Mechanistic analysis of a novel membrane-interacting variable loop in the pleckstrin-homology domain critical for dynamin function.

Classical dynamins are best understood for their ability to generate vesicles by membrane fission. During clathrin-mediated endocytosis (CME), dynamin is recruited to the membrane through multivalent protein and lipid interactions between its proline-rich domain (PRD) with SRC Homology 3 (SH3) domains in endocytic proteins and its pleckstrin-homology domain (PHD) with membrane lipids. Variable loops (VL) in the PHD bind lipids and partially insert into the membrane thereby anchoring the PHD to the membrane. Recent molecular dynamics (MD) simulations reveal a novel VL4 that interacts with the membrane. Importantly, a missense mutation that reduces VL4 hydrophobicity is linked to an autosomal dominant form of Charcot-Marie-Tooth (CMT) neuropathy. We analyzed the orientation and function of the VL4 to mechanistically link data from simulations with the CMT neuropathy. Structural modeling of PHDs in the cryo-electron microscopy (cryo-EM) cryoEM map of the membrane-bound dynamin polymer confirms VL4 as a membrane-interacting loop. In assays that rely solely on lipid-based membrane recruitment, VL4 mutants with reduced hydrophobicity showed an acute membrane curvature-dependent binding and a catalytic defect in fission. Remarkably, in assays that mimic a physiological multivalent lipid- and protein-based recruitment, VL4 mutants were completely defective in fission across a range of membrane curvatures. Importantly, expression of these mutants in cells inhibited CME, consistent with the autosomal dominant phenotype associated with the CMT neuropathy. Together, our results emphasize the significance of finely tuned lipid and protein interactions for efficient dynamin function.

miR-34a/DRP-1-mediated mitophagy participated in cisplatin-induced ototoxicity via increasing oxidative stress.

PURPOSE: Cisplatin is a widely used and effective chemotherapeutic agent for most solid malignant tumors. However, cisplatin-induced ototoxicity is a common adverse effect that limits the therapeutic efficacy of tumors in the clinic. To date, the specific mechanism of ototoxicity has not been fully elucidated, and the management of cisplatin-induced ototoxicity is also an urgent challenge. Recently, some authors believed that miR34a and mitophagy played a role in age-related and drug-induced hearing loss. Our study aimed to explore the involvement of miR-34a/DRP-1-mediated mitophagy in cisplatin-induced ototoxicity. METHODS: In this study, C57BL/6 mice and HEI-OC1 cells were treated with cisplatin. MiR-34a and DRP-1 levels were analyzed by qRT‒PCR and western blotting, and mitochondrial function was assessed via oxidative stress, JC-1 and ATP content. Subsequently, we detected DRP-1 levels and observed mitochondrial function by modulating miR-34a expression in HEI-OC1 cells to determine the effect of miR-34a on DRP-1-mediated mitophagy. RESULTS: MiR-34a expression increased and DRP-1 levels decreased in C57BL/6 mice and HEI-OC1 cells treated with cisplatin, and mitochondrial dysfunction was involved in this process. Furthermore, the miR-34a mimic decreased DRP-1 expression, enhanced cisplatin-induced ototoxicity and aggravated mitochondrial dysfunction. We further verified that the miR-34a inhibitor increased DRP-1 expression, partially protected against cisplatin-induced ototoxicity and improved mitochondrial function. CONCLUSION: MiR-34a/DRP-1-mediated mitophagy was related to cisplatin-induced ototoxicity and might be a novel target for investigating the treatment and protection of cisplatin-induced ototoxicity.

Exercise activates Sirt1-mediated Drp1 acetylation and inhibits hepatocyte apoptosis to improve nonalcoholic fatty liver disease.

