[The off-target effects of dynamin inhibitor Dyngo-4a during the internalization of dextran by bone marrow mesenchymal stem cells in rat].

Objective To investigate the possible off-target effects of dynamin (DNM) inhibitor Dyngo-4a in dynamin-dependent endocytic pathways. Methods Bone marrow mesenchymal stem cells (BMSCs) obtained from SD rats were isolated and cultured, and identified by flow cytometry. The cells were divided into inhibitor control group, Dyngo-4a-treated group, negative control siRNA (si-NC) transfection group, DNM2 siRNA transfection (si-DNM2) group, si-DNM2 and Dyngo-4a co-treated group. Real time quantitative PCR and Western blot analysis were used to verify the silencing efficiencies of DNM2 gene and CCK-8 assay were used to detect the cell viability after Dyngo-4a treatment. Confocal microscopy was used to detect the number and mean fluorescence intensity (MFI) of transferrin-Dylight649-positive and dextran-TMR-positive vesicles. Results The mRNA and protein expression levels of DNM2 were down-regulated using small interfering RNA. The number of transferrin-Dylight649-positive vesicles significantly decreased in si-DNM2 group compared with si-NC group. For the number and MFI of dextran-TMR-positive vesicles, no significant change was observed between the si-DNM2 group and the si-NC group, but there was a significant reduction in the si-DNM2 and Dyngo-4a co-treated group compared with the si-DNM2 group. A significant decrease was also found in the Dyngo-4a-treated group compared with the inhibitor control group. Conclusion The off-target effects of dynamin inhibitor Dyngo-4a presents in the internalization of dextran by BMSCs.

A novel small molecule inhibitor of human Drp1.

Mitochondrial dynamin-related protein 1 (Drp1) is a large GTPase regulator of mitochondrial dynamics and is known to play an important role in numerous pathophysiological processes. Despite being the most widely used Drp1 inhibitor, the specificity of Mdivi-1 towards human Drp1 has not been definitively proven and there have been numerous issues reported with its use including off-target effects. In our hands Mdivi-1 showed varying binding affinities toward human Drp1, potentially impacted by compound aggregation. Herein, we sought to identify a novel small molecule inhibitor of Drp1. From an initial virtual screening, we identified DRP1i27 as a compound which directly bound to the human isoform 3 of Drp1 via surface plasmon resonance and microscale thermophoresis. Importantly, DRP1i27 was found to have a dose-dependent increase in the cellular networks of fused mitochondria but had no effect in Drp1 knock-out cells. Further analogues of this compound were identified and screened, though none displayed greater affinity to human Drp1 isoform 3 than DRP1i27. To date, this is the first small molecule inhibitor shown to directly bind to human Drp1.

Inhibition of Drp1 ameliorates diabetic retinopathy by regulating mitochondrial homeostasis.

Diabetic retinopathy (DR) is a potentially blinding complication resulting from diabetes mellitus (DM). Retinal vascular endothelial cells (RMECs) dysfunction occupies an important position in the pathogenesis of DR, and mitochondrial disorders play a vital role in RMECs dysfunction. However, the detailed mechanisms underlying DR-induced mitochondrial disorders in RMECs remain elusive. In the present study, we used High glucose (HG)-induced RMECs in vitro and streptozotocin (STZ)-induced Sprague-Dawley rats in vivo to explore the related mechanisms. We found that HG-induced mitochondrial dysfunction via mitochondrial Dynamin-related protein 1(Drp1)-mediated mitochondrial fission. Drp1 inhibitor, Mdivi-1, rescued HG-induced mitochondrial dysfunction. Protein Kinase Cδ (PKCδ) could induce phosphorylation of Drp1, and we found that HG induced phosphorylation of PKCδ. PKCδ inhibitor (Go 6983) or PKCδ siRNA reversed HG-induced phosphorylation of Drp1 and further mitochondrial dysfunction. The above studies indicated that HG increases mitochondrial fission via promoting PKCδ/Drp1 signaling. Drp1 induces excessive mitochondrial fission and produces damaged mitochondrial, and mitophagy plays a key role in clearing damaged mitochondrial. Our study showed that HG suppressed mitophagy via inhibiting LC3B-II formation and p62 degradation. 3-MA (autophagy inhibitor) aggravated HG-induced RMECs damage, while rapamycin (autophagy agonist) rescued the above phenomenon. Further studies were identified that HG inhibited mitophagy by down-regulation of the PINK1/Parkin signaling pathway, and PINK1 siRNA aggravated HG-induced RMECs damage. Further in-depth study, we propose that Drp1 promotion of Hexokinase II (HK-II) separation from mitochondria, thus inhibiting HK-II-PINK1-mediated mitophagy. In vivo, we found that intraretinal microvascular abnormalities (IRMA), including retinal vascular leakage, acellular capillaries, and apoptosis were increased in STZ-induced DR rats, which were reversed by pretreatment with Mdivi-1 or Rapamycin. Altogether, our findings provide new insight into the mechanisms underlying the regulation of mitochondrial homeostasis and provide a potential treatment strategy for Diabetic retinopathy.

