Dynamin-related Protein 1 Inhibitor Research Articles

Decreasing mitochondrial fission ameliorates HIF-1α-dependent pathological retinal angiogenesis.

Angiogenesis plays a critical role in many pathological processes, including irreversible blindness in eye diseases such as retinopathy of prematurity. Endothelial mitochondria are dynamic organelles that undergo constant fusion and fission and are critical signalling hubs that modulate angiogenesis by coordinating reactive oxygen species (ROS) production and calcium signalling and metabolism. In this study, we investigated the role of mitochondrial dynamics in pathological retinal angiogenesis. We showed that treatment with vascular endothelial growth factor (VEGF; 20 ng/ml) induced mitochondrial fission in HUVECs by promoting the phosphorylation of dynamin-related protein 1 (DRP1). DRP1 knockdown or pretreatment with the DRP1 inhibitor Mdivi-1 (5 μM) blocked VEGF-induced cell migration, proliferation, and tube formation in HUVECs. We demonstrated that VEGF treatment increased mitochondrial ROS production in HUVECs, which was necessary for HIF-1α-dependent glycolysis, as well as proliferation, migration, and tube formation, and the inhibition of mitochondrial fission prevented VEGF-induced mitochondrial ROS production. In an oxygen-induced retinopathy (OIR) mouse model, we found that active DRP1 was highly expressed in endothelial cells in neovascular tufts. The administration of Mdivi-1 (10 mg·kg-1·d-1, i.p.) for three days from postnatal day (P) 13 until P15 significantly alleviated pathological angiogenesis in the retina. Our results suggest that targeting mitochondrial fission may be a therapeutic strategy for proliferative retinopathies and other diseases that are dependent on pathological angiogenesis.

Abstract P3178: Gja1-20k Is Downstream Of Drp1 And Can Rescue Drp1 Suppression In Mouse Heart

Dynamin-Related Protein 1 (DRP1) is a major mediator of mitochondrial fission and its mutation results with hyper-fused mitochondria and, in mice, a dilated cardiomyopathy. GJA1-20k is the most abundant N-terminus truncated isoform of Connexin 43 (Cx43) formed by internal translation and was originally identified as a trafficking subunit of Cx43. It has been recently identified that GJA1-20k is located on the mitochondrial membrane and plays a role in mediating ischemia preconditioning by promoting a protective mitochondrial fission. Interestingly, GJA1-20k mediated fission occurs even under DRP1 inhibition in vitro. Here, we hypothesize that GJA1-20k is downstream of DRP1 and that exogenous GJA1-20k administration rescues hyper-fusion of mitochondria in DRP1-suppressed mouse hearts. Using an AAV9-mediated DRP1 dominant negative mutant (Drp1-K38A) to suppress DRP1, we confirmed that the Drp1-K38A mouse hearts have an increase in cardiac mitochondrial size (158.9 ± 3.7% of control) and decrease in cardiac function (ejection fraction is 43.1 ± 1.5% versus 64.0 ± 4.1% in control animals), as assayed by electron microscopy and in vivo echocardiography, respectively. However, the administration of exogenous GJA1-20k via AAV9 transduction normalizes mitochondrial size (net 14.1 ± 3.3% decrease from control group) and rescues cardiac function (ejection fraction 61.6 ± 2.7%), suggesting that the progression to the cardiomyopathy is prevented by GJA1-20k administration. In summary, GJA1-20k functions downstream of DRP1 and can rescue the effect DRP1 suppression on mitochondrial size and cardiac function.

Discovery of Potent Allosteric DRP1 Inhibitors by Disrupting Protein-Protein Interaction with MiD49.

Mitochondrial dysfunction has been attributed to many disease indications, including metabolic, cardiovascular, neoplastic, and neurodegenerative diseases. Dynamin related protein 1 (DRP1) is crucial in regulating mitochondrial fission and maintaining mitochondrial homeostasis. MiD49 is a dynamic peripheral protein receptor on the surface of the mitochondrial membrane that recruits DRP1 protein to induce mitochondrial binary fission. By targeting the protein-protein interaction of DRP1/MiD49, we have discovered a novel and potent allosteric DRP1 inhibitor that inhibits mitochondria fragmentation in vitro. X-ray cocrystal structure revealed that it locked the closed DRP1 conformation by induced dimerization.