PURPOSE: Aerobic exercise has shown beneficial effects in the prevention and treatment of non-alcoholic fatty liver disease (NAFLD). Nevertheless, the regulatory mechanism is not turely clear. Therefore, we aim to clarify the possible mechanism by investigating the effects of aerobic exercise on NAFLD and its mitochondrial dysfunction. METHODS: NAFLD rat model was established by feeding high fat diet. and used oleic acid (OA) to treat HepG2 cells. Changes in histopathology, lipid accumulation, apoptosis, body weight, and biochemical parameters were assessed. In addition, antioxidants, mitochondrial biogenesis and mitochondrial fusion and division were assessed. RESULTS: The obtained in vivo results showed that aerobic exercise significantly improved lipid accumulation and mitochondrial dysfunction induced by HFD, activated the level of Sirtuins1 (Srit1), and weakened the acetylation and activity of dynamic-related protein 1 (Drp1). In vitro results showed that activation of Srit1 inhibited OA-induced apoptosis in HepG2 cells and alleviated OA-induced mitochondrial dysfunction by inhibiting Drp1 acetylation and reducing Drp1 expression. CONCLUSION: Aerobic exercise alleviates NAFLD and its mitochondrial dysfunction by activating Srit1 to regulate Drp1 acetylation. Our study clarifies the mechanism of aerobic exercise in alleviating NAFLD and its mitochondrial dysfunction and provides a new method for adjuvant treatment of NAFLD.

Impact of Acute High Glucose on Mitochondrial Function in a Model of Endothelial Cells: Role of PDGF-C.

An increase in plasma high glucose promotes endothelial dysfunction mainly through increasing mitochondrial ROS production. High glucose ROS-induced has been implicated in the fragmentation of the mitochondrial network, mainly by an unbalance expression of mitochondrial fusion and fission proteins. Mitochondrial dynamics alterations affect cellular bioenergetics. Here, we assessed the effect of PDGF-C on mitochondrial dynamics and glycolytic and mitochondrial metabolism in a model of endothelial dysfunction induced by high glucose. High glucose induced a fragmented mitochondrial phenotype associated with the reduced expression of OPA1 protein, high DRP1pSer616 levels and reduced basal respiration, maximal respiration, spare respiratory capacity, non-mitochondrial oxygen consumption and ATP production, regarding normal glucose. In these conditions, PDGF-C significantly increased the expression of OPA1 fusion protein, diminished DRP1pSer616 levels and restored the mitochondrial network. On mitochondrial function, PDGF-C increased the non-mitochondrial oxygen consumption diminished by high glucose conditions. These results suggest that PDGF-C modulates the damage induced by HG on the mitochondrial network and morphology of human aortic endothelial cells; additionally, it compensates for the alteration in the energetic phenotype induced by HG.

Fowl adenovirus serotype 4 enters leghorn male hepatocellular cells via the clathrin-mediated endocytosis pathway.

Hepatitis-hydropericardium syndrome (HHS) induced by fowl adenovirus serotype-4 (FAdV-4) has caused large economic losses to the world poultry industry in recent years. HHS is characterized by pericardial effusion and hepatitis, manifesting as a swollen liver with focal necroses and petechial haemorrhage. However, the process of FAdV-4 entry into hepatic cells remains largely unknown. In this paper, we present a comprehensive study on the entry mechanism of FAdV-4 into leghorn male hepatocellular (LMH) cells. We first observed that FAdV-4 internalization was inhibited by chlorpromazine and clathrin heavy chain (CHC) knockdown, suggesting that FAdV-4 entry into LMH cells depended on clathrin. By using the inhibitor dynasore, we showed that dynamin was required for FAdV-4 entry. In addition, we found that FAdV-4 entry was dependent on membrane cholesterol, while neither the knockdown of caveolin nor the inhibition of a tyrosine kinase-based signalling cascade affected FAdV-4 infection. These results suggested that FAdV-4 entry required cholesterol but not caveolae. We also found that macropinocytosis played a role, and phosphatidylinositol 3-kinase (PI3K) was required for FAdV-4 internalization. However, inhibitors of endosomal acidification did not prevent FAdV-4 entry. Taken together, our findings demonstrate that FAdV-4 enters LMH cells through dynamin- and cholesterol-dependent clathrin-mediated endocytosis, accompanied by the involvement of macropinocytosis requiring PI3K. Our work potentially provides insight into the entry mechanisms of other avian adenoviruses.

Rab32 promotes glioblastoma migration and invasion via regulation of ERK/Drp1-mediated mitochondrial fission.