N-acetyl-L-cysteine ameliorates mitochondrial dysfunction in ischemia/reperfusion injury via attenuating Drp-1 mediated mitochondrial autophagy.

BACKGROUND AND PURPOSE: Ischemic reperfusion (I/R) injury causes a wide array of functional and structure alternations of mitochondria, associated with oxidative stress and increased the severity of injury. Despite the previous evidence for N-acetyl-L-cysteine (NAC) provide neuroprotection after I/R injury, it is unknown to evaluate the effect of NAC on altered mitochondrial autophagy forms an essential axis to impaired mitochondrial quality control in cerebral I/R injury. METHODS: Male wistar rats subjected to I/R injury were used as transient Middle Cerebral Artery Occlusion (tMCAO) model. After I/R injury, the degree of cerebral tissue injury was detected by infarct volume, H&E staining and behavioral assessment. We also performed mitochondrial reactive oxygen species and mitochondrial membrane potential by flow cytometry and mitochondrial respiratory complexes to evaluate the mitochondrial dysfunction. Finally, we performed the western blotting analysis to measure the apoptotic and autophagic marker. RESULTS: We found that NAC administration significantly ameliorates brain injury, improves neurobehavioral outcome, decreases neuroinflammation and mitochondrial mediated oxidative stress. We evaluated the neuroprotective effect of NAC against neuronal apoptosis by assessing its ability to sustained mitochondrial integrity and function. Further studies revealed that beneficial effects of NAC is through targeting the mitochondrial autophagy via regulating the GSK-3ß/Drp1mediated mitochondrial fission and inhibiting the expression of beclin-1 and conversion of LC3, as well as activating the p-Akt pro-survival pathway. CONCLUSION: Our results suggest that NAC exerts neuroprotective effects to inhibit the altered mitochondrial changes and cell death in I/R injury via regulation of p-GSK-3ß mediated Drp-1 translocation to the mitochondria.

Emodin prevents renal ischemia-reperfusion injury via suppression of CAMKII/DRP1-mediated mitochondrial fission.

Acute kidney injury (AKI) is a serious threat to human health. Clinically, ischemia-reperfusion (I/R) injury is considered one of the most common contributors to AKI. Emodin has been reported to alleviate I/R injury in the heart, brain, and small intestine in rats and mice through its anti-inflammatory effects. The present study investigated whether emodin improved AKI induced by I/R and elucidated the molecular mechanisms. We used a mouse model of renal I/R injury and human renal tubular epithelial cell model of hypoxia/reoxygenation (H/R) injury. Ischemia/reperfusion resulted in renal dysfunction. Pretreatment with emodin ameliorated renal injury in mice following I/R injury. Emodin reduced mitochondrial-mediated apoptosis, suppressed the overproduction of mitochondrial reactive oxygen species and accelerated the recovery of adenosine triphosphate both in vivo and in vitro. Emodin prevented mitochondrial fission and restored the balance of mitochondrial dynamics. The phosphorylation of dynamin-related protein 1 (DRP1) at Ser616, a master regulator of mitochondrial fission, was upregulated in both models of I/R and H/R injury, and this upregulation was blocked by emodin. Using computational cognate protein kinase prediction and specific kinase inhibitors, we found that emodin inhibited the phosphorylation of calcium/calmodulin-dependent protein kinase II (https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1554), thereby inhibiting its kinase activity and reducing the phosphorylation of DRP1 at Ser616. The results demonstrated that emodin pretreatment could protect renal function by improving mitochondrial dysfunction induced by I/R.

Hydralazine protects the heart against acute ischaemia/reperfusion injury by inhibiting Drp1-mediated mitochondrial fission.