Prostacyclin mimetics inhibit DRP1-mediated pro-proliferative mitochondrial fragmentation in pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a rare cardiopulmonary disorder, involving the remodelling of the small pulmonary arteries. Underlying this remodelling is the hyper-proliferation of pulmonary arterial smooth muscle cells within the medial layers of these arteries and their encroachment on the lumen. Previous studies have demonstrated an association between excessive mitochondrial fragmentation, a consequence of increased expression and post-translational activation of the mitochondrial fission protein dynamin-related protein 1 (DRP1), and pathological proliferation in PASMCs derived from PAH patients. However, the impact of prostacyclin mimetics, widely used in the treatment of PAH, on this pathological mitochondrial fragmentation remains unexplored. We hypothesise that these agents, which are known to attenuate the proliferative phenotype of PAH PASMCs, do so in part by inhibiting mitochondrial fragmentation. In this study, we confirmed the previously reported increase in DRP1-mediated mitochondrial hyper-fragmentation in PAH PASMCs. We then showed that the prostacyclin mimetic treprostinil signals via either the Gs-coupled IP or EP2 receptor to inhibit mitochondrial fragmentation and the associated hyper-proliferation in a manner analogous to the DRP1 inhibitor Mdivi-1. We also showed that treprostinil recruits either the IP or EP2 receptor to activate PKA and induce the phosphorylation of DRP1 at the inhibitory residue S637 and inhibit that at the stimulatory residue S616, both of which are suggestive of reduced DRP1 fission activity. Like treprostinil, MRE-269, an IP receptor agonist, and butaprost, an EP2 receptor agonist, attenuated DRP1-mediated mitochondrial fragmentation through PKA. We conclude that prostacyclin mimetics produce their anti-proliferative effects on PAH PASMCs in part by inhibiting DRP1-mediated mitochondrial fragmentation.

Inhibition of DRP1-dependent mitochondrial fission by Mdivi-1 alleviates atherosclerosis through the modulation of M1 polarization

BackgroundInflammation and immune dysfunction with classically activated macrophages(M1) infiltration are important mechanisms in the progression of atherosclerosis (AS). Dynamin-related protein 1 (DRP1)-dependent mitochondrial fission is a novel target for alleviating inflammatory diseases. This study aimed to investigate the effects of DRP1 inhibitor Mdivi-1 on AS.MethodsApoE−/− mice were fed with a high-fat diet supplemented with or without Mdivi-1. RAW264.7 cells were stimulated by ox-LDL, pretreated with or without MCC950, Mito-TEMPO, or Mdivi-1. The burden of plaques and foam cell formation were determined using ORO staining. The blood lipid profles and inflammatory cytokines in serum were detected by commercial kits and ELISA, respectively. The mRNA expression of macrophage polarization markers, activation of NLRP3 and the phosphorylation state of DRP1 were detected. Mitochondrial reactive oxygen species (mito-ROS), mitochondrial staining, ATP level and mitochondrial membrane potential were detected by mito-SOX, MitoTracker, ATP determination kit and JC-1 staining, respectively.ResultsIn vivo, Mdivi-1 reduced the plaque areas, M1 polarization, NLRP3 activation and DRP1 phosphorylation at Ser616. In vitro, oxidized low-density lipoprotein (ox-LDL) triggered M1 polarization, NLRP3 activation and abnormal accumulation of mito-ROS. MCC950 and Mito-TEMPO suppressed M1 polarization mediated foam cell formation. Mito-TEMPO significantly inhibited NLRP3 activation. In addition, Mdivi-1 reduced foam cells by inhibiting M1 polarization. The possible mechanisms responsible for the anti-atherosclerotic effects of Mdivi-1 on reducing M1 polarization were associated with suppressing mito-ROS/NLRP3 pathway by inhibiting DRP1 mediated mitochondrial fission. In vitro, similar results were observed by DRP1 knockdown.ConclusionInhibition of DRP1-dependent mitochondrial fission by Mdivi-1 alleviated atherogenesis via suppressing mito-ROS/NLRP3-mediated M1 polarization, indicating DRP1-dependent mitochondrial fission as a potential therapeutic target for AS.

