injury in the diabetic brain. However, the dysfunctional mitochondria by Drp1 deletion in the diabetic brain are poorly understood. Here, we investigated the effects of neuron-specific Drp1 deletion on synaptic damage and mitophagy in the hippocampus of a high-fat diet (HFD)/streptozotocin (STZ)-induced diabetic mice. HFD/STZ-induced diabetic mice exhibited metabolic disturbances and synaptic damages. Floxed Drp1 mice were crossed with Ca²⁺/calmodulin-dependent protein kinase IIα (CaMKIIα)-Cre mice, to generate neuron-specific Drp1 knockout (Drp1cKO) mice, which showed marked mitochondrial swelling and dendritic spine loss in hippocampal neurons. In particular, diabetic Drp1cKO mice exhibited an increase in dendritic spine loss and higher levels of oxidative stress and neuroinflammation compared with diabetic wild-type (WT) mice. Diabetic WT mice generally displayed increased Drp1-induced small mitochondrial morphology in hippocampal neurons, but large mitochondria were prominently observed in diabetic Drp1cKO mice. The levels of microtubule-associated protein 1 light-chain 3 and lysosomal-associated membrane protein 1 proteins were significantly increased in the hippocampus of diabetic Drp1cKO mice compared with diabetic WT mice. The inhibition of Drp1 adversely promotes synaptic injury and neurodegeneration in the diabetic brain. The findings suggest that the exploratory mechanisms behind Drp1-mediated mitochondrial dysfunction could provide a possible therapeutic target for diabetic brain complications.Subject terms: Neurodegeneration, Neurodegeneration, Diabetes complications     

S6 kinase 1 plays a key role in mitochondrial morphology and cellular energy flow

Mitochondrial morphology, which is associated with changes in metabolism, cell cycle, cell development and cell death, is tightly regulated by the balance between fusion and fission. In this study, we found that S6 kinase 1 (S6K1) contributes to mitochondrial dynamics, homeostasis and function. Mouse embryo fibroblasts lacking S6K1 (S6K1-KO MEFs) exhibited more fragmented mitochondria and a higher level of Dynamin related protein 1 (Drp1) and active Drp1 (pS616) in both whole cell extracts and mitochondrial fraction. In addition, there was no evidence for autophagy and mitophagy induction in S6K1 depleted cells. Glycolysis and mitochondrial respiratory activity was higher in S6K1-KO MEFs, whereas OxPhos ATP production was not altered. However, inhibition of Drp1 by Mdivi1 (Drp1 inhibitor) resulted in higher OxPhos ATP production and lower mitochondrial membrane potential. Taken together the depletion of S6K1 increased Drp1-mediated fission, leading to the enhancement of glycolysis. The fission form of mitochondria resulted in lower yield for OxPhos ATP production as well as in higher mitochondrial membrane potential. Thus, these results have suggested a potential role of S6K1 in energy metabolism by modulating mitochondrial respiratory capacity and mitochondrial morphology.     

Dynasore Protects Mitochondria and Improves Cardiac Lusitropy in Langendorff Perfused Mouse Heart

Background

Heart failure due to diastolic dysfunction exacts a major economic, morbidity and mortality burden in the United States. Therapeutic agents to improve diastolic dysfunction are limited. It was recently found that Dynamin related protein 1 (Drp1) mediates mitochondrial fission during ischemia/reperfusion (I/R) injury, whereas inhibition of Drp1 decreases myocardial infarct size. We hypothesized that Dynasore, a small noncompetitive dynamin GTPase inhibitor, could have beneficial effects on cardiac physiology during I/R injury.

Methods and Results

In Langendorff perfused mouse hearts subjected to I/R (30 minutes of global ischemia followed by 1 hour of reperfusion), pretreatment with 1 µM Dynasore prevented I/R induced elevation of left ventricular end diastolic pressure (LVEDP), indicating a significant and specific lusitropic effect. Dynasore also decreased cardiac troponin I efflux during reperfusion and reduced infarct size. In cultured adult mouse cardiomyocytes subjected to oxidative stress, Dynasore increased cardiomyocyte survival and viability identified by trypan blue exclusion assay and reduced cellular Adenosine triphosphate(ATP) depletion. Moreover, in cultured cells, Dynasore pretreatment protected mitochondrial fragmentation induced by oxidative stress.