AIMS: Genetic and pharmacological inhibition of mitochondrial fission induced by acute myocardial ischaemia/reperfusion injury (IRI) has been shown to reduce myocardial infarct size. The clinically used anti-hypertensive and heart failure medication, hydralazine, is known to have anti-oxidant and anti-apoptotic effects. Here, we investigated whether hydralazine confers acute cardioprotection by inhibiting Drp1-mediated mitochondrial fission. METHODS AND RESULTS: Pre-treatment with hydralazine was shown to inhibit both mitochondrial fission and mitochondrial membrane depolarisation induced by oxidative stress in HeLa cells. In mouse embryonic fibroblasts (MEFs), pre-treatment with hydralazine attenuated mitochondrial fission and cell death induced by oxidative stress, but this effect was absent in MEFs deficient in the mitochondrial fission protein, Drp1. Molecular docking and surface plasmon resonance studies demonstrated binding of hydralazine to the GTPase domain of the mitochondrial fission protein, Drp1 (KD 8.6±1.0 µM), and inhibition of Drp1 GTPase activity in a dose-dependent manner. In isolated adult murine cardiomyocytes subjected to simulated IRI, hydralazine inhibited mitochondrial fission, preserved mitochondrial fusion events, and reduced cardiomyocyte death (hydralazine 24.7±2.5% vs. control 34.1±1.5%, P=0.0012). In ex vivo perfused murine hearts subjected to acute IRI, pre-treatment with hydralazine reduced myocardial infarct size (as % left ventricle: hydralazine 29.6±6.5% vs. vehicle control 54.1±4.9%, P=0.0083), and in the murine heart subjected to in vivo IRI, the administration of hydralazine at reperfusion, decreased myocardial infarct size (as % area-at-risk: hydralazine 28.9±3.0% vs. vehicle control 58.2±3.8%, P<0.001). CONCLUSION: We show that, in addition to its antioxidant and anti-apoptotic effects, hydralazine, confers acute cardioprotection by inhibiting IRI-induced mitochondrial fission, raising the possibility of repurposing hydralazine as a novel cardioprotective therapy for improving post-infarction outcomes.

IRF6 Regulates the Delivery of E-Cadherin to the Plasma Membrane.

IRF6 is a transcription factor that is required for craniofacial development and epidermal morphogenesis. Specifically, Irf6-deficient mice lack the terminally differentiated epidermal layers, leading to an absence of barrier function. This phenotype also includes intraoral adhesions due to the absence of the oral periderm, leading to the mislocalization of E-cadherin and other cell‒cell adhesion proteins of the oral epithelium. However, the mechanisms by which IRF6 controls the localization of cell adhesion proteins are not understood. In this study, we show that in human and murine keratinocytes, loss of IRF6 leads to a breakdown of epidermal sheets after mechanical stress. This defect is due to a reduction of adhesion proteins at the plasma membrane. Dynamin inhibitors rescued the IRF6-dependent resistance of epidermal sheets to mechanical stress, but only inhibition of clathrin-mediated endocytosis rescued the localization of junctional proteins at the membrane. Our data show that E-cadherin recycling but not its endocytosis is affected by loss of IRF6. Overall, we demonstrate a role for IRF6 in the delivery of adhesion proteins to the cell membrane.

Pyrimidyn-Based Dynamin Inhibitors as Novel Cytotoxic Agents.

Five focused libraries of pyrimidine-based dynamin GTPase inhibitors, in total 69 compounds were synthesised, and their dynamin inhibition and broad-spectrum cytotoxicity examined. Dynamin plays a crucial role in mitosis, and as such inhibition of dynamin was expected to broadly correlate with the observed cytotoxicity. The pyrimidines synthesised ranged from mono-substituted to trisubstituted. The highest levels of dynamin inhibition were noted with di- and tri- substituted pyrimidines, especially those with pendent amino alkyl chains. Short chains and simple heterocyclic rings reduced dynamin activity. There were three levels of dynamin activity noted: 1-10, 10-25 and 25-60 µM. Screening of these compounds in a panel of cancer cell lines: SW480 (colon), HT29 (colon), SMA (spontaneous murine astrocytoma), MCF-7 (breast), BE2-C (glioblastoma), SJ-G2 (neuroblastoma), MIA (pancreas), A2780 (ovarian), A431 (skin), H460 (lung), U87 (glioblastoma) and DU145 (prostate) cell lines reveal a good correlation between the observed dynamin inhibition and the observed cytotoxicity. The most active analogues (31 a,b) developed returned average GI50 values of 1.0 and 0.78 µM across the twelve cell lines examined. These active analogues were: N2 -(3-dimethylaminopropyl)-N4 -dodecyl-6-methylpyrimidine-2,4-diamine (31 a) and N4 -(3-dimethylaminopropyl)-N2 -dodecyl-6-methylpyrimidine-2,4-diamine (31 b).

Dynamin inhibition causes context-dependent cell death of leukemia and lymphoma cells.

