Dynamin Research Articles

Analysis of DNM2, EPN2 and EXOC4 relative gene expression levels in peripheral blood from Parkinson’s disease patients

Analysis of DNM2, EPN2 and EXOC4 relative gene expression levels in peripheral blood from Parkinson’s disease patients

A case of de novo dynamin 2 (DNM2)-related centronuclear myopathy with electrical but not clinical myotonia

A case of de novo dynamin 2 (DNM2)-related centronuclear myopathy with electrical but not clinical myotonia

BMP-9 and LDL crosstalk regulates ALK-1 endocytosis and LDL transcytosis in endothelial cells

Bone morphogenetic protein-9 (BMP-9) is a circulating cytokine that is known to play an essential role in the endothelial homeostasis and the binding of BMP-9 to the receptor activin-like kinase 1 (ALK-1) promotes endothelial cell quiescence. Previously, using an unbiased screen, we identified ALK-1 as a high-capacity receptor for low-density lipoprotein (LDL) in endothelial cells that mediates its transcytosis in a nondegradative manner. Here we examine the crosstalk between BMP-9 and LDL and how it influences their interactions with ALK-1. Treatment of endothelial cells with BMP-9 triggers the extensive endocytosis of ALK-1, and it is mediated by caveolin-1 (CAV-1) and dynamin-2 (DNM2) but not clathrin heavy chain. Knockdown of CAV-1 reduces BMP-9–mediated internalization of ALK-1, BMP-9–dependent signaling and gene expression. Similarly, treatment of endothelial cells with LDL reduces BMP-9–induced SMAD1/5 phosphorylation and gene expression and silencing of CAV-1 and DNM2 diminishes LDL-mediated ALK-1 internalization. Interestingly, BMP-9–mediated ALK-1 internalization strongly re-duces LDL transcytosis to levels seen with ALK-1 deficiency. Thus, BMP-9 levels can control cell surface levels of ALK-1, via CAV-1, to regulate both BMP-9 signaling and LDL transcytosis.

Small molecule inhibition of Dynamin-dependent endocytosis targets multiple niche signals and impairs leukemia stem cells

Intensive chemotherapy for acute leukemia can usually induce complete remission, but fails in many patients to eradicate the leukemia stem cells responsible for relapse. There is accumulating evidence that these relapse-inducing cells are maintained and protected by signals provided by the microenvironment. Thus, inhibition of niche signals is a proposed strategy to target leukemia stem cells but this requires knowledge of the critical signals and may be subject to compensatory mechanisms. Signals from the niche require receptor-mediated endocytosis, a generic process dependent on the Dynamin family of large GTPases. Here, we show that Dynole 34-2, a potent inhibitor of Dynamin GTPase activity, can block transduction of key signalling pathways and overcome chemoresistance of leukemia stem cells. Our results provide a significant conceptual advance in therapeutic strategies for acute leukemia that may be applicable to other malignancies in which signals from the niche are involved in disease progression and chemoresistance.

Osteopontin and LDLR Are Upregulated in Hearts of Sudden Cardiac Death Victims With Heart Failure With Preserved Ejection Fraction and Diabetes Mellitus.

Background: Diabetes mellitus (DM) is associated with increased risk of sudden cardiac death (SCD), particularly in patients with heart failure with preserved ejection fraction (HFpEF). However, there are no known biomarkers in the population with DM and HFpEF to predict SCD risk.Objectives: This study was designed to test the hypothesis that osteopontin (OPN) and some proteins previously correlated with OPN, low-density lipoprotein receptor (LDLR), dynamin 2 (DNM2), fibronectin-1 (FN1), and 2-oxoglutarate dehydrogenase-like (OGDHL), are potential risk markers for SCD, and may reflect modifiable molecular pathways in patients with DM and HFpEF.Methods: Heart tissues were obtained at autopsy from 9 SCD victims with DM and HFpEF and 10 age and gender-matched accidental death control subjects from a Finnish SCD registry and analyzed for the expression of OPN and correlated proteins, including LDLR, DNM2, FN1, and OGDHL by immunohistochemistry.Results: We observed a significant upregulation in the expression of OPN, LDLR, and FN1, and a marked downregulation of DNM2 in heart tissues of SCD victims with DM and HFpEF as compared to control subjects (p < 0.01).Conclusions: The dysregulated protein expression of OPN, LDLR, FN1, and DNM2 in patients with DM and HFpEF who experienced SCD provides novel potential modifiable molecular pathways that may be implicated in the pathogenesis of SCD in these patients. Since secreted OPN and soluble LDLR can be measured in plasma, these results support the value of further prospective studies to assess the predictive value of these plasma biomarkers and to determine whether tuning expression levels of OPN and LDLR alters SCD risk in patients with DM and HFpEF.