Conclusion

Dynasore protects cardiac lusitropy and limits cell damage through a mechanism that maintains mitochondrial morphology and intracellular ATP in stressed cells. Mitochondrial protection through an agent such as Dynasore can have clinical benefit by positively influencing the energetics of diastolic dysfunction.     

NR4A1 Promotes Cerebral Ischemia Reperfusion Injury by Repressing Mfn2-Mediated Mitophagy and Inactivating the MAPK–ERK–CREB Signaling Pathway

Mitochondrial dysfunction has been acknowledged as the key pathogenic mechanism in cerebral ischemia–reperfusion (IR) injury. Mitophagy is the protective system used to sustain mitochondrial homeostasis. However, the upstream regulator of mitophagy in response to brain IR injury is not completely understood. Nuclear receptor subfamily 4 group A member 1 (NR4A1) has been found to be associated with mitochondrial protection in a number of diseases. The aim of our study is to explore the functional role of NR4A1 in cerebral IR injury, with a particular focus on its influence on mitophagy. Wild-type mice and NR4A1-knockout mice were used to generate cerebral IR injury in vivo. Mitochondrial function and mitophagy were detected via immunofluorescence assays and western blotting. Cellular apoptosis was determined via MTT assays, caspase-3 activity and western blotting. Our data revealed that NR4A1 was significantly increased in the reperfused brain tissues. Genetic ablation of NR4A1 reduced the cerebral infarction area and repressed neuronal apoptosis. The functional study demonstrated that NR4A1 modulated cerebral IR injury by inducing mitochondrial damage. Higher NR4A1 promoted mitochondrial potential reduction, evoked cellular oxidative stress, interrupted ATP generation, and initiated caspase-9-dependent apoptosis. Mechanistically, NR4A1 induced mitochondrial damage by disrupting Mfn2-mediated mitophagy. Knockdown of NR4A1 elevated Mfn2 expression and therefore reversed mitophagic activity, sending a prosurvival signal for mitochondria in the setting of cerebral IR injury. Further, we demonstrated that NR4A1 modulated Mfn2 expression via the MAPK–ERK–CREB signaling pathway. Blockade of the ERK pathway could abrogate the permissive effect of NR4A1 deletion on mitophagic activation, contributing to neuronal mitochondrial apoptosis. Overall, our results demonstrate that the pathogenesis of cerebral IR injury is closely associated with a drop in protective mitophagy due to increased NR4A1 through the MAPK–ERK–CREB signaling pathway.     

Mitochondrial PKC‐ε deficiency promotes I/R‐mediated myocardial injury via GSK3β‐dependent mitochondrial permeability transition pore opening

Mitochondrial fission is critically involved in cardiomyocyte apoptosis, which has been considered as one of the leading causes of ischaemia/reperfusion (I/R)‐induced myocardial injury. In our previous works, we demonstrate that aldehyde dehydrogenase‐2 (ALDH2) deficiency aggravates cardiomyocyte apoptosis and cardiac dysfunction. The aim of this study was to elucidate whether ALDH2 deficiency promotes mitochondrial injury and cardiomyocyte death in response to I/R stress and the underlying mechanism. I/R injury was induced by aortic cross‐clamping for 45 min. followed by unclamping for 24 hrs in ALDH2 knockout (ALDH2^?/?) and wild‐type (WT) mice. Then myocardial infarct size, cell apoptosis and cardiac function were examined. The protein kinase C (PKC) isoform expressions and their mitochondrial translocation, the activity of dynamin‐related protein 1 (Drp1), caspase9 and caspase3 were determined by Western blot. The effects of N‐acetylcysteine (NAC) or PKC‐δ shRNA treatment on glycogen synthase kinase‐3β (GSK‐3β) activity and mitochondrial permeability transition pore (mPTP) opening were also detected. The results showed that ALDH2^?/? mice exhibited increased myocardial infarct size and cardiomyocyte apoptosis, enhanced levels of cleaved caspase9, caspase3 and phosphorylated Drp1. Mitochondrial PKC‐ε translocation was lower in ALDH2^?/? mice than in WT mice, and PKC‐δ was the opposite. Further data showed that mitochondrial PKC isoform ratio was regulated by cellular reactive oxygen species (ROS) level, which could be reversed by NAC pre‐treatment under I/R injury. In addition, PKC‐ε inhibition caused activation of caspase9, caspase3 and Drp1Ser⁶¹⁶ in response to I/R stress. Importantly, expression of phosphorylated GSK‐3β (inactive form) was lower in ALDH2^?/? mice than in WT mice, and both were increased by NAC pre‐treatment. I/R‐induced mitochondrial translocation of GSK‐3β was inhibited by PKC‐δ shRNA or NAC pre‐treatment. In addition, mitochondrial membrane potential (?Ψ_m) was reduced in ALDH2^?/? mice after I/R, which was partly reversed by the GSK‐3β inhibitor (SB216763) or PKC‐δ shRNA. Collectively, our data provide the evidence that abnormal PKC‐ε/PKC‐δ ratio promotes the activation of Drp1 signalling, caspase cascades and GSK‐3β‐dependent mPTP opening, which results in mitochondrial injury‐triggered cardiomyocyte apoptosis and myocardial dysfuction in ALDH2^?/? mice following I/R stress.     