Role of KLF4/NDRG1/DRP1 axis in hypoxia-induced pulmonary hypertension

N-myc downstream regulated gene 1 (NDRG1) has recently drawn increasing attention because of its involvement in angiogenesis, cell proliferation, and differentiation. We used in vitro [human pulmonary artery smooth muscle cells (hPASMCs)] and in vivo (rat) models under hypoxic conditions and found a vital role of NDRG1 in reducing apoptosis and increasing proliferation and migration by overexpressing and knocking down NDRG1. We also proved that hypoxia induced the protein expression of dynamin-related protein 1 (DRP1) and stimulated The phosphatidylinositol-3-kinase (PI3K)/ Protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathways, and these effects were reversed by NDRG1 knockdown. The relationship between NDRG1 and DRP1 and the PI3K/Akt/mTOR pathway was further evaluated by adding mdivi-1 (DRP1 inhibitor) or LY294002 (PI3K inhibitor). NDRG1 was found to regulate the proliferation, apoptosis, and migration of hypoxia-treated hPASMCs via DRP1 and PI3K/Akt/mTOR signaling pathways. We explored the upstream regulators of NDRG1 using in vivo and in vitro hypoxia models. Hypoxia was found to upregulate and downregulate KLF transcription factor 4 (KLF4) protein expression in the cytoplasm and nucleus, respectively. Further, we showed that KLF4 regulated the proliferation and migration of hypoxia-treated hPASMCs via NDRG1. These results indicated a link between KLF4, NDRG1, and DRP1 for the first time, providing new ideas for treating hypoxic pulmonary hypertension.

Limiting mitochondrial plasticity by targeting DRP1 induces metabolic reprogramming and reduces breast cancer brain metastases.

Disseminated tumor cells with metabolic flexibility to utilize available nutrients in distal organs persist, but the precise mechanisms that facilitate metabolic adaptations remain unclear. Here we show fragmented mitochondrial puncta in latent brain metastatic (Lat) cells enable fatty acid oxidation (FAO) to sustain cellular bioenergetics and maintain redox homeostasis. Depleting the enriched dynamin-related protein 1 (DRP1) and limiting mitochondrial plasticity in Lat cells results in increased lipid droplet accumulation, impaired FAO and attenuated metastasis. Likewise, pharmacological inhibition of DRP1 using a small-molecule brain-permeable inhibitor attenuated metastatic burden in preclinical models. In agreement with these findings, increased phospho-DRP1 expression was observed in metachronous brain metastasis compared with patient-matched primary tumors. Overall, our findings reveal the pivotal role of mitochondrial plasticity in supporting the survival of Lat cells and highlight the therapeutic potential of targeting cellular plasticity programs in combination with tumor-specific alterations to prevent metastatic recurrences.

Abstract 4831: Mitochondrial plasticity enables metabolic reprogramming and metastatic latency