CONGENITAL MYOPATHIES 2: P.102 MiRNAs as biomarkers in myotubular myopathy

Myotubular myopathy (MTM) is a devastating childhood muscle disease associated with severe disabilities and early death. Maani et al (2018)., recently identified tamoxifen as a novel therapeutic candidate for MTM that improves muscle structure, strength and prolongs survival in MTM mice through modulation of dynamin-2 (DNM2), a known disease modifier. As clinical trials for tamoxifen in MTM are imminent, there remains a need for a reliable, non-invasive biomarker to reflect disease severity and treatment response, facilitating disease monitoring and testing of novel therapies.

CONGENITAL MYOPATHIES 2: P.98 A dog model for centronuclear myopathy carrying the most common DNM2 mutation

Centronuclear myopathies (CNM) are clinically and genetically heterogeneous disorders characterized by muscle atrophy and abnormal nuclear centralization in muscle fibers. Autosomal dominant CNM primarily results from mutations in DNM2, encoding the GTPase dynamin 2, a key regulator of membrane remodelling and endocytosis. The clinical presentation of the patients correlates with the position of the mutation, and ranges from severe neonatal hypotonia and respiratory distress to milder adult-onset forms. The most common mutation c.1393C>T (R465W) is found in 25% of all cases, and affected individuals typically show moderate generalized muscle weakness, ptosis, and ophthalmoplegia. Functional investigations suggest that the DNM2 mutations induce a gain-of-function, and the genetic or pharmacological downregulation of dynamin 2 efficiently antagonizes the disease development in the mouse model. Here we characterize a spontaneous CNM dog model, and we provide phenotypical, genetic, histological, and ultrastructural data through a longitudinal study over a period of 18 months. We identified the common DNM2 mutation R465W in a Border collie, and crossing with a healthy Beagle dam resulted in four affected offspring carrying the mutation, and a single healthy dog without the mutation. The affected dogs presented with mildly progressive muscle weakness from the age of 4 months, and MRI revealed muscle atrophy in particular of the paravertebral and masticatory muscles. Noteworthy, histological anomalies preceded the clinical signs, and the CNM-typical hallmarks myofiber atrophy, endomysial fibrosis, central nuclei, necklace fibers, and mitochondrial clustering were seen in biopsies at 2 months of age. Electron microscopy performed at 6 months of age detected mitochondrial anomalies, and functional investigations confirmed mitochondrial dysfunction. Overall, DNM2 dogs manifested a slowly progressive congenital myopathy and recapitulated the histological picture of the human disorder. The present study also provides insight into the sequence of events leading to muscle dysfunction. There is currently no treatment for DNM2-related CNM, and the canine model represents a valuable tool to validate pre-clinical treatment options on a large mammal based on proof-of-concepts recently obtained in the mouse.

CONGENITAL MYOPATHIES 2: P.99 Phenotyping by NMR of a novel dog model of centronuclear myopathy

A new dog model was generated with a genetic background based on a DNM2 mutation identified in a male Border Collie. The heterozygous R465W mutation of the DNM2 gene is known in humans to be responsible for a form of centronuclear myopathy (CNM). Pups developed from zygotes obtained after fertilization of Beagle oocytes with spermatozoa from the affected Border Collie. The outbred nature and the large size of the dogs contribute to their added value because they help better predict safety of translated treatments.

Dynamin 2 Is Correlated with Recurrence and Poor Prognosis of Papillary Thyroid Cancer.

BackgroundPapillary thyroid cancer (PTC) is the most common histological type of thyroid cancer. Most PTC patients have favorable outcomes, but 10% of patients still have distant metastases at presentation or during follow-up. Dynamin 2 (DNM2) is the only DNM ubiquitously expressed in human tissues, but its expression and clinical significance in PTC is still unknown.Material/MethodsIn our study, we investigated the expression of DNM2 in 112 cases of PTC and classified the patients into low and high expression of DNM2. The clinical significance of DNM2 was evaluated by assessing its correlation with the clinicopathological parameters with the chi-square method. The correlations between DNM2 expression and the disease-free survival rate or overall survival rate were assessed with the Kaplan-Meier method and the log-rank test. The independent prognostic factors of PTC were determined by the Cox-regression hazard model.ResultsPatients with low and high DNM2 expression accounted for 75% and 25% respectively in the 112 patients with PTC. High DNM2 expression was significantly associated with recurrence (P=0.014) and poor prognosis (P=0.004). In addition to tumor stage, DNM2 expression was an independent prognostic biomarker of PTC, indicating an unfavorable prognosis.ConclusionsDNM2 was an independent PTC biomarker indicating more likely recurrence and poorer prognosis. Detecting DNM2 expression may help to select the high-risk patients for adjuvant therapy.

