MAP4K4 exacerbates cardiac microvascular injury in diabetes by facilitating S-nitrosylation modification of Drp1.

Dynamin-related protein 1 (Drp1) is a crucial regulator of mitochondrial dynamics, the overactivation of which can lead to cardiovascular disease. Multiple distinct posttranscriptional modifications of Drp1 have been reported, among which S-nitrosylation was recently introduced. However, the detailed regulatory mechanism of S-nitrosylation of Drp1 (SNO-Drp1) in cardiac microvascular dysfunction in diabetes remains elusive. The present study revealed that mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) was consistently upregulated in diabetic cardiomyopathy (DCM) and promoted SNO-Drp1 in cardiac microvascular endothelial cells (CMECs), which in turn led to mitochondrial dysfunction and cardiac microvascular disorder. Further studies confirmed that MAP4K4 promoted SNO-Drp1 at human C644 (mouse C650) by inhibiting glutathione peroxidase 4 (GPX4) expression, through which MAP4K4 stimulated endothelial ferroptosis in diabetes. In contrast, inhibition of MAP4K4 via DMX-5804 significantly reduced endothelial ferroptosis, alleviated cardiac microvascular dysfunction and improved cardiac dysfunction in db/db mice by reducing SNO-Drp1. In parallel, the C650A mutation in mice abolished SNO-Drp1 and the role of Drp1 in promoting cardiac microvascular disorder and cardiac dysfunction. In conclusion, our findings demonstrate that MAP4K4 plays an important role in endothelial dysfunction in DCM and reveal that SNO-Drp1 and ferroptosis activation may act as downstream targets, representing potential therapeutic targets for DCM.

lncRNA Oip5-as1 inhibits excessive mitochondrial fission in myocardial ischemia/reperfusion injury by modulating DRP1 phosphorylation.

BACKGROUND: Aberrant mitochondrial fission, a critical pathological event underlying myocardial ischemia/reperfusion (MI/R) injury, has emerged as a potential therapeutic target. The long non-coding RNA (lncRNA) Oip5-as1 is increasingly recognized for its regulatory roles, particularly in MI/R injury. However, its precise mechanistic role in modulating mitochondrial dynamics remains elusive. This study aims to elucidate the mechanistic role of Oip5-as1 in regulating mitochondrial fission and evaluate its therapeutic potential against MI/R injury. METHODS: To simulate in vitro MI/R injury, HL-1 cardiomyocytes were subjected to hypoxia/reoxygenation (H/R). Lentiviral vectors were employed to achieve overexpression or knockdown of Oip5-as1 in HL-1 cells by expressing Oip5-as1 or shRNA targeting Oip5-as1, respectively. The impact of Oip5-as1 on mitochondrial dynamics in HL-1 cells was assessed using CCK-8 assay, flow cytometry, immunofluorescence staining, and biochemical assays. MI/R injury was induced in mice by ligating the left anterior descending coronary artery. Conditional knockout mice for Oip5-as1 were generated using the CRISPR/Cas9 genome editing technology, while overexpression of Oip5-as1 in mice was achieved via intramyocardial administration of AAV9 vectors. In mice, the role of Oip5-as1 was evaluated through echocardiographic assessment, histopathological staining, and transmission electron microscopy. Furthermore, Western blotting, RNA pull-down, RNA immunoprecipitation, and co-immunoprecipitation assays were conducted to investigate Oip5-as1's underlying mechanisms. RESULTS: The expression levels of Oip5-as1 are significantly decreased in MI/R-injured HL-1 cells and myocardium. In HL-1 cells undergoing H/R injury, overexpression of Oip5-as1 attenuated excessive mitochondrial fission, preserved mitochondrial functionality, and reduced cellular apoptosis, while knockdown of Oip5-as1 exhibited the opposite effects. Furthermore, in a mouse model of MI/R injury, overexpression of Oip5-as1 diminished mitochondrial fission, myocardial infarct size and improved cardiac function. However, knockout of Oip5-as1 exacerbated myocardial injury and cardiac dysfunction, which were significantly reversed by treatment with a mitochondrial division inhibitor-1 (Mdivi-1). Mechanistically, Oip5-as1 selectively interacts with AKAP1 and CaN proteins, inhibiting CaN activation and subsequent DRP1 dephosphorylation at Ser637, thereby constraining DRP1's translocation to the mitochondria and its involvement in mitochondrial fission. CONCLUSIONS: Our study underscores the pivotal role of Oip5-as1 in mitigating excessive mitochondrial fission during MI/R injury. The findings not only enhance our comprehension of the molecular mechanisms underlying MI/R injury but also identify Oip5-as1 as a potential therapeutic target for ameliorating MI/R injury.