Current chemotherapy for treatment of pediatric acute leukemia, although generally successful, is still a matter of concern due to treatment resistance, relapses and life-long side effects for a subset of patients. Inhibition of dynamin, a GTPase involved in clathrin-mediated endocytosis and regulation of the cell cycle, has been proposed as a potential anti-cancer regimen, but the effects of dynamin inhibition on leukemia cells has not been extensively addressed. Here we adopted single cell and whole-population analysis by flow cytometry and live imaging, to assess the effect of dynamin inhibition (Dynasore, Dyngo-4a, MitMAB) on pediatric acute leukemia cell lines (CCRF-CEM and THP-1), human bone marrow biopsies from patients diagnosed with acute lymphoblastic leukemia (ALL), as well as in a model of lymphoma (EL4)-induced tumor growth in mice. All inhibitors suppressed proliferation and induced pronounced caspase-dependent apoptotic cell death in CCRF-CEM and THP-1 cell lines. However, the inhibitors showed no effect on bone marrow biopsies, and did not prevent EL4-induced tumor formation in mice. We conclude that dynamin inhibition affects highly proliferating human leukemia cells. These findings form a basis for evaluation of the potential, and constraints, of employing dynamin inhibition in treatment strategies against leukemia and other malignancies.

Mdivi-1 alleviates atopic dermatitis through the inhibition of NLRP3 inflammasome.

Atopic dermatitis (AD) is a chronic inflammatory cutaneous disorder with few treatment options. Dynamin-related protein 1 (Drp1)-dependent mitochondrial fission contributes to the activation of NLRP3 inflammasome, and inhibiting Drp1 has been become an attractive therapeutic strategy for inflammatory diseases. This study aimed to investigate the effects of Drp1 inhibitor mdivi-1 on experimental AD. We firstly detected the effects of mdivi-1 on primary human keratinocytes in an inflammatory cocktail-induced AD-related inflammation in vitro. Results showed that mdivi-1 inhibited NLRP3 inflammasome activation and pyroptosis which were evidenced by decreased expression of NLRP3, ASC, cleavage of caspase-1, GSDMD-NT, mature interleukin (IL)-1ß and IL-18 in keratinocytes under AD-like inflammation. Next, mouse model of AD-like skin lesions was induced by epicutaneous application of 2,4-dinitrochlorobenzene (DNCB) and mdivi-1 (25 mg/kg/day, days 5-33 during construction of AD model) was intraperitoneally injected into DNCB-induced mice. AD mice with mdivi-1 treatment exhibited ameliorated AD symptoms, lower serum IgE level, and reduced epidermal thickening, mast cells infiltration, and production of IL-4, IL-5 and IL-13 in the lesional tissues. Indeed, mdivi-1 significantly inhibited NLRP3 inflammasome activation and pyroptotic injury occurred in DNCB-treated skin tissues. Mechanically, mdivi-1 regulated the expression of mitochondrial dynamic proteins and suppressed the activation of NF-κB signal pathway which is an upstream of NLRP3 inflammasome both in vitro and in vivo. This study demonstrated that mdivi-1 could protect against experimental AD through inhibiting the activation of NLRP3 inflammasome and subsequent inflammatory cytokine release, and mdivi-1 might exert this function by inhibiting mitochondrial fission and subsequently blocking NF-κB pathway.

Drp1-dependent peptide reverse mitochondrial fragmentation, a homeostatic response in Friedreich ataxia.

Friedreich ataxia is an autosomal recessive, neurodegenerative disease characterized by the deficiency of the iron-sulfur cluster assembly protein frataxin. Loss of this protein impairs mitochondrial function. Mitochondria alter their morphology in response to various stresses; however, such alterations to morphology may be homeostatic or maladaptive depending upon the tissue and disease state. Numerous neurodegenerative diseases exhibit excessive mitochondrial fragmentation, and reversing this phenotype improves bioenergetics for diseases in which mitochondrial dysfunction is a secondary feature of the disease. This paper demonstrates that frataxin deficiency causes excessive mitochondrial fragmentation that is dependent upon Drp1 activity in Friedreich ataxia cellular models. Drp1 inhibition by the small peptide TAT-P110 reverses mitochondrial fragmentation but also decreases ATP levels in frataxin-knockdown fibroblasts and FRDA patient fibroblasts, suggesting that fragmentation may provide a homeostatic pathway for maintaining cellular ATP levels. The cardiolipin-stabilizing compound SS-31 similarly reverses fragmentation through a Drp1-dependent mechanism, but it does not affect ATP levels. The combination of TAT-P110 and SS-31 does not affect FRDA patient fibroblasts differently from SS-31 alone, suggesting that the two drugs act through the same pathway but differ in their ability to alter mitochondrial homeostasis. In approaching potential therapeutic strategies for FRDA, an important criterion for compounds that improve bioenergetics should be to do so without impairing the homeostatic response of mitochondrial fragmentation.

Pharmacological inhibition of dynamin-related protein 1 attenuates skeletal muscle insulin resistance in obesity.