Dynasore protects ocular surface mucosal epithelia subjected to oxidative stress by maintaining UPR and calcium homeostasis

The mucosal epithelia of the ocular surface protect against external threats to the eye. Using a model of human stratified corneal epithelial cells with mucosal differentiation, we previously demonstrated that a small molecule inhibitor of dynamin GTPases, dynasore, prevents damage to cells and their transcellular barriers when subjected to oxidative stress. Investigating mechanisms, we now report the novel finding that dynasore acts by maintaining Ca+2 homeostasis, thereby inhibiting the PERK branch of the unfolded protein response (UPR) that promotes cell death. Dynasore was found to protect mitochondria by preventing mitochondrial permeability transition pore opening (mPTP), but, unlike reports using other systems, this was not mediated by dynamin family member DRP1. Necrostatin-1, an inhibitor of RIPK1 and lytic forms of programmed cell death, also inhibited mPTP opening and further protected the plasma membrane barrier. Significantly, necrostatin-1 did not protect the mucosal barrier. Oxidative stress increased mRNA for sXBP1, a marker of the IRE1 branch of the UPR, and CHOP, a marker of the PERK branch. It also stimulated phosphorylation of eIF2α, the upstream regulator of CHOP, as well as an increase in intracellular Ca2+. Dynasore selectively inhibited the increase in PERK branch markers, and also prevented the increase intracellular Ca2+ in response to oxidative stress. The increase in PERK branch markers were also inhibited when cells were treated with the cell permeable Ca2+ chelator, BAPTA-AM. To our knowledge, this is the first time that dynasore has been shown to have an effect on the UPR and suggests therapeutic applications.

DRP1 deficiency induces mitochondrial dysfunction and oxidative stress-mediated apoptosis during porcine oocyte maturation

BackgroundEnvironmental pollution induces oxidative stress and apoptosis in mammalian oocytes, which can cause defects in reproduction; however, the molecular regulation of oxidative stress in oocytes is still largely unknown. In the present study, we identified that dynamin-related protein 1 (DRP1) is an important molecule regulating oocyte mitochondrial function and preventing oxidative stress/apoptosis. DRP1 is a member of the dynamin GTPase superfamily localized at the mitochondrial-endoplasmic reticulum interaction site, where it regulates the fission of mitochondria and other related cellular processes.ResultsOur results show that DRP1 was stably expressed during different stages of porcine oocyte meiosis, and might have a potential relationship with mitochondria as it exhibited similar localization. Loss of DRP1 activity caused failed porcine oocyte maturation and cumulus cell expansion, as well as defects in polar body extrusion. Further analysis indicated that a DRP1 deficiency caused mitochondrial dysfunction and induced oxidative stress, which was confirmed by increased reactive oxygen species levels. Moreover, the incidence of early apoptosis increased as detected by positive Annexin-V signaling.ConclusionsTaken together, our results indicate that DRP1 is essential for porcine oocyte maturation and that a DRP1 deficiency could induce mitochondrial dysfunction, oxidative stress, and apoptosis.

Molecular Mechanisms of Intercellular Dissemination of Bacterial Pathogens.

Several intracellular bacterial pathogens, including Listeria monocytogenes, Shigella flexerni, and Rickettsia spp. use an actin-based motility process to spread in mammalian cell monolayers. Cell-to-cell spread is mediated by protrusive structures that contain bacteria encased in the host cell plasma membrane. These protrusions, which form in infected host cells, are internalized by neighboring cells. In this review, we summarize key findings on cell-to-cell spread, focusing on recent work on mechanisms of protrusion formation and internalization. We also discuss the dynamic behavior of bacterial populations during spread, and highlight recent findings showing that intercellular spread by an extracellular bacterial pathogen.

Dynamin regulates the dynamics and mechanical strength of the actin cytoskeleton as a multifilament actin-bundling protein.