Abstract Introduction: Metastatic relapses are common in epidermal growth factor receptor 2 (HER2) amplified breast cancer patients considered disease-free after primary diagnosis and treatment. Stem-like disseminated tumor cells with specialized metastatic traits adapt and survive as latent metastases in distal organs by overcoming oxidative stress, nutrient limitation, microenvironmental and immune defenses. These latent subclinical metastatic cells are responsible for late recurrences. Understanding the traits and vulnerabilities of these cells is critical for developing strategies to prevent metastatic relapse. Method: Here, we employed mouse models and patient samples along with RNA sequencing, Steady-state metabolite analysis, Lipid profiling, C13-isotope tracing, C12-BODIPY pulse chase, Lentiviral shRNA Knockdown, Transmission electron microscopy etc. to investigate how disseminated latent metastatic cells meet their cellular energetic demands and initiate overt metastasis in the lipid-rich brain microenvironment. Result: The ability to utilize available nutrients in distal organs and rewire metabolism is critical for survival of disseminated tumor cells. We report that latent metastatic cells in the brain uptake, store, and utilize fatty acids secreted by tumor-associated reactive astrocytes to meet their cellular energetic needs. Fatty acid oxidation (FAO) and dynamin-related protein 1(DRP1) driven punctate mitochondria enabled utilization of fatty acids and promote survival of latent cells by maintaining cellular bioenergetics and redox homeostasis. Attenuating fatty acid oxidation by genetic and pharmacological inhibition disrupted mitochondrial dynamics and limits metastatic incidence. Likewise, depleting DRP1 altered mitochondrial dynamics that results in reduced FAO, increased reactive oxygen species and attenuated metastatic latency and relapse in HER2+ breast cancer brain metastatic models. Furthermore, comparison with patient-matched primary tumor and brain metastasis identified increased pDRP1 expression in brain metastatic lesions. Moreover, pharmacological inhibition of DRP1 reduced brain metastatic burden in these preclinical models. Conclusion: Despite significant advances in targeted therapies, HER2+ breast cancer patients often develop brain metastasis and their prevention and management remain an unmet need. Our findings in latent HER2+ breast cancer brain metastatic models demonstrate mitochondrial plasticity and altered fatty acid metabolism are key determinants of metastatic adaptation to the brain and establish DRP1 as a promising therapeutic target for treating brain metastasis. Key Words: Breast Cancer Brain Metastasis, Mitochondrial Plasticity, DRP1, Metastatic Latency, Redox Homeostasis. Citation Format: Pravat Kumar Parida, Srinivas Malladi. Mitochondrial plasticity enables metabolic reprogramming and metastatic latency. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4831.

Increased mitochondrial fission induces NLRP3/cGAS-STING mediated pro-inflammatory pathways and apoptosis in UVB-irradiated immortalized human keratinocyte HaCaT cells

Ultraviolet B (UVB) irradiation causes skin inflammation and apoptosis. Mitochondria are highly dynamic and undergo constant fusion and fission that are essential for maintaining physiological functions of cells. Although dysfunction of mitochondria has been implicated in skin damages, little is known about the roles of mitochondrial dynamics in these processes. UVB irradiation increases abnormal mitochondrial content but decreases mitochondrial volume in immortalized human keratinocyte HaCaT cells. UVB irradiation resulted in marked upregulation of mitochondrial fission protein dynamin-related protein 1 (DRP1) and downregulation of mitochondrial outer membrane fusion proteins 1 and 2 (MFN1 and MFN2) in HaCaT cells. Mitochondrial dynamics was discovered to be crucial for NLRP3 inflammasome and cGAS-STING pathway activation, as well as the induction of apoptosis. Inhibition of mitochondrial fission by treatments with a DRP1 inhibitor, mdivi-1, or with DRP1-targeted siRNA, efficiently prevented UVB-induced NLRP3/cGAS-STING mediated pro-inflammatory pathways or apoptosis in the HaCaT cells, whereas inhibition of mitochondrial fusion with MFN1and 2 siRNA increased these pro-inflammatory pathways or apoptosis. The enhanced mitochondrial fission and reduced fusion caused the up-regulation of reactive oxygen species (ROS). Application of an antioxidant, N-acetyl-l-cysteine (NAC), which scavenges excessive ROS, attenuated inflammatory responses through suppressing NLRP3 inflammasome and cGAS-STING pathway activation, and rescued cells from apoptosis caused by UVB-irradiation. Together, our findings revealed the regulation of NLRP3/cGAS-STING inflammatory pathways and apoptosis by mitochondrial fission/fusion dynamics in UVB-irradiated HaCaT cells, providing a new strategy for the therapy of UVB skin injury.

DRP1 Inhibition Enhances Venetoclax-Induced Mitochondrial Apoptosis in TP53-Mutated Acute Myeloid Leukemia Cells through BAX/BAK Activation.