The combination of Panax ginseng and Angelica sinensis alleviates ischemia brain injury by suppressing NLRP3 inflammasome activation and microglial pyroptosis

BackgroundThe combination of Panax ginseng and Angelica sinensis (CPA) has been used to treat stroke for one thousand years and demonstrated clinically to have satisfied effects. However, the underlying mechanism remains unknown.PurposeWe investigate whether CPA has neuroprotective effects via suppressing Nod-like receptor protein 3 (NLRP3) inflammasome and microglial pyroptosis against ischemic injury in transient middle cerebral artery occlusion (MCAO) rats.MethodsMale rats were divided randomly into sham operated, MCAO, MCC950 (NLRP3-specific inhibitor) and CPA groups. Neurological deficits, glucose uptake, infarct size, activation of NLRP3 inflammasomes, microglial pyroptosis and related signaling pathways were detected. BV-2 microglial cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) were used in in vitro experiments.ResultsCompared with sham rats, elevated level of proinflammatory interleukin-1β (IL-1β) in plasma, neurological function deficit, reduced glucose uptake in ipsilateral hemisphere, obvious infarct size, the activation of NLRP3 inflammasomes and enhanced microglial pyroptosis were presented in MCAO rats. The administrations of MCC950 and CPA respectively reversed the results. In vitro OGD/R induced the release of lactate dehydrogenase, promoted NLRP3 inflammasomes activation and pyroptosis in BV-2 cells, which was significantly suppressed by treatment with ginsenoside Rd (Rd) and Z-ligustilide (LIG). Mechanistically, OGD/R induced high expression of dynamin-related protein 1 (Drp1) and mitochondrial fission, as well as NLRP3 inflammasomes activation and pyroptosis in BV-2 cells, which was attenuated by treatment with Rd and LIG. Moreover, the increased expression of Drp1 was validated in MCAO rats, and also abolished by MCC950 or CPA treatments.ConclusionCPA treatment attenuates cerebral injury via inhibition of NLRP3 inflammasomes activation and microglial pyroptosis after stroke, which at least partially involved in the amelioration of Drp1-mediated mitochondrial fission.     

Regulator of calcineurin 1 deletion attenuates mitochondrial dysfunction and apoptosis in acute kidney injury through JNK/Mff signaling pathway

Ischemia-reperfusion (I/R) induced acute kidney injury (AKI), characterized by excessive mitochondrial damage and cell apoptosis, remains a clinical challenge. Recent studies suggest that regulator of calcineurin 1 (RCAN1) regulates mitochondrial function in different cell types, but the underlying mechanisms require further investigation. Herein, we aim to explore whether RCAN1 involves in mitochondrial dysfunction in AKI and the exact mechanism. In present study, AKI was induced by I/R and cisplatin in RCAN1^flox/flox mice and mice with renal tubular epithelial cells (TECs)-specific deletion of RCAN1. The role of RCAN1 in hypoxia-reoxygenation (HR) and cisplatin-induced injury in human renal proximal tubule epithelial cell line HK-2 was also examined by overexpression and knockdown of RCAN1. Mitochondrial function was assessed by transmission electron microscopy, JC-1 staining, MitoSOX staining, ATP production, mitochondrial fission and mitophagy. Apoptosis was detected by TUNEL assay, Annexin V-FITC staining and Western blotting analysis of apoptosis-related proteins. It was found that protein expression of RCAN1 was markedly upregulated in I/R- or cisplatin-induced AKI mouse models, as well as in HR models in HK-2 cells. RCAN1 deficiency significantly reduced kidney damage, mitochondrial dysfunction, and cell apoptosis, whereas RCAN1 overexpression led to the opposite phenotypes. Our in-depth mechanistic exploration demonstrated that RCAN1 increases the phosphorylation of mitochondrial fission factor (Mff) by binding to downstream c-Jun N-terminal kinase (JNK), then promotes dynamin related protein 1 (Drp1) migration to mitochondria, ultimately leads to excessive mitochondrial fission of renal TECs. In conclusion, our study suggests that RCAN1 could induce mitochondrial dysfunction and apoptosis by activating the downstream JNK/Mff signaling pathway. RCAN1 may be a potential therapeutic target for conferring protection against I/R- or cisplatin-AKI. Subject terms: Pathogenesis, Medical research     