The CaMK Family Differentially Promotes Necroptosis and Mouse Cardiac Graft Injury and Rejection.

Organ transplantation is associated with various forms of programmed cell death which can accelerate transplant injury and rejection. Targeting cell death in donor organs may represent a novel strategy for preventing allograft injury. We have previously demonstrated that necroptosis plays a key role in promoting transplant injury. Recently, we have found that mitochondria function is linked to necroptosis. However, it remains unknown how necroptosis signaling pathways regulate mitochondrial function during necroptosis. In this study, we investigated the receptor-interacting protein kinase 3 (RIPK3) mediated mitochondrial dysfunction and necroptosis. We demonstrate that the calmodulin-dependent protein kinase (CaMK) family members CaMK1, 2, and 4 form a complex with RIPK3 in mouse cardiac endothelial cells, to promote trans-phosphorylation during necroptosis. CaMK1 and 4 directly activated the dynamin-related protein-1 (Drp1), while CaMK2 indirectly activated Drp1 via the phosphoglycerate mutase 5 (PGAM5). The inhibition of CaMKs restored mitochondrial function and effectively prevented endothelial cell death. CaMKs inhibition inhibited activation of CaMKs and Drp1, and cell death and heart tissue injury (n = 6/group, p < 0.01) in a murine model of cardiac transplantation. Importantly, the inhibition of CaMKs greatly prolonged heart graft survival (n = 8/group, p < 0.01). In conclusion, CaMK family members orchestrate cell death in two different pathways and may be potential therapeutic targets in preventing cell death and transplant injury.

TROP2 promotes PINK1-mediated mitophagy and apoptosis to accelerate the progression of senile chronic obstructive pulmonary disease by up-regulating DRP1 expression.

Chronic obstructive pulmonary disease (COPD) is a chronic airway inflammatory disease characterised by irreversible airflow limitation. The elderly are a vulnerable population for developing COPD. With the growth of age, physiological degenerative changes occur in the thorax, bronchus, lung and vascular wall, which can lead to age-related physiological attenuation of lung function in the elderly, so the prevalence of COPD increases with age. Its pathogenesis has not yet been truly clarified. Mitophagy plays an important role in maintaining the stability of mitochondrial function and intracellular environment by scavenging damaged mitochondria. Currently, studies have shown that trophoblast antigen 2 (TROP2) expression is up-regulated in airway basal cells of patients with COPD, suggesting that TROP2 is involved in the progression of COPD. However, whether it is involved in disease progression by regulating mitochondrial function remains unclear. In this study, compared with non-smoking non-COPD patients, the expression of TROP2 in lung tissues of smoking non-COPD patients and patients with COPD increased, and TROP2 expression in patients with COPD was higher than that in smoking non-COPD patients. To further explore the role of TROP2, we stimulated BEAS-2B with cigarette smoke to construct an in vitro model. We found that TROP2 expression increased, whereas TROP2 silencing reversed the cigarette smoke extract-induced decrease in mitochondrial membrane potential, increased reactive oxygen species content, decreased adenosine triphosphate (ATP) production, increased inflammatory factor secretion and increased apoptosis. In addition, we searched online bioinformatics and screened the gene dynamin-related protein 1 (DRP1) related to mitophagy as the research object. Co-IP assay verified the binding relationship between DRP1 and TROP2. Further study found that TROP2 promoted mitophagy and apoptosis of BEAS-2B cells by up-regulating the expression of DRP1. In addition, PTEN-induced putative kinase 1 (PINK1) is a potential binding protein of DRP1, and DRP1 accelerated mitophagy and apoptosis of BEAS-2B cells by promoting the expression of PINK1. We established a COPD SD rat model by cigarette smoke exposure and LPS instillation and treated it by intraperitoneal injection of si-TROP2. The results showed that TROP2 silencing restored lung function and reduced the secretion of inflammatory factors in bronchoalveolar lavage fluid. In conclusion, TROP2 can be used as a new reference for COPD treatment.