Although TP53 mutations in acute myeloid leukemia (AML) are associated with poor response to venetoclax, the underlying resistance mechanism remains unclear. Herein, we investigated the functional role of dynamin-related protein 1 (DRP1) in venetoclax sensitivity in AML cells with respect to TP53 mutation status. Effects of DRP1 inhibition on venetoclax-induced cell death were compared in TP53-mutated (THP-1 and Kasumi-1) and TP53 wild-type leukemia cell lines (MOLM-13 and MV4-11), as well as in primary AML cells obtained from patients. Venetoclax induced apoptosis in TP53 wild-type AML cells but had limited effects in TP53-mutated AML cells. DRP1 expression was downregulated in MOLM-13 cells after venetoclax treatment but was unaffected in THP-1 cells. Cotreatment of THP-1 cells with venetoclax and a TP53 activator NSC59984 downregulated DRP1 expression and increased apoptosis. Combination treatment with the DRP1 inhibitor Mdivi-1 and venetoclax significantly increased mitochondria-mediated apoptosis in TP53-mutated AML cells. The combination of Mdivi-1 and venetoclax resulted in noticeable downregulation of MCL-1 and BCL-xL, accompanied by the upregulation of NOXA, PUMA, BAK, and BAX. These findings suggest that DRP1 is functionally associated with venetoclax sensitivity in TP53-mutated AML cells. Targeting DRP1 may represent an effective therapeutic strategy for overcoming venetoclax resistance in TP53-mutated AML.

STING signaling sensing of DRP1-dependent mtDNA release in kupffer cells contributes to lipopolysaccharide-induced liver injury in mice

Aberrant pro-inflammatory activation of Kupffer cells (KCs) is strongly involved in the pathogenesis of septic liver injury. Recent evidence indicates the crucial roles of excessive stimulator of interferon genes (STING) signaling activation during sepsis. However, the role of STING signaling in septic liver injury remains unclear. In this study, we demonstrated that STING signaling was markedly activated in KCs isolated from wild type mice after lipopolysaccharide (LPS) treatment. STING deficiency effectively protected liver function, attenuated systemic inflammatory response and decreased mortality in LPS-treated mice, which were aggravated by STING agonist (DMXAA). Importantly, STING signaling activation in KCs contributed to LPS-induced liver injury through promoting hepatocyte death. Mechanistically, STING signaling could be activated by release of mitochondrial DNA (mtDNA) through dynamin-related protein 1 (DRP1)-dependent mitochondrial fission in LPS-treated KCs. Additionally, LPS stimulation enhanced DRP1-dependent mitochondrial ROS production, which promoted the leak of mtDNA into the cytosol and subsequent STING signaling activation in KCs. The in vivo experiments showed that pharmacological inhibition of DRP1 with Mdivi-1 partially prevented the activation of STING signaling in KCs isolated from LPS-challenged mice, as well as alleviated liver injury and inhibited systemic inflammatory response. In summary, our study comprehensively confirmed that STING signaling senses the DRP1-dependent release of mtDNA in KCs and its activation might play a key role in LPS-induced liver injury, which offers new sights and therapeutic targets for management of septic liver injury.

Mitochondrial Micropeptide STMP1 Enhances Mitochondrial Fission to Promote Tumor Metastasis.

This study identifies the mitochondrial micropeptide STMP1 as a regulator of metastasis that promotes mitochondrial fission and tumor cell migration via DRP1 and MYH9.

Downregulation of Uncoupling Protein 2(UCP2) Mediated by MicroRNA-762 Confers Cardioprotection and Participates in the Regulation of Dynamic Mitochondrial Homeostasis of Dynamin Related Protein1 (DRP1) After Myocardial Infarction in Mice.

Acute myocardial infarction (MI) is one of the leading causes of death in the world, and its pathophysiological mechanisms have not been fully elucidated. The purpose of this study was to investigate the role and mechanism of uncoupling protein 2 (UCP2) after MI in mouse heart. Here, we examined the expression and role of UCP2 in mouse heart 4 weeks after MI. The expression of UCP2 was detected by RT-PCR and western blotting. Cardiac function, myocardial fibrosis, and cardiomyocyte apoptosis were assessed by echocardiography and immunohistochemistry. Phosphatase dynamin-related protein1 (P-DRP1) and myocardial fibrosis-related proteins were measured. Cardiomyocytes were exposed to hypoxia for 6 h to mimic the model of MI. Mdivi, an inhibitor of P-DRP1, was used to inhibit DRP1-dependent mitochondrial fission. Mitochondrial superoxide, membrane potential, oxygen consumption rate, and cardiomyocyte apoptosis were detected after hypoxia. It is shown mitochondrial superoxide, membrane potential, oxygen consumption rate, and cardiomyocyte apoptosis were dependent on the level of P-DRP1. UCP2 overexpression reduced cardiomyocyte apoptosis (fibrosis), improved cardiac function and inhibit the phosphorylation of DRP1 and the ratio of P-DRP1/DRP1. However, inhibition of DRP1 by mdivi did not further reduce cell apoptosis rate and cardiac function in UCP2 overexpression group. In addition, bioinformatics analysis, luciferase activity, and western blot assay proved UCP2 was a direct target gene of microRNA-762, a up-regulated microRNA after MI. In conclusion, UCP2 plays a protective role after MI and the mechanism is involved in microRNA-762 upstream and DRP1-dependent mitochondrial fission downstream.