The dynamin GTPase is known to bundle actin filaments, but the underlying molecular mechanism and physiological relevance remain unclear. Our genetic analyses revealed a function of dynamin in propelling invasive membrane protrusions during myoblast fusion in vivo. Using biochemistry, total internal reflection fluorescence microscopy, electron microscopy and cryo-electron tomography, we show that dynamin bundles actin while forming a helical structure. At its full capacity, each dynamin helix captures 12-16 actin filaments on the outer rim of the helix. GTP hydrolysis by dynamin triggers disassembly of fully assembled dynamin helices, releasing free dynamin dimers/tetramers and facilitating Arp2/3-mediated branched actin polymerization. The assembly/disassembly cycles of dynamin promote continuous actin bundling to generate mechanically stiff actin super-bundles. Super-resolution and immunogold platinum replica electron microscopy revealed dynamin along actin bundles at the fusogenic synapse. These findings implicate dynamin as a unique multifilament actin-bundling protein that regulates the dynamics and mechanical strength of the actin cytoskeletal network.

Epsin but not AP-2 supports reconstitution of endocytic clathrin-coated vesicles.

Formation of clathrin-coated vesicles (CCVs) in receptor-mediated endocytosis is a mechanistically well-established process, in which clathrin, the adaptor protein complex AP-2, and the large GTPase dynamin play crucial roles. In order to obtain more mechanistic insight into this process, here we established a giant unilamellar vesicle (GUV)-based invitro CCV reconstitution system with chemically defined components and the full-length recombinant proteins clathrin, AP-2, epsin-1, and dynamin-2. Our results support the predominant model in which hydrolysis of GTP by dynamin is a prerequisite to generate CCVs. Strikingly, in this system at near physiological concentrations of reagents, epsin-1 alone does not have the propensity for scission but is required for bud formation, whereas AP-2 and clathrin are not sufficient. Thus, our study reveals that epsin-1 is an important factor for the maturation of clathrin coated buds, a prerequisite for vesicle generation.

RNAi-Based Gene Therapy Rescues Developmental and Epileptic Encephalopathy in a Genetic Mouse Model

Developmental and epileptic encephalopathy (DEE) associated with de novo variants in the gene encoding dynamin-1 (DNM1) is a severe debilitating disease with no pharmacological remedy. Like most genetic DEEs, the majority of DNM1 patients suffer from therapy-resistant seizures and comorbidities such as intellectual disability, developmental delay, and hypotonia. We tested RNAi gene therapy in the Dnm1 fitful mouse model of DEE using a Dnm1-targeted therapeutic microRNA delivered by a self-complementary adeno-associated virus vector. Untreated or control-injected fitful mice have growth delay, severe ataxia, and lethal tonic-clonic seizures by 3 weeks of age. These major impairments are mitigated following a single treatment in newborn mice, along with key underlying cellular features including gliosis, cell death, and aberrant neuronal metabolic activity typically associated with recurrent seizures. Our results underscore the potential for RNAi gene therapy to treat DNM1 disease and other genetic DEEs where treatment would require inhibition of the pathogenic gene product.

Fenretinide induces a new form of dynamin-dependent cell death in pediatric sarcoma

Alveolar rhabdomyosarcoma (aRMS) is a highly malicious childhood malignancy characterized by specific chromosomal translocations mostly encoding the oncogenic transcription factor PAX3-FOXO1 and therefore also referred to as fusion-positive RMS (FP-RMS). Previously, we have identified fenretinide (retinoic acid p-hydroxyanilide) to affect PAX3-FOXO1 expression levels as well as FP-RMS cell viability. Here, we characterize the mode of action of fenretinide in more detail. First, we demonstrate that fenretinide-induced generation of reactive oxygen species (ROS) depends on complex II of the mitochondrial respiratory chain, since ROS scavenging as well as complexing of iron completely abolished cell death. Second, we co-treated cells with a range of pharmacological inhibitors of specific cell death pathways including z-vad (apoptosis), necrostatin-1 (necroptosis), 3-methyladenine (3-MA) (autophagy), and ferrostatin-1 (ferroptosis) together with fenretinide. Surprisingly, none of these inhibitors was able to prevent cell death. Also genetic depletion of key players in the apoptotic and necroptotic pathway (BAK, BAX, and RIPK1) confirmed the pharmacological data. Interestingly however, electron microscopy of fenretinide-treated cells revealed an excessive accumulation of cytoplasmic vacuoles, which were distinct from autophagosomes. Further flow cytometry and fluorescence microscopy experiments suggested a hyperstimulation of macropinocytosis, leading to an accumulation of enlarged early and late endosomes. Surprisingly, pharmacological inhibition as well as genetic depletion of large dynamin GTPases completely abolished fenretinide-induced vesicle formation and subsequent cell death, suggesting a new form of dynamin-dependent programmed cell death. Taken together, our data identify a new form of cell death mediated through the production of ROS by fenretinide treatment, highlighting the value of this compound for treatment of sarcoma patients including FP-RMS.