QiDongNing induces lung cancer cell apoptosis via triggering P53/DRP1-mediated mitochondrial fission.

Non-small-cell lung cancer (NSCLC) is a major cause of worldwide cancer death, posing a challenge for effective treatment. Our previous findings showed that Chinese herbal medicine (CHM) QiDongNing (QDN) could upregulate the expression of p53 and trigger cell apoptosis in NSCLC. Here, our objective was to investigate the mechanisms of QDN-induced apoptosis enhancement. We chose A549 and NCI-H460 cells for validation in vitro, and LLC cells were applied to form a subcutaneous transplantation tumour model for validation in more depth. Our findings indicated that QDN inhibited multiple biological behaviours, including cell proliferation, cloning, migration, invasion and induction of apoptosis. We further discovered that QDN increased the pro-apoptotic BAX while inhibiting the anti-apoptotic Bcl2. QDN therapy led to a decline in adenosine triphosphate (ATP) and a rise in reactive oxygen species (ROS). Furthermore, QDN elevated the levels of the tumour suppressor p53 and the mitochondrial division factor DRP1 and FIS1, and decreased the mitochondrial fusion molecules MFN1, MFN2, and OPA1. The results were further verified by rescue experiments, the p53 inhibitor Pifithrin-α and the mitochondrial division inhibitor Mdivi1 partially inhibited QDN-induced apoptosis and mitochondrial dysfunction, whereas overexpression of p53 rather increased the efficacy of the therapy. Additionally, QDN inhibited tumour growth with acceptable safety in vivo. In conclusion, QDN induced apoptosis via triggering p53/DRP1-mediated mitochondrial fission in NSCLC cells.

Intertwined relationship of dynamin-related protein 1, mitochondrial metabolism and circadian rhythm.

In recent years, mitochondria have gained significant interest in the field of biomedical research due to their impact on human health and ageing. As mitochondrial dynamics are strongly controlled by clock genes, misalignment of the circadian rhythm leads to adverse metabolic health effects. In this review, by exploring various aspects of research and potential links, we hope to update the current understanding of the intricate relationship between DRP1-mediated mitochondrial dynamics and changes in circadian rhythmicity leading to health issues. Thus, this review addresses the potential bidirectional relationships between DRP1-linked mitochondrial function and circadian rhythm misalignment, their impact on different metabolic pathways, and the potential therapeutics for metabolic and systemic disorders.

GTPBP8 modulates mitochondrial fission through a Drp1-dependent process.