Drp1-dependent mitochondrial fission via MiD49/51 is essential for apoptotic cristae remodeling

Mitochondrial fission facilitates cytochrome c release from the intracristae space into the cytoplasm during intrinsic apoptosis, although how the mitochondrial fission factor Drp1 and its mitochondrial receptors Mff, MiD49, and MiD51 are involved in this reaction remains elusive. Here, we analyzed the functional division of these receptors with their knockout (KO) cell lines. In marked contrast to Mff-KO cells, MiD49/MiD51-KO and Drp1-KO cells completely resisted cristae remodeling and cytochrome c release during apoptosis. This phenotype in MiD49/51-KO cells, but not Drp1-KO cells, was completely abolished by treatments disrupting cristae structure such as OPA1 depletion. Unexpectedly, OPA1 oligomers generally thought to resist cytochrome c release by stabilizing the cristae structure were similarly disassembled in Drp1-KO and MiD49/51-KO cells, indicating that disassembly of OPA1 oligomers is not directly linked to cristae remodeling for cytochrome c release. Together, these results indicate that Drp1-dependent mitochondrial fission through MiD49/MiD51 regulates cristae remodeling during intrinsic apoptosis.     

Mitochondrial division ensures the survival of postmitotic neurons by suppressing oxidative damage

Mitochondria divide and fuse continuously, and the balance between these two processes regulates mitochondrial shape. Alterations in mitochondrial dynamics are associated with neurodegenerative diseases. Here we investigate the physiological and cellular functions of mitochondrial division in postmitotic neurons using in vivo and in vitro gene knockout for the mitochondrial division protein Drp1. When mouse Drp1 was deleted in postmitotic Purkinje cells in the cerebellum, mitochondrial tubules elongated due to excess fusion, became large spheres due to oxidative damage, accumulated ubiquitin and mitophagy markers, and lost respiratory function, leading to neurodegeneration. Ubiquitination of mitochondria was independent of the E3 ubiquitin ligase parkin in Purkinje cells lacking Drp1. Treatment with antioxidants rescued mitochondrial swelling and cell death in Drp1KO Purkinje cells. Moreover, hydrogen peroxide converted elongated tubules into large spheres in Drp1KO fibroblasts. Our findings suggest that mitochondrial division serves as a quality control mechanism to suppress oxidative damage and thus promote neuronal survival.     

MCU Up‐regulation contributes to myocardial ischemia‐reperfusion Injury through calpain/OPA‐1–mediated mitochondrial fusion/mitophagy Inhibition

Mitochondrial dynamic disorder is involved in myocardial ischemia/reperfusion (I/R) injury. To explore the effect of mitochondrial calcium uniporter (MCU) on mitochondrial dynamic imbalance under I/R and its related signal pathways, a mouse myocardial I/R model and hypoxia/reoxygenation model of mouse cardiomyocytes were established. The expression of MCU during I/R increased and related to myocardial injury, enhancement of mitochondrial fission, inhibition of mitochondrial fusion and mitophagy. Suppressing MCU functions by Ru360 during I/R could reduce myocardial infarction area and cardiomyocyte apoptosis, alleviate mitochondrial fission and restore mitochondrial fusion and mitophagy. However, spermine administration, which could enhance MCU function, deteriorated the above‐mentioned myocardial cell injury and mitochondrial dynamic imbalanced. In addition, up‐regulation of MCU promoted the expression and activation of calpain‐1/2 and down‐regulated the expression of Optic atrophy type 1 (OPA1). Meantime, in transgenic mice (overexpression calpastatin, the endogenous inhibitor of calpain) I/R model and OPA1 knock‐down cultured cell. In I/R models of transgenic mice over‐expressing calpastatin, which is the endogenous inhibitor of calpain, and in H/R models with siOPA1 transfection, inhibition of calpains could enhance mitochondrial fusion and mitophagy, and inhibit excessive mitochondrion fission and apoptosis through OPA1. Therefore, we conclude that during I/R, MCU up‐regulation induces calpain activation, which down‐regulates OPA1, consequently leading to mitochondrial dynamic imbalance.     