The highly widespread and infiltrative nature of glioblastoma multiforme (GBM) makes complete surgical resection hard, causing high recurrence rate and poor patients' prognosis. However, the mechanism underlying GBM migration and invasion is still unclear. In this study, we investigated the role of a Ras-related protein Rab32 on GBM and uncovered its underlying molecular and subcellular mechanisms that contributed to GBM aggressiveness. The correlation of Rab32 expression with patient prognosis and tumor grade was investigated by public dataset analysis and clinical specimen validation. The effect of Rab32 on migration and invasion of GBM had been evaluated using wound healing assay, cell invasion assay, as well as protein analysis upon Rab32 manipulations. Mitochondrial dynamics of cells upon Rab32 alterations were detected by immunofluorescence staining and western blotting. Both the subcutaneous and intracranial xenograft tumor model were utilized to evaluate the effect of Rab32 on GBM in vivo. The expression level of Rab32 is significantly elevated in the GBM, especially in the most malignant mesenchymal subtype, and is positively correlated with tumor pathological grade and poor prognosis. Knockdown of Rab32 attenuated the capability of GBM's migration and invasion. It also suppressed the expression levels of invasion-related proteins (MMP2 and MMP9) as well as mesenchymal transition markers (N-cadherin, vimentin). Interestingly, Rab32 transported Drp1 to mitochondrial from the cytoplasm and modulated mitochondrial fission in an ERK1/2 signaling-dependent manner. Furthermore, silencing of Rab32 in vivo suppressed tumor malignancy via ERK/Drp1 axis. Rab32 regulates ERK1/2/Drp1-dependent mitochondrial fission and causes mesenchymal transition, promoting migration and invasion of GBM. It serves as a novel therapeutic target for GBM, especially for the most malignant mesenchymal subtype. Schematic of Rab32 promotes GBM aggressiveness via regulation of ERK/Drp1-mediated mitochondrial fission. Rab32 transports Drp1 from the cytoplasm to the mitochondria and recruits ERK1/2 to activate the ser616 site of Drp1, which in turn mediates mitochondrial fission and promotes mesenchymal transition, migration and invasion of GBM.

Chemical inhibition of mitochondrial fission via targeting the DRP1-receptor interaction.

Mitochondrial fission is critical for mitochondrial dynamics and homeostasis. The dynamin superfamily GTPase DRP1 is recruited by three functionally redundant receptors, MFF, MiD49, and MiD51, to mitochondria to drive fission. Here, we exploit high-content live-cell imaging to screen for mitochondrial fission inhibitors and have developed a covalent compound, mitochondrial division inhibitor (MIDI). MIDI treatment potently blocks mitochondrial fragmentation induced by mitochondrial toxins and restores mitochondrial morphology in fusion-defective cells carrying pathogenic mitofusin and OPA1 mutations. Mechanistically, MIDI does not affect DRP1 tetramerization nor DRP1 GTPase activity but does block DRP1 recruitment to mitochondria. Subsequent biochemical and cellular characterizations reveal an unexpected mechanism that MIDI targets DRP1 interaction with multiple receptors via covalent interaction with DRP1-C367. Taken together, beyond developing a potent mitochondrial fission inhibitor that profoundly impacts mitochondrial morphogenesis, our study establishes proof of concept for developing protein-protein interaction inhibitors targeting DRP1.

Human atlastins are sufficient to drive the fusion of liposomes with a physiological lipid composition.

The dynamin-like GTPase atlastin is believed to be the minimal machinery required for homotypic endoplasmic reticulum (ER) membrane fusion, mainly because Drosophila atlastin is sufficient to drive liposome fusion. However, it remains unclear whether mammalian atlastins, including the three human atlastins, are sufficient to induce liposome fusion, raising doubts about their major roles in mammalian cells. Here, we show that all human atlastins are sufficient to induce fusion when reconstituted into liposomes with a lipid composition mimicking that of the ER. Although the fusogenic activity of ATL1, which is predominantly expressed in neuronal cells, was weaker than that of ATL2 or ATL3, the addition of M1-spastin, a neuron-specific factor, markedly increased ATL1-mediated liposome fusion. Although we observed efficient fusion between ER microsomes isolated from cultured, non-neuronal cells that predominantly express ATL2-1, an autoinhibited isoform of ATL2, ATL2-1 failed to support liposome fusion by itself as reported previously, indicating that cellular factors enable ATL2-1 to mediate ER fusion in vivo.