Mitochondrial Fragmentation Triggers Ineffective Hematopoiesis in Myelodysplastic Syndromes.

Ineffective hematopoiesis is a fundamental process leading to the pathogenesis of myelodysplastic syndromes (MDS). However, the pathobiological mediators of ineffective hematopoiesis in MDS remain unclear. Here, we demonstrated that overwhelming mitochondrial fragmentation in mutant hematopoietic stem cells and progenitors (HSC/P) triggers ineffective hematopoiesis in MDS. Mouse modeling of CBL exon deletion with RUNX1 mutants, previously unreported comutations in patients with MDS, recapitulated not only clinically relevant MDS phenotypes but also a distinct MDS-related gene signature. Mechanistically, dynamin-related protein 1 (DRP1)-dependent excessive mitochondrial fragmentation in HSC/Ps led to excessive reactive oxygen species production, induced inflammatory signaling activation, and promoted subsequent dysplasia formation and impairment of granulopoiesis. Mitochondrial fragmentation was generally observed in patients with MDS. Pharmacologic inhibition of DRP1 attenuated mitochondrial fragmentation and rescued ineffective hematopoiesis phenotypes in mice with MDS. These findings provide mechanistic insights into ineffective hematopoiesis and indicate that dysregulated mitochondrial dynamics could be a therapeutic target for bone marrow failure in MDS. SIGNIFICANCE: We demonstrated that excessive mitochondrial fragmentation is a fundamental pathobiological phenomenon that could trigger dysplasia formation and ineffective hematopoiesis in MDS. Our findings provide mechanistic insights into ineffective hematopoiesis and suggest dysregulated mitochondrial dynamics as a therapeutic target for treating MDS.This article is highlighted in the In This Issue feature, p. 1.

Aerobic exercise suppresses hepatocellular carcinoma by downregulating dynamin-related protein 1 through PI3K/AKT pathway

ObjectiveExercise, as a common non-drug intervention, is one of several lifestyle choices known to reduce the risk of cancer. Mitochondrial division has been reported to play a key role in the occurrence and transformation of hepatocellular carcinoma (HCC). This study investigated whether exercise could regulate the occurrence and development of HCC through mitosis. MethodsBioinformatics technology was used to analyze the expression level of dynamin-related protein 1 (DRP1), a key protein of mitochondrial division. The effects of DRP1 and DRP1 inhibitor (mdivi-1) on the proliferation and migration of liver cancer cells BEL-7402 were observed using cell counting kit-8, plate colony formation, transwell cell migration, and scratch experiments. Enzyme-linked immunosorbent assay, Western blot and real-time polymerase chain reaction were used to detect the expression of DRP1 and its downstream phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway. A treadmill exercise intervention was tested in a nude mouse human liver cancer subcutaneous tumor model expressing different levels of DRP1. The size and weight of subcutaneous tumors in mice were detected before and after exercise. ResultsThe expression of DRP1 in liver cancer tissues was significantly upregulated compared with normal liver tissues (P < 0.001). The proliferation rate and the migration of BEL-7402 cells in the DRP1 over-expression group were higher than that in the control group. The mdivi-1 group showed an inhibitory effect on the proliferation and migration of BEL-7402 cells at 50 μmol/L. Aerobic exercise was able to inhibit the expression of DRP1 and decrease the size and weight of subcutaneous tumors. Moreover, the expression of phosphorylated PI3K (p-PI3K) and phosphorylated AKT (p-AKT) decreased in the exercise group. However, exercise could not change p-PI3K and p-AKT levels after knocking down DRP1 or using mdivi-1 on subcutaneous tumor. ConclusionAerobic exercise can suppress the development of tumors partially by regulating DRP1 through PI3K/AKT pathway.