Dynamin-related protein-1 (Drp1) is a key regulator in mitochondrial fission. Excessive Drp1-mediated mitochondrial fission in skeletal muscle under the obese condition is associated with impaired insulin action. However, it remains unknown whether pharmacological inhibition of Drp1, using the Drp1-specific inhibitor Mitochondrial Division Inhibitor 1 (Mdivi-1), is effective in alleviating skeletal muscle insulin resistance and improving whole-body metabolic health under the obese and insulin-resistant condition. We subjected C57BL/6J mice to a high-fat diet (HFD) or low-fat diet (LFD) for 5-weeks. HFD-fed mice received Mdivi-1 or saline injections for the last week of the diet intervention. Additionally, myotubes derived from obese insulin-resistant humans were treated with Mdivi-1 or saline for 12 h. We measured glucose area under the curve (AUC) from a glucose tolerance test (GTT), skeletal muscle insulin action, mitochondrial dynamics, respiration, and H2 O2 content. We found that Mdivi-1 attenuated impairments in skeletal muscle insulin signaling and blood glucose AUC from a GTT induced by HFD feeding (p < 0.05). H2 O2 content was elevated in skeletal muscle from the HFD group (vs. LFD, p < 0.05), but was reduced with Mdivi-1 treatment, which may partially explain the improvement in skeletal muscle insulin action. Similarly, Mdivi-1 enhanced the mitochondrial network structure, reduced reactive oxygen species, and improved insulin action in myotubes from obese humans (vs. saline, p < 0.05). In conclusion, inhibiting Drp1 with short-term Mdivi-1 administration attenuates the impairment in skeletal muscle insulin signaling and improves whole-body glucose tolerance in the setting of obesity-induced insulin resistance. Targeting Drp1 may be a viable approach to treat obesity-induced insulin resistance.

Chemical Inhibitors of Dynamin Exert Differential Effects in VEGF Signaling.

VEGFR2 is the main receptor and mediator of the vasculogenic and angiogenic activity of VEGF. Activated VEGFR2 internalizes through clathrin-mediated endocytosis and macropinocytosis. As dynamin is a key regulator of the clathrin pathway, chemical inhibitors of dynamin are commonly used to assess the role of the clathrin route in receptor signaling. However, drugs may also exert off-target effects. Here, we compare the effects of three dynamin inhibitors, dynasore, dyngo 4a and dynole, on VEGFR2 internalization and signaling. Although these drugs consistently inhibit clathrin-mediated endocytosis of both transferrin (a typical cargo of this route) and VEGFR2, surprisingly, they exert contradictory effects in receptor signaling. Thus, while dynasore has no effect on phosphorylation of VEGFR2, the other two drugs are strong inhibitors. Furthermore, although dyngo does not interfere with phosphorylation of Akt, dynasore and dynole have a strong inhibitory effect. These inconsistent effects suggest that the above dynamin blockers, besides inhibiting dynamin-dependent endocytosis of VEGFR2, exert additional inhibitory effects on signaling that are independent of endocytosis; i.e., they are due to off-target effects. Using a recently developed protocol, we comparatively validate the specificity of two endocytic inhibitors, dynasore and EIPA. Our findings highlight the importance of assessing whether the effect of an endocytic drug on signaling is specifically due to its interference with endocytosis or due to off-targets.

Seizures are a druggable mechanistic link between TBI and subsequent tauopathy.

Traumatic brain injury (TBI) is a prominent risk factor for dementias including tauopathies like chronic traumatic encephalopathy (CTE). The mechanisms that promote prion-like spreading of Tau aggregates after TBI are not fully understood, in part due to lack of tractable animal models. Here, we test the putative role of seizures in promoting the spread of tauopathy. We introduce 'tauopathy reporter' zebrafish expressing a genetically encoded fluorescent Tau biosensor that reliably reports accumulation of human Tau species when seeded via intraventricular brain injections. Subjecting zebrafish larvae to a novel TBI paradigm produced various TBI features including cell death, post-traumatic seizures, and Tau inclusions. Bath application of dynamin inhibitors or anticonvulsant drugs rescued TBI-induced tauopathy and cell death. These data suggest a role for seizure activity in the prion-like seeding and spreading of tauopathy following TBI. Further work is warranted regarding anti-convulsants that dampen post-traumatic seizures as a route to moderating subsequent tauopathy.