Mitochondrial fission is a tightly regulated process involving multiple proteins and cell signaling. Despite extensive studies on mitochondrial fission factors, our understanding of the regulatory mechanisms remains limited. This study shows the critical role of a mitochondrial GTPase, GTPBP8, in orchestrating mitochondrial fission in mammalian cells. Depletion of GTPBP8 resulted in drastic elongation and interconnectedness of mitochondria. Conversely, overexpression of GTPBP8 shifted mitochondrial morphology from tubular to fragmented. Notably, the induced mitochondrial fragmentation from GTPBP8 overexpression was inhibited in cells either depleted of the mitochondrial fission protein Drp1 (also known as DNM1L) or carrying mutated forms of Drp1. Importantly, downregulation of GTPBP8 caused an increase in oxidative stress, modulating cell signaling involved in the increased phosphorylation of Drp1 at Ser637. This phosphorylation hindered the recruitment of Drp1 to mitochondria, leading to mitochondrial fission defects. By contrast, GTPBP8 overexpression triggered enhanced recruitment and assembly of Drp1 at mitochondria. In summary, our study illuminates the cellular function of GTPBP8 as a pivotal modulator of the mitochondrial division apparatus, inherently reliant on its influence on Drp1.

Deoxynivalenol Inhibits Progenitor Leydig Cell Development by Stimulating Mitochondrial Fission in Rats.

Deoxynivalenol (DON) is a common food contaminant that can impair male reproductive function. This study investigated the effects and mechanisms of DON exposure on progenitor Leydig cell (PLC) development in prepubertal male rats. Rats were orally administrated DON (0-4 mg/kg) from postnatal days 21-28. DON increased PLC proliferation but inhibited PLC maturation and function, including reducing testosterone levels and downregulating biomarkers like HSD11B1 and INSL3 at ≥2 mg/kg. DON also stimulated mitochondrial fission via upregulating DRP1 and FIS1 protein levels and increased oxidative stress by reducing antioxidant capacity (including NRF2, SOD1, SOD2, and CAT) in PLCs in vivo. In vitro, DON (2-4 µM) inhibited PLC androgen biosynthesis, increased reactive oxygen species production and protein levels of DRP1, FIS1, MFF, and pAMPK, decreased mitochondrial membrane potential and MFN1 protein levels, and caused mitochondrial fragmentation. The mitochondrial fission inhibitor mdivi-1 attenuated DON-induced impairments in PLCs. DON inhibited PLC steroidogenesis, increased oxidative stress, perturbed mitochondrial homeostasis, and impaired maturation. In conclusion, DON disrupts PLC development in prepubertal rats by stimulating mitochondrial fission.

C5a-C5aR1 axis controls mitochondrial fission to promote podocyte injury in lupus nephritis.

Podocytes are essential to maintaining the integrity of the glomerular filtration barrier, but they are frequently affected in lupus nephritis (LN). Here, we show that the significant upregulation of Drp1S616 phosphorylation in podocytes promotes mitochondrial fission, leading to mitochondrial dysfunction and podocyte injury in LN. Inhibition or knockdown of Drp1 promotes mitochondrial fusion and protects podocytes from injury induced by LN serum. In vivo, pharmacological inhibition of Drp1 reduces the phosphorylation of Drp1S616 in podocytes in lupus-prone mice. Podocyte injury is reversed when Drp1 is inhibited, resulting in the alleviation of proteinuria. Mechanistically, complement component C5a (C5a) upregulates the phosphorylation of Drp1S616 and promotes mitochondrial fission in podocytes. Moreover, the expression of C5a receptor 1 (C5aR1) is notably upregulated in podocytes in LN. C5a-C5aR1 axis-controlled phosphorylation of Drp1S616 and mitochondrial fission are substantially suppressed when C5aR1 is knocked down by siRNA. Moreover, lupus-prone mice treated with C5aR inhibitor show reduced phosphorylation of Drp1S616 in podocytes, resulting in significantly less podocyte damage. Together, this study uncovers a novel mechanism by which the C5a-C5aR1 axis promotes podocyte injury by enhancing Drp1-mediated mitochondrial fission, which could have significant implications for the treatment of LN.

NME3 is a gatekeeper for DRP1-dependent mitophagy in hypoxia.