Ginsenoside Rg1 alleviates lipopolysaccharide-induced pyroptosis in human periodontal ligament cells via inhibiting Drp1-mediated mitochondrial fission.

OBJECTIVE: The present study aimed to investigate whether Ginsenoside Rg1 alleviated lipopolysaccharide (LPS) - induced pyroptosis of human periodontal ligament cells (HPDLCs) and further explore the underlying mechanism. DESIGN: Cell viability was detected using the CCK-8 assay. Proinflammatory cytokine secretion and lactate dehydrogenase release were examined by ELISA. Flow cytometry analysis was conducted to determine the pyroptosis ratio, and ATP production was estimated using the ATP assay kit. Fluorescence staining was utilized to visualize mitochondrial morphology and analyze mitochondrial reactive oxygen species (mtROS), and the mitochondrial membrane potential level. Western blot and qRT-PCR were used to determine the expression of signaling pathway-related proteins and mRNA, respectively. RESULTS: The results discovered that Ginsenoside Rg1 treatment enhanced cell viability in comparison to LPS stimulation, attenuated pyroptosis in HPDLCs, and reduced the release of lactate dehydrogenase, IL-1ß, and IL-18 significantly. Additionally, we found that Ginsenoside Rg1 upregulated ATP content and mitochondrial membrane potential level while reducing aberrant mitochondrial fission and mtROS production. Mechanistically, we found that Ginsenoside Rg1 upregulated dynamin-related protein 1 (Drp1) phosphorylation at Ser 637 in an AMP-activated protein kinase (AMPK)-dependent manner, and reduced pyroptosis-related proteins expression, including NLRP3, ASC, Caspase-1, and GSDMD-NT. CONCLUSIONS: These findings demonstrate that Ginsenoside Rg1 treatment attenuates LPS-induced pyroptosis and inflammation damage in HPDLCs, which may connect to the activation of the AMPK/Drp1/NLRP3 signaling pathway. Moreover, the results offer a potential theoretical foundation for applying Ginsenoside Rg1 in inflammatory diseases such as periodontitis.

DRP1 mutations associated with EMPF1 encephalopathy alter mitochondrial membrane potential and metabolic programs.

Mitochondria and peroxisomes are dynamic signaling organelles that constantly undergo fission, driven by the large GTPase dynamin-related protein 1 (DRP1; encoded by DNM1L). Patients with de novo heterozygous missense mutations in DNM1L present with encephalopathy due to defective mitochondrial and peroxisomal fission (EMPF1) - a devastating neurodevelopmental disease with no effective treatment. To interrogate the mechanisms by which DRP1 mutations cause cellular dysfunction, we used human-derived fibroblasts from patients who present with EMPF1. In addition to elongated mitochondrial morphology and lack of fission, patient cells display lower coupling efficiency, increased proton leak and upregulation of glycolysis. Mitochondrial hyperfusion also results in aberrant cristae structure and hyperpolarized mitochondrial membrane potential. Peroxisomes show a severely elongated morphology in patient cells, which is associated with reduced respiration when cells are reliant on fatty acid oxidation. Metabolomic analyses revealed impaired methionine cycle and synthesis of pyrimidine nucleotides. Our study provides insight into the role of mitochondrial dynamics in cristae maintenance and the metabolic capacity of the cell, as well as the disease mechanism underlying EMPF1.

Mitochondria-associated endoplasmic reticulum membranes promote mitochondrial fission through AKAP1-Drp1 pathway in podocytes under high glucose conditions.

Excessive mitochondrial fission in podocytes is a critical feature of diabetic nephropathy (DN). Mitochondria-associated endoplasmic reticulum membranes (MAMs) are contact sites between the endoplasmic reticulum (ER) and mitochondria, which are suggested to be related to mitochondrial function. However, the role of MAMs in mitochondrial dynamics disorder in podocytes remains unknown. Here, we firstly reported a novel mechanism of MAMs' effects on mitochondrial dynamics in podocytes under diabetic conditions. Increased MAMs were found in diabetic podocytes in vivo and in vitro, which were positively correlated with excessive mitochondrial fission. What's more, we also found that A-kinase anchoring protein 1 (AKAP1) was located in MAMs, and its translocation to MAMs was increased in podocytes cultured with high glucose (HG). In addition, AKAP1 knockdown significantly reduced mitochondrial fission and attenuated high glucose induced-podocyte injury through regulating phosphorylation of dynamin-related protein 1 (Drp1) and its subsequent mitochondrial translocation. On the contrary, AKAP1 overexpression in these podocytes showed the opposite effect. Finally, pharmacological inhibition of Drp1 alleviated excessive mitochondrial fission and podocyte damage in AKAP1 overexpressed podocytes. Our data suggest that MAMs were increased in podocytes under diabetic conditions, leading to excessive mitochondrial fission and podocyte damage through AKAP1-Drp1 signaling.