Mice carrying an analogous heterozygous dynamin 2 K562E mutation that causes neuropathy in humans develop predominant characteristics of a primary myopathy.

Some mutations affecting dynamin 2 (DNM2) can cause dominantly inherited Charcot–Marie–Tooth (CMT) neuropathy. Here, we describe the analysis of mice carrying the DNM2 K562E mutation which has been associated with dominant-intermediate CMT type B (CMTDIB). Contrary to our expectations, heterozygous DNM2 K562E mutant mice did not develop definitive signs of an axonal or demyelinating neuropathy. Rather, we found a primary myopathy-like phenotype in these mice. A likely interpretation of these results is that the lack of a neuropathy in this mouse model has allowed the unmasking of a primary myopathy due to the DNM2 K562E mutation which might be overshadowed by the neuropathy in humans. Consequently, we hypothesize that a primary myopathy may also contribute to the disease mechanism in some CMTDIB patients. We propose that these findings should be considered in the evaluation of patients, the determination of the underlying disease processes and the development of tailored potential treatment strategies.

Dynamin 2-dependent endocytosis is essential for mouse oocyte development and fertility.

During folliculogenesis, oocytes are dependent on metabolic and molecular support from surrounding somatic cells. Here, we examined the role of the dynamin (DNM) family of mechanoenzymes in mediating endocytotic uptake into growing follicular oocytes. We found DNM1 and DNM2 to be highly expressed in growing follicular oocytes as well as in mature germinal vesicle (GV) and metaphase II (MII) stage oocytes. Moreover, oocyte-specific conditional knockout (cKO) of DNM2 (DNM2Δ) led to complete sterility, with follicles arresting at the preantral stage of development. In addition, DNM2Δ ovaries were characterized by disrupted follicular growth as well as oocyte and follicle apoptosis. Further, the loss of DNM activity, either through DNM2 cKO or through pharmacological inhibition (Dyngo 6a) led to the impairment of endocytotic pathways in preantral oocytes as well as in mature GV and MII oocytes, respectively. Loss of DNM activity resulted in the redistribution of endosomes and the misslocalization of clathrin and actin, suggesting dysfunctional endocytosis. Notably, there was no observable effect on the fertility of DNM1Δ females. Our study has provided new insight into the complex and dynamic nature of oocyte growth during folliculogenesis, suggesting a role for DNM2 in mediating the endocytotic events that are essential for oocyte development.

Integrating Optical and Electrochemical Approaches to Assess the Actions of Dynamin at the Fusion Pore.

Of the techniques currently available to monitor dense core granule exocytosis in adrenal chromaffin cells, two have proven particularly useful: carbon-fiber amperometry and total internal reflection fluorescence (TIRF) microscopy. Amperometry enables the detection of oxidizable catecholamines escaping a fusion pore with millisecond time resolution. TIRF microscopy, and its variant polarized-TIRF (pTIRF) microscopy, provides information on the characteristics of fusion pores at temporally later stages. Used in conjunction, amperometry and TIRF microscopy allow an investigator to follow the fate of a fusion pore from its formation to expansion orreclosure. The properties of fusion pores, including theirstructure and dynamics, have been shown by multiple groups to be modified by the dynamin GTPase (Dyn1). In this chapter, we describe how amperometry and TIRF microscopy enableinsights into dynamin-dependent effects on exocytosis in primary cultures of bovine adrenal chromaffin cells.

NEW LEADERSHIP DYNAMICS IN THE INFORMATION AGE: LATERAL LEADERSHIP AND THOUGHT LEADERSHIP

Given the emerging challenges in a globalized and integrated world, achievement of organizational goals requires new kinds of leadership beyond classical thinking, centered on creativity, innovation, insight, logical sense, and acknowledging customer demands and preferences. Thus, this paper analyzes some emerging leadership styles in globalized at information age. In this context, lateral and thought leadership will be examined from various perspectives. In this study, after a brief theoretical analysis, focused on the evolution and the main characteristics of both leadership styles. Next, the similarities and differences of the leadership with each other and with conventional leadership will be studied. The paper will then be summarized achieved findings at conclusion. The major finding is that both leaderships, as a relatively new approach, focuses on meeting the challenges of corporate business emerging in a globalized world with a slight difference methods and implementation.