NME3 is a member of the nucleoside diphosphate kinase (NDPK) family localized on the mitochondrial outer membrane (MOM). Here, we report a role of NME3 in hypoxia-induced mitophagy dependent on its active site phosphohistidine but not the NDPK function. Mice carrying a knock-in mutation in the Nme3 gene disrupting NME3 active site histidine phosphorylation are vulnerable to ischemia/reperfusion-induced infarction and develop abnormalities in cerebellar function. Our mechanistic analysis reveals that hypoxia-induced phosphatidic acid (PA) on mitochondria is essential for mitophagy and the interaction of DRP1 with NME3. The PA binding function of MOM-localized NME3 is required for hypoxia-induced mitophagy. Further investigation demonstrates that the interaction with active NME3 prevents DRP1 susceptibility to MUL1-mediated ubiquitination, thereby allowing a sufficient amount of active DRP1 to mediate mitophagy. Furthermore, MUL1 overexpression suppresses hypoxia-induced mitophagy, which is reversed by co-expression of ubiquitin-resistant DRP1 mutant or histidine phosphorylatable NME3. Thus, the site-specific interaction with active NME3 provides DRP1 a microenvironment for stabilization to proceed the segregation process in mitophagy.

Altered Grooming Cycles in Transgenic Drosophila.

Head grooming in Drosophila consists of repeated sweeps of the legs across the head, comprising regular cycles. We used the GAL4-UAS system to study the effects of overexpressing shibirets1 and of Adar knockdown via RNA interference, on the period of head-grooming cycles in Drosophila. Overexpressing shibirets1 interferes with synaptic vesicle recycling and thus with cell communication, while Adar knockdown reduces RNA editing of neuronal transcripts for a large number of genes. All transgenic flies and their controls were tested at 22° to avoid temperature effects; in wild type, cycle frequency varied with temperature with a Q10 of 1.3. Two experiments were performed with transgenic shibirets1: (1) each fly was heat-shocked for 10 min at 30° immediately before testing at 22° and (2) flies were not heat shocked. In both experiments, cycle period was increased when shibirets1 was overexpressed in all neurons, but was not increased when shibirets1 was overexpressed in motoneurons alone. We hypothesize that grooming cycles in flies overexpressing shibirets1 are lengthened because of synaptic impairment in neural circuits that control head-grooming cycles. In flies with constitutive, pan-neuronal Adar knockdown, cycle period was more variable within individuals, but mean cycle period was not significantly altered. We conclude that RNA editing is essential for the maintenance of within-individual stereotypy of head-grooming cycles.

A partial Drp1 knockout improves autophagy flux independent of mitochondrial function.

BACKGROUND: Dynamin-related protein 1 (Drp1) plays a critical role in mitochondrial dynamics. Partial inhibition of this protein is protective in experimental models of neurological disorders such as Parkinson's disease and Alzheimer's disease. The protective mechanism has been attributed primarily to improved mitochondrial function. However, the observations that Drp1 inhibition reduces protein aggregation in such neurological disorders suggest the involvement of autophagy. To investigate this potential novel protective mechanism of Drp1 inhibition, a model with impaired autophagy without mitochondrial involvement is needed. METHODS: We characterized the effects of manganese (Mn), which causes parkinsonian-like symptoms in humans, on autophagy and mitochondria by performing dose-response studies in two cell culture models (stable autophagy HeLa reporter cells and N27 rat immortalized dopamine neuronal cells). Mitochondrial function was assessed using the Seahorse Flux Analyzer. Autophagy flux was monitored by quantifying the number of autophagosomes and autolysosomes, as well as the levels of other autophagy proteins. To strengthen the in vitro data, multiple mouse models (autophagy reporter mice and mutant Drp1+/- mice and their wild-type littermates) were orally treated with a low chronic Mn regimen that was previously reported to increase α-synuclein aggregation and transmission via exosomes. RNAseq, laser captured microdissection, immunofluorescence, immunoblotting, stereological cell counting, and behavioural studies were used. RESULTS IN VITRO: data demonstrate that at low non-toxic concentrations, Mn impaired autophagy flux but not mitochondrial function and morphology. In the mouse midbrain, RNAseq data further confirmed autophagy pathways were dysregulated but not mitochondrial related genes. Additionally, Mn selectively impaired autophagy in the nigral dopamine neurons but not the nearby nigral GABA neurons. In cells with a partial Drp1-knockdown and Drp1+/- mice, Mn induced autophagic impairment was significantly prevented. Consistent with these observations, Mn increased the levels of proteinase-K resistant α-synuclein and Drp1-knockdown protected against this pathology. CONCLUSIONS: This study demonstrates that improved autophagy flux is a separate mechanism conferred by Drp1 inhibition independent of its role in mitochondrial fission. Given that impaired autophagy and mitochondrial dysfunction are two prominent features of neurodegenerative diseases, the combined protective mechanisms targeting these two pathways conferred by Drp1 inhibition make this protein an attractive therapeutic target.