The dynamin-related protein Vps1 and the peroxisomal membrane protein Pex27 function together during peroxisome fission.

Dynamin-related proteins (Drps) mediate a variety of membrane remodelling processes. The Saccharomyces cerevisiae Drp, Vps1, is required for endocytosis, endosomal sorting, vacuole fusion, and peroxisome fission and breakdown. How Drps, and in particular Vps1, can function at so many different subcellular locations is of interest to our understanding of cellular organisation. We found that the peroxisomal membrane protein Pex27 is specifically required for Vps1-dependent peroxisome fission in proliferating cells but is not required for Dnm1-dependent peroxisome fission. Pex27 accumulates in constricted regions of peroxisomes and affects peroxisome geometry upon overexpression. Moreover, Pex27 physically interacts with Vps1 in vivo and is required for the accumulation of a GTPase-defective Vps1 mutant (K42A) on peroxisomes. During nitrogen starvation, a condition that halts cell division and induces peroxisome breakdown, Vps1 associates with the pexophagophore. Pex27 is neither required for Vps1 recruitment to the pexophagophore nor for pexophagy. Our study identifies Pex27 as a Vps1-specific partner for the maintenance of peroxisome number in proliferating yeast cells.

Ovarian Cancer: A Landscape of Mitochondria with Emphasis on Mitochondrial Dynamics.

Ovarian cancer (OC) represents the main cause of death from gynecological malignancies in western countries. Altered cellular and mitochondrial metabolism are considered hallmarks in cancer disease. Several mitochondrial aspects have been found altered in OC, such as the oxidative phosphorylation system, oxidative stress and mitochondrial dynamics. Mitochondrial dynamics includes cristae remodeling, fusion, and fission processes forming a dynamic mitochondrial network. Alteration of mitochondrial dynamics is associated with metabolic change in tumour development and, in particular, the mitochondrial shaping proteins appear also to be responsible for the chemosensitivity and/or chemoresistance in OC. In this review a focus on the mitochondrial dynamics in OC cells is presented.

Mito-TEMPO protects preimplantation porcine embryos against mitochondrial fission-driven apoptosis through DRP1/PINK1-mediated mitophagy.

AIMS: Mdivi-1 (Md-1) is a well-known inhibitor of mitochondrial fission and mitophagy. The mitochondrial superoxide scavenger Mito-TEMPO (MT) exerts positive effects on the developmental competence of pig embryos. This study aimed to explore the adverse effects of Md-1 on developmental capacity in porcine embryos and the protective effects of MT against Md-1-induced injury. MAIN METHODS: We exposed porcine embryos to Md-1 (10 and 50 µM) for 2 days after in vitro fertilization (IVF). MT (0.1 µM) treatment was applied for 4 days after exposing embryos to Md-1. We assessed blastocyst development, DNA damage, mitochondrial superoxide production, and mitochondrial distribution using TUNEL assay, Mito-SOX, and Mito-tracker, respectively. Subsequently, the expression of PINK1, DRP1, and p-DRP1Ser616 was evaluated via immunofluorescence staining and Western blot analysis. KEY FINDINGS: Md-1 compromised the developmental competence of blastocysts. Apoptosis and mitochondrial superoxide production were significantly upregulated in 50 µM Md-1-treated embryos, accompanied by a downregulation of p-DRP1Ser616, PINK1, and LC3B levels and lower mitophagy activity at the blastocyst stage. We confirmed the protective effects of MT against the detrimental effect of Md-1 on blastocyst developmental competence, mitochondrial fission, and DRP1/PINK1-mediated mitophagy activation. Eventually, MT recovered DRP1/PINK1-mediated mitophagy and mitochondrial fission by inhibiting superoxide production in Md-1-treated embryos. SIGNIFICANCE: MT protects against detrimental effects of Md-1 on porcine embryos by suppressing superoxide production. These findings expand available scientific knowledge on improving outcomes of IVF.