Phosphorylation of Dynamin-Related Protein 1 (DRP1) Regulates Mitochondrial Dynamics and Skeletal Muscle Wasting in Cancer Cachexia.

BackgroundCancer-associated cachexia (CAC) is a syndrome characterized by skeletal muscle atrophy, and the underlying mechanisms are still unclear. Recent research studies have shed light on a noteworthy link between mitochondrial dynamics and muscle physiology. In the present study, we investigate the role of dynamin-related protein 1 (DRP1), a pivotal factor of mitochondrial dynamics, in myotube atrophy during cancer-associated cachexia.MethodsSeventy-six surgical patients, including gastrointestinal tumor and benign disease, were enrolled in the study and divided to three groups: control, non-cachexia, and cancer-associated cachexia. Demographic data were collected. Their rectus abdominis samples were acquired intraoperatively. Muscle fiber size, markers of ubiquitin proteasome system (UPS), mitochondrial ultrastructure, and markers of mitochondrial function and dynamics were assayed. A cachexia model in vitro was established via coculturing a C2C12 myotube with media from C26 colon cancer cells. A specific DRP1 inhibitor, Mdivi-1, and a lentivirus of DRP1 knockdown/overexpression were used to regulate the expression of DRP1. Muscle diameter, mitochondrial morphology, mass, reactive oxygen species (ROS), membrane potential, and markers of UPS, mitochondrial function, and dynamics were determined.ResultsPatients of cachexia suffered from a conspicuous worsened nutrition status and muscle loss compared to patients of other groups. Severe mitochondrial swelling and enlarged area were observed, and partial alterations in mitochondrial function were found in muscle. Analysis of mitochondrial dynamics indicated an upregulation of phosphorylated DRP1 at the ser616 site. In vitro, cancer media resulted in the atrophy of myotube. This was accompanied with a prominent unbalance of mitochondrial dynamics, as well as enhanced mitochondrial ROS and decreased mitochondrial function and membrane potential. However, certain concentrations of Mdivi-1 and DRP1 knockdown rebalanced the mitochondrial dynamics, mitigating this negative phenotype caused by cachexia. Moreover, overexpression of DRP1 aggravated these phenomena.ConclusionIn clinical patients, cachexia induces abnormal mitochondrial changes and possible fission activation for the atrophied muscle. Our cachexia model in vitro further demonstrates that unbalanced mitochondrial dynamics contributes to this atrophy and mitochondrial impairment, and rebuilding the balance by regulating of DRP1 could ameliorate these alterations.

DRP1 Inhibition Rescues Mitochondrial Integrity and Excessive Apoptosis in CS-A Disease Cell Models.

Cockayne syndrome group A (CS-A) is a rare recessive progeroid disorder characterized by sun sensitivity and neurodevelopmental abnormalities. Cells derived from CS-A patients present as pathological hallmarks excessive oxidative stress, mitochondrial fragmentation and apoptosis associated with hyperactivation of the mitochondrial fission dynamin related protein 1 (DRP1). In this study, by using human cell models we further investigated the interplay between DRP1 and CSA and we determined whether pharmacological or genetic inhibition of DRP1 affects disease progression. Both reactive oxygen and nitrogen species are in excess in CS-A cells and when the mitochondrial translocation of DRP1 is inhibited a reduction of these species is observed together with a recovery of mitochondrial integrity and a significant decrease of apoptosis. This study indicates that the CSA-driven modulation of DRP1 pathway is key to control mitochondrial homeostasis and apoptosis and suggests DRP1 as a potential target in the treatment of CS patients.

Glutamine deficiency promotes stemness and chemoresistance in tumor cells through DRP1-induced mitochondrial fragmentation.