Drp1-dependent mitochondrial fragmentation mediates photoreceptor abnormalities in type 1 diabetic retina.

Recent studies have highlighted that retinal neurodegeneration precedes microvascular changes in diabetic retinopathy (DR), but the specific mechanisms remain unclear. Given the pivotal role of dysfunctional mitochondria and oxidative stress in early DR, our objective was to observe mitochondria-related alterations in the neural retina of type one diabetic mellitus mice with no evidence of DR (T1DM-NDR). We aimed to identify the key mitochondrial-related proteins contributing to mitochondrial injury. Our study revealed that T1DM-NDR mice exhibited outer retina thinning, including the ellipsoid zone, inner segment, and outer segment. Additionally, there was an impaired amplitude of the b-wave in electroretinogram (ERG) and a disorganized arrangement of the photoreceptor layer. In both the retina of DM mice and high glucose (HG)-treated 661w cells, mitochondria appeared swollen and fragmented, with disrupted cristae, disorganized or shortened branches in the mitochondrial network, and decreased mitochondrial membrane potential. Among the mitochondrial-related proteins, dynamin-related protein 1 (Drp1) was upregulated, and the ratio of phosphorylated Drp1 protein at serine 616 (S616) and serine 637 (S637) sites significantly increased in the retina of DM mice. The administration of Mdivi-1 ameliorated high-glucose-induced dysfunctional mitochondria, thereby protecting T1DM-NDR mice retina from morphological and functional injuries. Our findings suggest that hyperglycemia promotes Drp1-mediated mitochondrial dysfunction, which may be a significant factor in the development of DR. The inhibition of high-glucose-induced mitochondrial fission emerges as a potential and innovative intervention strategy for preventing DR.

ISGylation of DRP1 closely balances other post-translational modifications to mediate mitochondrial fission.

Dynamin related protein 1 (DRP1), a pivotal mitochondrial fission protein, is post-translationally modified by multiple mechanisms. Here we identify a new post-translational modification of DRP1 by the ubiquitin-like protein, interferon-stimulated gene 15 (ISG15). DRP1 ISGylation is mediated by ISG15 E3 ligase, HERC5; this promotes mitochondrial fission. DeISGylation of DRP1 however leads to hyperfusion. Heterologous expression of SARS-CoV2 PLpro, a deISGylating enzyme, results in similar mitochondrial filamentation, significant decrease in total DRP1 protein levels and efflux of mtDNA. We report that deISGylated DRP1 gets ubiquitylated and degraded by TRIM25, instead of PARKIN and MITOL. While the cytosolic pool of DRP1 is primarily ISGylated, both mitochondrial and cytosolic fractions may be ubiquitylated. It is known that phosphorylation of DRP1 at S616 residue regulates its mitochondrial localisation; we show that ISGylation of phospho-DRP1 (S616) renders fission competence at mitochondria. This is significant because DRP1 ISGylation affects its functionality and mitochondrial dynamics in Alzheimer's disease pathophysiology.