Traumatic brain injury can result from direct head concussions, rapid head movements, or a blast wave generated by an explosion. Traumatic brain injury often causes seizures in the short term and is a risk factor for certain dementias, including Alzheimer's disease and chronic traumatic encephalopathy in the long term. A protein called Tau undergoes a series of chemical changes in these dementias that makes it accumulate, form toxic filaments and kill neurons. The toxic abnormal Tau proteins are initially found only in certain regions of the brain, but they spread as the disease progresses. Previous studies in Alzheimer's disease and other diseases where Tau proteins are abnormal suggest that Tau can spread between neighboring neurons and this can be promoted by neuron activity. However, scientists do not know whether similar mechanisms are at work following traumatic brain injury. Given that seizures are very common following traumatic brain injury, could they be partly responsible for promoting dementia? To investigate this, researchers need animal models in which they can measure neural activity associated with traumatic brain injury and observe the spread of abnormal Tau proteins. Alyenbaawi et al. engineered zebrafish so that their Tau proteins would be fluorescent, making it possible to track the accumulation of aggregated Tau protein in the brain. Next, they invented a simple way to perform traumatic brain injury on zebrafish larvae by using a syringe to produce a pressure wave. After this procedure, many of the fish exhibited features consistent with progression towards dementia, and seizure-like behaviors. The results showed that post-traumatic seizures are linked to the spread of aggregates of abnormal Tau following traumatic brain injury. Alyenbaawi et al. also found that anticonvulsant drugs can lower the levels of abnormal Tau proteins in neurons, preventing cell death, and could potentially ameliorate dementias associated with traumatic brain injury. These drugs are already being used to prevent post-traumatic epilepsy, but more research is needed to confirm whether they reduce the risk or severity of Tau-related neurodegeneration.

Dynamin-related protein 1 inhibition reduces hepatic PCSK9 secretion.

AIMS: Proteostasis maintains protein homeostasis and participates in regulating critical cardiometabolic disease risk factors including proprotein convertase subtilisin/kexin type 9 (PCSK9). Endoplasmic reticulum (ER) remodeling through release and incorporation of trafficking vesicles mediates protein secretion and degradation. We hypothesized that ER remodeling that drives mitochondrial fission participates in cardiometabolic proteostasis. METHODS AND RESULTS: We used in vitro and in vivo hepatocyte inhibition of a protein involved in mitochondrial fission, dynamin-related protein 1 (DRP1). Here, we show that DRP1 promotes remodeling of select ER microdomains by tethering vesicles at ER. A DRP1 inhibitor, mitochondrial division inhibitor 1 (mdivi-1) reduced ER localization of a DRP1 receptor, mitochondrial fission factor, suppressing ER remodeling-driven mitochondrial fission, autophagy, and increased mitochondrial calcium buffering and PCSK9 proteasomal degradation. DRP1 inhibition by CRISPR/Cas9 deletion or mdivi-1 alone or in combination with statin incubation in human hepatocytes and hepatocyte-specific Drp1-deficiency in mice reduced PCSK9 secretion (-78.5%). In HepG2 cells, mdivi-1 increased low-density lipoprotein receptor via c-Jun transcription and reduced PCSK9 mRNA levels via suppressed sterol regulatory binding protein-1c. Additionally, mdivi-1 reduced macrophage burden, oxidative stress, and advanced calcified atherosclerotic plaque in aortic roots of diabetic Apoe-deficient mice and inflammatory cytokine production in human macrophages. CONCLUSIONS: We propose a novel tethering function of DRP1 beyond its established fission function, with DRP1-mediated ER remodeling likely contributing to ER constriction of mitochondria that drives mitochondrial fission. We report that DRP1-driven remodeling of select ER micro-domains may critically regulate hepatic proteostasis and identify mdivi-1 as a novel small molecule PCSK9 inhibitor.

Anti-inflammatory and neuroprotective effects of natural cordycepin in rotenone-induced PD models through inhibiting Drp1-mediated mitochondrial fission.

Accumulating evidences suggest that inflammation-mediated neurons dysfunction participates in the initial and development of Parkinson's disease (PD), whereas mitochondria have been recently recognized as crucial regulators in NLRP3 inflammasome activation. Cordycepin, a major component of cordyceps militaris, has been shown to possess neuroprotective and anti-inflammatory activity. However, the effects of cordycepin in rotenone-induced PD models and the possible mechanisms are still not fully understood. Here, we observed that motor dysfunction and dopaminergic neurons loss induced by rotenone exposure were ameliorated by cordycepin. Cordycepin also reversed Drp1-mediated aberrant mitochondrial fragmentation through increasing AMPK phosphorylation and maintained normal mitochondrial morphology. Additionally, cordycepin effectively increased adenosine 5'-triphosphate (ATP) content, mitochondrial membrane potential (MMP), and reduced mitochondrial ROS levels, as well as inhibited complex 1 activity. More importantly, cordycepin administration inhibited the expression of NLRP3 inflammasome components and the release of pro-inflammatory cytokine in rotenone-induced rats and cultured neuronal PC12 cells. Moreover, we demonstrated that the activation of NLRP3 inflammasome within neurons could be suppressed by the mitochondrial division inhibitor (Mdivi-1). Collectively, the present study provides evidence that cordycepin exerts neuroprotective effects partially through preventing neural NLRP3 inflammasome activation induced by Drp1-dependent mitochondrial fragmentation in rotenone-injected PD models.