Inhibition of Drp1-dependent mitochondrial fission by natural compounds as a therapeutic strategy for organ injuries.

Mitochondria are morphologically dynamic organelles frequently undergoing fission and fusion processes that regulate mitochondrial integrity and bioenergetics. These processes are considered critical for cell survival. The mitochondrial fission process regulates mitochondrial biogenesis and mitophagy. It is associated with apoptosis, while mitochondrial fusion controls the accurate distribution of mitochondrial DNA and metabolic substances across the mitochondria. Excessive mitochondrial fission results in mitochondrial structural changes, dysfunction, and cell damage. Accumulating evidence demonstrates that mitochondrial dynamics affect neurodegenerative and cardiovascular diseases along with several other diseases. Biological molecules regulating the process of mitochondrial fission are potential targets for developing therapeutic agents. Many natural products target the dynamin-related protein 1 (Drp1)-dependent mitochondrial fission pathway, and their inhibitory effects ameliorate mitochondrial fragmentation. In this article, we reviewed the research literature that describes Drp1-dependent inhibition as a mechanism for the protective effects of natural compounds.

Structural and functional insights into the chloroplast division site regulators PARC6 and PDV1 in the intermembrane space.

Chloroplast division involves the coordination of protein complexes from the stroma to the cytosol. The Min system of chloroplasts includes multiple stromal proteins that regulate the positioning of the division site. The outer envelope protein PLASTID DIVISION1 (PDV1) was previously reported to recruit the cytosolic chloroplast division protein ACCUMULATION AND REPLICATION OF CHLOROPLAST5 (ARC5). However, we show here that PDV1 is also important for the stability of the inner envelope chloroplast division protein PARALOG OF ARC6 (PARC6), a component of the Min system. We solved the structure of both the C-terminal domain of PARC6 and its complex with the C terminus of PDV1. The formation of an intramolecular disulfide bond within PARC6 under oxidized conditions prevents its interaction with PDV1. Interestingly, this disulfide bond can be reduced by light in planta, thus promoting PDV1-PARC6 interaction and chloroplast division. Interaction with PDV1 can induce the dimerization of PARC6, which is important for chloroplast division. Magnesium ions, whose concentration in chloroplasts increases upon light exposure, also promote the PARC6 dimerization. This study highlights the multilayer regulation of the PDV1-PARC6 interaction as well as chloroplast division.

[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.

The ß2-adrenergic receptor agonist formoterol restores mitochondrial homeostasis in glucose-induced renal proximal tubule injury through separate integrated pathways.

Mitochondrial dysfunction drives the development and progression of diabetic kidney disease (DKD). Previously, we discovered that the ß2-adrenergic receptor (AR) agonist formoterol regulates mitochondrial dynamics in the hyperglycemic renal proximal tubule. The goal of this study was to identify signaling mechanisms through which formoterol restores the mitochondrial fission/fusion proteins Drp1 and Mfn1. Using primary renal proximal tubule cells (RPTC), the effect of chronic high glucose on RhoA/ROCK1/Drp1 and Raf/MEK1/2/ERK1/2/Mfn1 signaling was determined. In glucose-treated RPTC, RhoA became hyperactive, leading to ROCK1-induced activation of Drp1. Treatment with formoterol and/or pharmacological inhibitors targeting RhoA, ROCK1 and Drp1 blocked RhoA and Drp1 hyperactivity. Inhibiting this pathway also restored maximal mitochondrial respiration. By preventing Gßγ signaling with gallein, we determined that formoterol signals through the Gßγ subunit of the ß2-AR to restore RhoA and Drp1. Furthermore, formoterol restored this pathway by blocking binding of RhoA with the guanine nucleotide exchange factor p114RhoGEF. Formoterol also restored the mitochondrial fusion protein Mfn1 through a second Gßγ-dependent mechanism composed of Raf/MEK1/2/ERK1/2/Mfn1. Glucose-treated RPTC exhibited decreased Mfn1 activity, which was restored with formoterol. Pharmacological inhibition of Gßγ, Raf and MEK1/2 also restored Mfn1 activity. We demonstrate that glucose promotes the interaction between RhoA and p114RhoGEF, leading to increased RhoA and ROCK1-mediated activation of Drp1, and decreases Mfn1 activity through Raf/MEK1/2/ERK1/2. Formoterol restores these pathways and mitochondrial function in response to elevated glucose by activating separate yet integrative pathways that promote mitochondrial biogenesis, decreased fission and increased fusion in RPTC, further supporting its potential as a therapeutic for DKD.