Mitochondrial morphology and activity regulate furrow ingression and contractile ring dynamics in Drosophila cellularization

Mitochondria are maternally inherited in many organisms. Mitochondrial morphology and activity regulation is essential for cell survival, differentiation, and migration. An analysis of mitochondrial dynamics and function in morphogenetic events in early metazoan embryogenesis has not been carried out. In our study we find a crucial role of mitochondrial morphology regulation in cell formation in Drosophila embryogenesis. We find that mitochondria are small and fragmented and translocate apically on microtubules and distribute progressively along the cell length during cellularization. Embryos mutant for the mitochondrial fission protein, Drp1 (dynamin-related protein 1), die in embryogenesis and show an accumulation of clustered mitochondria on the basal side in cellularization. Additionally, Drp1 mutant embryos contain lower levels of reactive oxygen species (ROS). ROS depletion was previously shown to decrease myosin II activity. Drp1 loss also leads to myosin II depletion at the membrane furrow, thereby resulting in decreased cell height and larger contractile ring area in cellularization similar to that in myosin II mutants. The mitochondrial morphology and cellularization defects in Drp1 mutants are suppressed by reducing mitochondrial fusion and increasing cytoplasmic ROS in superoxide dismutase mutants. Our data show a key role for mitochondrial morphology and activity in supporting the morphogenetic events that drive cellularization in Drosophila embryos.     

Parkin and Mitofusins Reciprocally Regulate Mitophagy and Mitochondrial Spheroid Formation

Mitochondrial homeostasis via mitochondrial dynamics and quality control is crucial to normal cellular functions. Mitophagy (mitochondria removed by autophagy) stimulated by a mitochondrial uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP), requires Parkin, but it is not clear why Parkin is crucial to this process. We found that in the absence of Parkin, carbonyl cyanide m-chlorophenylhydrazone induced the formation of mitochondrial spheroids. Mitochondrial spheroid formation is also induced in vivo in the liver by acetaminophen overdose, a condition causing severe oxidative mitochondrial damages and liver injury. Mitochondrial spheroids could undergo a maturation process by interactions with acidic compartments. The formation of this new structure required reactive oxygen species and mitofusins. Parkin suppressed these mitochondrial dynamics by promoting mitofusin degradation. Consistently, genetic deletion of mitofusins without concomitant expression of Parkin was sufficient to prevent mitochondrial spheroid formation and resumed mitophagy. Mitochondrial spheroid formation and mitophagy could represent different strategies of mitochondrial homeostatic response to oxidative stress and are reciprocally regulated by mitofusins and Parkin.     

Inhibition of Drp1 provides neuroprotection in vitro and in vivo

Impaired regulation of mitochondrial dynamics, which shifts the balance towards fission, is associated with neuronal death in age-related neurodegenerative diseases, such as Alzheimer's disease or Parkinson's disease. A role for mitochondrial dynamics in acute brain injury, however, has not been elucidated to date. Here, we investigated the role of dynamin-related protein 1 (Drp1), one of the key regulators of mitochondrial fission, in neuronal cell death induced by glutamate toxicity or oxygen-glucose deprivation (OGD) in vitro, and after ischemic brain damage in vivo. Drp1 siRNA and small molecule inhibitors of Drp1 prevented mitochondrial fission, loss of mitochondrial membrane potential (MMP), and cell death induced by glutamate or tBid overexpression in immortalized hippocampal HT-22 neuronal cells. Further, Drp1 inhibitors protected primary neurons against glutamate excitotoxicity and OGD, and reduced the infarct volume in a mouse model of transient focal ischemia. Our data indicate that Drp1 translocation and associated mitochondrial fission are key features preceding the loss of MMP and neuronal cell death. Thus, inhibition of Drp1 is proposed as an efficient strategy of neuroprotection against glutamate toxicity and OGD in vitro and ischemic brain damage in vivo.