Ophiopogonin D alleviates diabetic myocardial injuries by regulating mitochondrial dynamics.

ETHNOPHARMACOLOGICAL RELEVANCE: Ophiopogonin D (OP-D) is a steroidal saponin extracted from Ophiopogon japonicus (Thunb.) Ker Gawl. (Liliaceae), that has been traditionally used to treat cough, sputum, and thirst in some Asian countries. Recently, various pharmacological roles of OP-D have been identified, including anti-inflammatory, cardioprotective, and anti-cancer effects. However, whether OP-D can prevent diabetic myocardial injury remains unknown. AIM OF THE STUDY: In this study, we aimed to observe the effects of OP-D on the diabetic myocardium. MATERIALS AND METHODS: Leptin receptor-deficient db/db mice were used as an animal model for type 2 diabetes. The effects of OP-D on blood glucose, blood lipids, myocardial ultrastructure, and mitochondrial function in mice were observed after four weeks of intragastric administration. Palmitic acid was used to stimulate cardiomyocytes to establish a myocardial lipotoxicity model. Cell apoptosis, mitochondrial morphology, and function were observed. RESULTS: Blood glucose and blood lipid levels were significantly increased in db/db mice, accompanied by myocardial mitochondrial injury and dysfunction. OP-D treatment reduced blood lipid levels in db/db mice and relieved mitochondrial injury and dysfunction. OP-D inhibited palmitic acid induced-mitochondrial fission and dysfunction, reduced endogenous apoptosis, and improved cell survival rate in H9C2 cardiomyocytes. Both in vivo and in vitro models showed increased phosphorylation of DRP1 at Ser-616, reduced phosphorylation of DRP1 at Ser-637, and reduced expression of fusion proteins MFN1/2 and OPA1. Meanwhile, immunofluorescence co-localization analysis revealed that palmitic acid stimulated the translocation of DRP1 protein from the cytoplasm to the mitochondria in H9C2 cardiomyocytes. The imbalance of mitochondrial dynamics, protein expression, and translocation of DRP1 were effectively reversed by OP-D treatment. In isolated mice ventricular myocytes, palmitic acid enhanced cytoplasmic Ca2+ levels and suppressed contractility in ventricular myocytes, accompanied by activation of calcineurin, a key regulator of DRP1 dephosphorylation at Ser-637. OP-D reversed the changes caused by palmitic acid. CONCLUSIONS: Our findings indicate that OP-D intervention could alleviate lipid accumulation and mitochondrial injury in diabetic mouse hearts and palmitic acid-stimulated cardiomyocytes. The cardioprotective effect of OP-D may be mediated by the regulation of mitochondrial dynamics.

Preservation of mitochondrial homeostasis is responsible for the ameliorative effects of Suhuang antitussive capsule on non-resolving inflammation via inhibition of NF-κB signaling and NLRP3 inflammasome activation.

ETHNOPHARMACOLOGICAL RELEVANCE: Suhuang antitussive capsule (Suhuang), one of traditional antitussive Chinese patent medicines, has been used for the treatment of post-infectious cough and cough variant asthma in clinical practice. It has been demonstrated to show numerous biological actions including antitussive and anti-inflammatory effects. AIM OF THE STUDY: This study aims to investigate the effects of Suhuang on non-resolving inflammation and its underlying molecular mechanism. MATERIAL AND METHODS: In vitro, mitochondrial membrane potential and ROS were detected by flow cytometry analysis. mtDNA release and mPTP fluorescence were determined by Q-PCR and fluorescence microplate reader analysis. Cytochrome C release and 8-OHdG levels were evaluated by ELISA. Additionally, the effects of Suhuang on Drp1, MMP9, IκBα/NF-κB and NLRP3/ASC/Caspase-1 expression were determined by Q-PCR, gelatin zymography or immunoblot analysis. In vivo, C57/BL6 mice were orally administrated for 2 weeks with Suhuang, then lung injury was induced by LPS. Inflammatory mediators mRNA, histological assessment and NF-κB/Caspase-1/IL-1ß levels were evaluated by Q-PCR, H&E staining and immunoblot analysis. Two sepsis models of mice were further used to evaluate its anti-inflammatory effects. RESULTS: Suhuang restored mitochondrial homeostasis by inhibiting Drp1 activation and mitochondrial fission. Besides, Suhuang reduced mPTP opening, mitochondrial membrane potential collapse, ROS overproduction and mtDNA release. Moreover, Suhuang down-regulated MMP9 expression. As a consequence of preserved mitochondrial homeostasis, Suhuang inhibited NF-κB pathway activation by prevention of NF-κB-p65 phosphorylation and IκBα degradation. Suhuang also limited NLRP3 inflammasome activation by blocking NLRP3-ASC interaction and promoting NLRP3 ubiquitination degradation. Drp1 knockdown in vitro diminished the inhibitory effects of Suhuang on inflammatory responses, indicating the essential role of Drp1 in the Suhuang's activity. Consistently, the therapeutic effects of Suhuang were confirmed in LPS-inhaled mice, which recapitulated the protective actions of Suhuang in mitochondrial homeostasis in vitro. Additionally, two sepsis models of mice confirmed the inhibitory effects of Suhuang on uncontrolled inflammation. CONCLUSIONS: Altogether, our work reveals that Suhuang inhibits non-resolving inflammation through inhibition of NF-κB signaling and NLRP3 inflammasome activation by preserving mitochondrial homeostasis, providing new pharmacological data for the clinical use of Suhuang. Our study also suggests mitochondrial homeostasis as a potential intrinsic regulatory strategy for treating inflammatory diseases.