Wiskostatin and other carbazole scaffolds as off target inhibitors of dynamin I GTPase activity and endocytosis.

Wiskostatin (1-(3,6-dibromo-9H-carbazol-9-yl)-3-(dimethylamino)propan-2-ol) (1) is a carbazole-based compound reported as a specific and relatively potent inhibitor of the N-WASP actin remodelling complex (S-isomer EC50 = 4.35 µM; R-isomer EC50 = 3.44 µM). An NMR solution structure showed that wiskostatin interacts with a cleft in the regulatory GTPase binding domain of N-WASP. However, numerous studies have reported wiskostatin's actions on membrane transport and cytokinesis that are independent of the N-WASP-Arp2/3 complex pathway, but offer limited alternative explanation. The large GTPase, dynamin has established functional roles in these pathways. This study reveals that wiskostatin and its analogues, as well as other carbazole-based compounds, are inhibitors of helical dynamin GTPase activity and endocytosis. We characterise the effects of wiskostatin on in vitro dynamin GTPase activity, in-cell endocytosis, and determine the importance of wiskostatin functional groups on these activities through design and synthesis of libraries of wiskostatin analogues. We also examine whether other carbazole-based scaffolds frequently used in research or the clinic also modulate dynamin and endocytosis. Understanding off-targets for compounds used as research tools is important to be able to confidently interpret their action on biological systems, particularly when the target and off-targets affect overlapping mechanisms (e.g. cytokinesis and endocytosis). Herein we demonstrate that wiskostatin is a dynamin inhibitor (IC50 20.7 ± 1.2 µM) and a potent inhibitor of clathrin mediated endocytosis (IC50 = 6.9 ± 0.3 µM). Synthesis of wiskostatin analogues gave rise to 1-(9H-carbazol-9-yl)-3-((4-methylbenzyl)amino)propan-2-ol (35) and 1-(9H-carbazol-9-yl)-3-((4-chlorobenzyl)amino)propan-2-ol (43) as potent dynamin inhibitors (IC50 = 1.0 ± 0.2 µM), and (S)-1-(3,6-dibromo-9H-carbazol-9-yl)-3-(dimethylamino)propan-2-ol (8a) and (R)-1-(3,6-dibromo-9H-carbazol-9-yl)-3-(dimethylamino)propan-2-ol (8b) that are amongst the most potent inhibitors of clathrin mediated endocytosis yet reported (IC50 = 2.3 ± 3.3 and 2.1 ± 1.7 µM, respectively).

Ginsenoside CK improves skeletal muscle insulin resistance by activating DRP1/PINK1-mediated mitophagy.

Skeletal muscle insulin resistance is the main cause of type 2 diabetes, and mitochondria play a key role. Ginsenoside CK is the main active compound of ginseng with a variety of therapeutic effects, but few studies have reported on its mechanism towards skeletal muscle insulin resistance. Here, we found that CK significantly increased skeletal muscle insulin sensitivity, thereby alleviating hyperglycemia and insulin resistance. Furthermore, the effects of CK on skeletal muscle were associated with an improved mitochondrial fusion/fission dynamics balance and fatty acid oxidation. In fatty acid (FA)-induced C2C12 cells, CK promoted the translocation of GLUT4 to the cell membrane to improve glucose uptake and glycogen synthesis and also enhanced the mitochondrial quality. CK ameliorated the damaged mitochondrial membrane potential (ΔΨm), which was based on mitophagy activation. After the knockdown of mitophagy-related receptors, we found that DRP1/PINK1 was the key pathway of CK-induced mitophagy. These findings indicated that ginsenoside CK is a promising lead compound against diabetes.