Force-induced tail-autotomy mitochondrial fission and biogenesis of matrix-excluded mitochondrial-derived vesicles for quality control.

Mitochondria constantly fuse and divide for mitochondrial inheritance and functions. Here, we identified a distinct type of naturally occurring fission, tail-autotomy fission, wherein a tail-like thin tubule protrudes from the mitochondrial body and disconnects, resembling autotomy. Next, utilizing an optogenetic mitochondria-specific mechanostimulator, we revealed that mechanical tensile force drives tail-autotomy fission. This force-induced fission involves DRP1/MFF and endoplasmic reticulum tubule wrapping. It redistributes mitochondrial DNA, producing mitochondrial fragments with or without mitochondrial DNA for different fates. Moreover, tensile force can decouple outer and inner mitochondrial membranes, pulling out matrix-excluded tubule segments. Subsequent tail-autotomy fission separates the matrix-excluded tubule segments into matrix-excluded mitochondrial-derived vesicles (MDVs) which recruit Parkin and LC3B, indicating the unique role of tail-autotomy fission in segregating only outer membrane components for mitophagy. Sustained force promotes fission and MDV biogenesis more effectively than transient one. Our results uncover a mechanistically and functionally distinct type of fission and unveil the role of tensile forces in modulating fission and MDV biogenesis for quality control, underscoring the heterogeneity of fission and mechanoregulation of mitochondrial dynamics.

Circ_0098823 binding with IGF2BP3 regulates DNM1L stability to promote metastasis of hepatocellular carcinoma via mitochondrial fission.

Hepatocellular carcinoma (HCC) is highly metastatic and invasive. CircRNA participates in gene regulation of multiple tumor metastases, but little is known whether it is a bystander or an actual player in HCC metastasis. We aim to explore the molecular mechanisms of novel circRNAs in HCC metastasis. RT-qPCR was used to detect the expression of 13 circRNAs derived by the ERBB3 gene. The function of circ_0098823 and DNM1L in HCC cells were estimated by CCK-8, transwell assays, flow cytometry, electron microscope, and in vivo experiments. RNA binding protein of circ_0098823 was confirmed by RNA pull-down, mass spectrometry, and RNA immunoprecipitation. The expression of DNM1L after IGF2BP3 knockdown was detected by RT-qPCR and western blot. Circ_0098823 was significantly up-regulated both in HCC tissues and HGF induced cell lines. Circ_0098823 overexpression significantly enhanced proliferation, migration, and invasion but decreased apoptosis of HCC cells, particularly promoted mitochondrial fission. Compared with the control group, the tumors in the circ_0098823 knockdown mice were significantly smaller and lighter. Circ_0098823 silencing suppressed DNM1L expression, a key molecule for fission, which enhanced proliferation, migration and invasion, and inhibited apoptosis of HCC cell. IGF2BP3 was a binding protein of circ_0098823. The expression and mRNA stability of DNM1L were down-regulated by IGF2BP3 knockdown. IGF2BP3 knockdown significantly alleviated the excessive migration, invasion and apoptosis of HCC cells caused by circ_0098823 overexpression. This study uncovered a novel circ_0098823 with tumor-promoting effect, and the mechanism by which circ_0098823 participates in HCC progression through IGF2BP3-guided DNM1L. Our study broadens molecular understanding of HCC progression.

Insulin induces bioenergetic changes and alters mitochondrial dynamics in podocytes.