Tyrosine kinase Fyn promotes apoptosis after intracerebral hemorrhage in rats by activating Drp1 signaling.

Tyrosine kinase Fyn is a member of the Src kinase family, which is involved in neuroinflammation, apoptosis, and oxidative stress. Its role in intracerebral hemorrhage (ICH) is not fully understood. In this study, we found that Fyn was significantly elevated in human brain tissue after ICH. Accordingly, we investigated the role of Fyn in a rat ICH model, which was constructed by injecting blood into the right basal ganglia. In this model, Fyn expression was significantly upregulated in brain tissue adjacent to the hematoma. SiRNA-induced Fyn knockdown was neuroprotective for secondary cerebral damage, as demonstrated by reduced brain edema, suppression of the modified neurological severity score, and mitigation of blood-brain barrier permeability and neuronal damage. Fyn downregulation reduced apoptosis following ICH, as indicated by downregulation of apoptosis-related proteins AIF, Cyt.c, caspase 3, and Bax; upregulation of anti-apoptosis-related protein Bcl-2; and decreased tunnel staining. Mdivi-1, a Drp1 inhibitor, reversed Fyn overexpression induced pro-apoptosis. However, Fyn did not significantly affect inflammation-related proteins NF-κB, TNF-α, caspase 1, MPO, IL-1ß, or IL-18 after ICH. Fyn activated Drp1 signaling by phosphorylating Drp1 at serine 616, which increased apoptosis after ICH in rats. This study clarifies the relationship between Fyn, apoptosis, and inflammation following ICH and provides a new strategy for exploring the prevention and treatment of ICH. KEY MESSAGES: ICH induced an increase in Fyn expression in human and rat cerebral tissues. Knockdown of Fyn prevented cerebral damage following ICH. Inhibition of Fyn had no significant effects on inflammatory responses. However, the downregulation of Fyn exerted neuroprotective effects on apoptosis. Fyn perturbed ICH-induced cell apoptosis by interacting with and phosphorylating (Ser616) Drp1 in a rat ICH model.

Drp1-dependent mitochondrial fission in cardiovascular disease.

Mitochondria are highly dynamic organelles undergoing cycles of fusion and fission to modulate their morphology, distribution, and function, which are referred as 'mitochondrial dynamics'. Dynamin-related protein 1 (Drp1) is known as the major pro-fission protein whose activity is tightly regulated to clear the damaged mitochondria via mitophagy, ensuring a strict control over the intricate process of cellular and organ dynamics in heart. Various posttranslational modifications (PTMs) of Drp1 have been identified including phosphorylation, SUMOylation, palmitoylation, ubiquitination, S-nitrosylation, and O-GlcNAcylation, which implicate a role in the regulation of mitochondrial dynamics. An intact mitochondrial homeostasis is critical for heart to fuel contractile function and cardiomyocyte metabolism, while defects in mitochondrial dynamics constitute an essential part of the pathophysiology underlying various cardiovascular diseases (CVDs). In this review, we summarize current knowledge on the critical role of Drp1 in the pathogenesis of CVDs including endothelial dysfunction, smooth muscle remodeling, cardiac hypertrophy, pulmonary arterial hypertension, myocardial ischemia-reperfusion, and myocardial infarction. We also highlight how the targeting of Drp1 could potentially contribute to CVDs treatments.