Glutamine is essential for maintaining the TCA cycle in cancer cells yet they undergo glutamine starvation in the core of tumors. Cancer stem cells (CSCs), responsible for tumor recurrence are often found in the nutrient limiting cores. Our study uncovers the molecular basis and cellular links between glutamine deprivation and stemness in the cancer cells. We showed that glutamine is dispensable for the survival of ovarian and colon cancer cells while it is required for their proliferation. Glutamine starvation leads to the metabolic reprogramming in tumor cells with enhanced glycolysis and unaltered oxidative phosphorylation. Production of reactive oxygen species (ROS) in glutamine limiting condition induces MAPK-ERK1/2 signaling pathway to phosphorylate dynamin-related protein-1(DRP1) at Ser616. Moreover, p-DRP1 promotes mitochondrial fragmentation and enhances numbers of CD44 and CD117/CD45 positive CSCs. Besides the established features of cancer stem cells, glutamine deprivation induces perinuclear localization of fragmented mitochondria and reduction in proliferation rate which are usually observed in CSCs. Treatment with glutaminase inhibitor (L-DON) mimics the effects of glutamine starvation without altering cell survival in in vitro as well as in in vivo model. Interestingly, the combinatorial treatment of L-DON with DRP1 inhibitor (MDiVi-1) reduces the stem cell population in tumor tissue in mouse model. Collectively our data suggest that glutamine deficiency in the core of tumors can increase the cancer stem cell population and the combination therapy with MDiVi-1 and L-DON is a useful approach to reduce CSCs population in tumor.

Dynamin-related protein 1 inhibition reduces hepatic PCSK9 secretion.

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. 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. 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.

Mitochondrial Fission and Mitophagy Coordinately Restrict High Glucose Toxicity in Cardiomyocytes.

Hyperglycemia-induced mitochondrial dysfunction plays a key role in the pathogenesis of diabetic cardiomyopathy. Injured mitochondrial segments are separated by mitochondrial fission and eliminated by autophagic sequestration and subsequent degradation in the lysosome, a process termed mitophagy. However, it remains poorly understood how high glucose affects the activities of, and the relationship between, mitochondrial fission and mitophagy in cardiomyocytes. In this study, we determined the functional roles of mitochondrial fission and mitophagy in hyperglycemia-induced cardiomyocyte injury. High glucose (30 mM, HG) reduced mitochondrial connectivity and particle size and increased mitochondrial number in neonatal rat ventricular cardiomyocytes, suggesting an enhanced mitochondrial fragmentation. SiRNA knockdown of the pro-fission factor dynamin-related protein 1 (DRP1) restored mitochondrial size but did not affect HG toxicity, and Mdivi-1, a DRP1 inhibitor, even increased HG-induced cardiomyocyte injury, as shown by superoxide production, mitochondrial membrane potential and cell death. However, DRP1 overexpression triggered mitochondrial fragmentation and mitigated HG-induced cardiomyocyte injury, suggesting that the increased mitochondrial fission is beneficial, rather than detrimental, to cardiomyocytes cultured under HG conditions. This is in contrast to the prevailing hypothesis that mitochondrial fragmentation mediates or contributes to HG cardiotoxicity. Meanwhile, HG reduced mitophagy flux as determined by the difference in the levels of mitochondria-associated LC3-II or the numbers of mitophagy foci indicated by the novel dual fluorescent reporter mt-Rosella in the absence and presence of the lysosomal inhibitors. The ability of HG to induce mitochondrial fragmentation and inhibit mitophagy was reproduced in adult mouse cardiomyocytes. Overexpression of Parkin, a positive regulator of mitophagy, or treatment with CCCP, a mitochondrial uncoupler, induced mitophagy and attenuated HG-induced cardiomyocyte death, while Parkin knockdown had opposite effects, suggesting an essential role of mitophagy in cardiomyocyte survival under HG conditions. Strikingly, Parkin overexpression increased mitochondrial fragmentation, while DRP1 overexpression accelerated mitophagy flux, demonstrating a reciprocal activation loop that controls mitochondrial fission and mitophagy. Thus, strategies that promote the mutual positive interaction between mitochondrial fission and mitophagy while simultaneously maintain their levels within the physiological range would be expected to improve mitochondrial health, alleviating hyperglycemic cardiotoxicity.