Diabetic nephropathy (DN) is one of the most frequent complications of diabetes. Early stages of DN are associated with hyperinsulinemia and progressive insulin resistance in insulin-sensitive cells, including podocytes. The diabetic environment induces pathological changes, especially in podocyte bioenergetics, which is tightly linked with mitochondrial dynamics. The regulatory role of insulin in mitochondrial morphology in podocytes has not been fully elucidated. Therefore, the main goal of the present study was to investigate effects of insulin on the regulation of mitochondrial dynamics and bioenergetics in human podocytes. Biochemical analyses were performed to assess oxidative phosphorylation efficiency by measuring the oxygen consumption rate (OCR) and glycolysis by measuring the extracellular acidification rate (ECAR). mRNA and protein expression were determined by real-time polymerase chain reaction and Western blot. The intracellular mitochondrial network was visualized by MitoTracker staining. All calculations were conducted using CellProfiler software. Short-term insulin exposure exerted inhibitory effects on various parameters of oxidative respiration and adenosine triphosphate production, and glycolysis flux was elevated. After a longer time of treating cells with insulin, an increase in mitochondrial size was observed, accompanied by a reduction of expression of the mitochondrial fission markers DRP1 and FIS1 and an increase in mitophagy. Overall, we identified a previously unknown role for insulin in the regulation of oxidative respiration and glycolysis and elucidated mitochondrial dynamics in human podocytes. The present results emphasize the importance of the duration of insulin stimulation for its metabolic and molecular effects, which should be considered in clinical and experimental studies of DN.

Role of the dynamin-related protein 2 family and SH3P2 in clathrin-mediated endocytosis in Arabidopsis thaliana.

Clathrin-mediated endocytosis (CME) is vital for the regulation of plant growth and development through controlling plasma membrane protein composition and cargo uptake. CME relies on the precise recruitment of regulators for vesicle maturation and release. Homologues of components of mammalian vesicle scission are strong candidates to be part of the scission machinery in plants, but the precise roles of these proteins in this process are not fully understood. Here, we characterised the roles of the plant dynamin-related protein 2 (DRP2) family (hereafter DRP2s) and SH3-domain containing protein 2 (SH3P2), the plant homologue to recruiters of dynamins, such as endophilin and amphiphysin, in CME by combining high-resolution imaging of endocytic events in vivo and characterisation of the purified proteins in vitro. Although DRP2s and SH3P2 arrive similarly late during CME and physically interact, genetic analysis of the sh3p123 triple mutant and complementation assays with non-SH3P2-interacting DRP2 variants suggest that SH3P2 does not directly recruit DRP2s to the site of endocytosis. These observations imply that, despite the presence of many well-conserved endocytic components, plants have acquired a distinct mechanism for CME.

Rapamycin alleviates mitochondrial dysfunction in anti-NMDAR encephalitis mice.

Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is one of the most prevalent forms of autoimmune encephalitis, characterized by a series of neurological and psychiatric symptoms, including cognitive impairment, seizures and psychosis. The underlying mechanism of anti-NMDAR encephalitis remains unclear. In the current study, the mouse model of anti-NMDAR encephalitis with active immunization was performed. We first uncovered excessive mitochondrial fission in the hippocampus and temporal cortex of anti-NMDAR encephalitis mice, indicated by elevated level of Phospho-DRP1 (Ser616) (p-Drp1-S616). Moreover, blockade of the autophagic flux was also demonstrated, leading to the accumulation of fragmented mitochondria, and elevated levels of mitochondrial reactive oxygen species (mtROS) and mitochondrial DNA (mtDNA) in anti-NMDAR encephalitis. More importantly, we found that the mTOR signaling pathway was overactivated, which could aggravate mitochondrial fission and inhibit autophagy, resulting in mitochondrial dysfunction. While rapamycin, the specific inhibitor of the mTOR signaling pathway, significantly alleviated mitochondrial dysfunction by inhibiting mitochondrial fission and enhancing autophagy. Levels of mtROS and mtDNA were markedly reduced after the treatment of rapamycin. In addition, rapamycin also significantly alleviated cognitive dysfunction and anxious behaviors found in anti-NMDAR encephalitis mice. Thus, our study reveals the vital role of mitochondrial dysfunction in pathological mechanism of anti-NMDAR encephalitis and lays a theoretical foundation for rapamycin to become a clinically targeted drug for anti-NMDAR encephalitis.