A centronuclear myopathy-causing mutation in dynamin-2 disrupts neuronal morphology and excitatory synaptic transmission in a murine model of the disease.

AIMS: Dynamin-2 is a large GTPase, a member of the dynamin superfamily that regulates membrane remodelling and cytoskeleton dynamics. Mutations in the dynamin-2 gene (DNM2) cause autosomal dominant centronuclear myopathy (CNM), a congenital neuromuscular disorder characterised by progressive weakness and atrophy of the skeletal muscles. Cognitive defects have been reported in some DNM2-linked CNM patients suggesting that these mutations can also affect the central nervous system (CNS). Here we studied how a dynamin-2 CNM-causing mutation influences the CNS function. METHODS: Heterozygous mice harbouring the p.R465W mutation in the dynamin-2 gene (HTZ), the most common causing autosomal dominant CNM, were used as disease model. We evaluated dendritic arborisation and spine density in hippocampal cultured neurons, analysed excitatory synaptic transmission by electrophysiological field recordings in hippocampal slices, and evaluated cognitive function by performing behavioural tests. RESULTS: HTZ hippocampal neurons exhibited reduced dendritic arborisation and lower spine density than WT neurons, which was reversed by transfecting an interference RNA against the dynamin-2 mutant allele. Additionally, HTZ mice showed defective hippocampal excitatory synaptic transmission and reduced recognition memory compared to the WT condition. CONCLUSION: Our findings suggest that the dynamin-2 p.R465W mutation perturbs the synaptic and cognitive function in a CNM mouse model and support the idea that this GTPase plays a key role in regulating neuronal morphology and excitatory synaptic transmission in the hippocampus.

Gain-of-Function Dynamin-2 Mutations Linked to Centronuclear Myopathy Impair Ca2+-Induced Exocytosis in Human Myoblasts.

Gain-of-function mutations of dynamin-2, a mechano-GTPase that remodels membrane and actin filaments, cause centronuclear myopathy (CNM), a congenital disease that mainly affects skeletal muscle tissue. Among these mutations, the variants p.A618T and p.S619L lead to a gain of function and cause a severe neonatal phenotype. By using total internal reflection fluorescence microscopy (TIRFM) in immortalized human myoblasts expressing the pH-sensitive fluorescent protein (pHluorin) fused to the insulin-responsive aminopeptidase IRAP as a reporter of the GLUT4 vesicle trafficking, we measured single pHluorin signals to investigate how p.A618T and p.S619L mutations influence exocytosis. We show here that both dynamin-2 mutations significantly reduced the number and durations of pHluorin signals induced by 10 µM ionomycin, indicating that in addition to impairing exocytosis, they also affect the fusion pore dynamics. These mutations also disrupt the formation of actin filaments, a process that reportedly favors exocytosis. This altered exocytosis might importantly disturb the plasmalemma expression of functional proteins such as the glucose transporter GLUT4 in skeletal muscle cells, impacting the physiology of the skeletal muscle tissue and contributing to the CNM disease.

Knockin' on Egg's Door: Maternal Control of Egg Activation That Influences Cortical Granule Exocytosis in Animal Species.

Fertilization by multiple sperm leads to lethal chromosomal number abnormalities, failed embryo development, and miscarriage. In some vertebrate and invertebrate eggs, the so-called cortical reaction contributes to their activation and prevents polyspermy during fertilization. This process involves biogenesis, redistribution, and subsequent accumulation of cortical granules (CGs) at the female gamete cortex during oogenesis. CGs are oocyte- and egg-specific secretory vesicles whose content is discharged during fertilization to block polyspermy. Here, we summarize the molecular mechanisms controlling critical aspects of CG biology prior to and after the gametes interaction. This allows to block polyspermy and provide protection to the developing embryo. We also examine how CGs form and are spatially redistributed during oogenesis. During egg activation, CG exocytosis (CGE) and content release are triggered by increases in intracellular calcium and relies on the function of maternally-loaded proteins. We also discuss how mutations in these factors impact CG dynamics, providing unprecedented models to investigate the genetic program executing fertilization. We further explore the phylogenetic distribution of maternal proteins and signaling pathways contributing to CGE and egg activation. We conclude that many important biological questions and genotype-phenotype relationships during fertilization remain unresolved, and therefore, novel molecular players of CG biology need to be discovered. Future functional and image-based studies are expected to elucidate the identity of genetic candidates and components of the molecular machinery involved in the egg activation. This, will open new therapeutic avenues for treating infertility in humans.

Hypoxic Regulation of the Large-Conductance, Calcium and Voltage-Activated Potassium Channel, BK.

Hypoxia is a condition characterized by a reduction of cellular oxygen levels derived from alterations in oxygen balance. Hypoxic events trigger changes in cell-signaling cascades, oxidative stress, activation of pro-inflammatory molecules, and growth factors, influencing the activity of various ion channel families and leading to diverse cardiovascular diseases such as myocardial infarction, ischemic stroke, and hypertension. The large-conductance, calcium and voltage-activated potassium channel (BK) has a central role in the mechanism of oxygen (O2) sensing and its activity has been related to the hypoxic response. BK channels are ubiquitously expressed, and they are composed by the pore-forming α subunit and the regulatory subunits ß (ß1-ß4), γ (γ1-γ4), and LINGO1. The modification of biophysical properties of BK channels by ß subunits underly a myriad of physiological function of these proteins. Hypoxia induces tissue-specific modifications of BK channel α and ß subunits expression. Moreover, hypoxia modifies channel activation kinetics and voltage and/or calcium dependence. The reported effects on the BK channel properties are associated with events such as the increase of reactive oxygen species (ROS) production, increases of intracellular Calcium ([Ca2+]i), the regulation by Hypoxia-inducible factor 1α (HIF-1α), and the interaction with hemeproteins. Bronchial asthma, chronic obstructive pulmonary diseases (COPD), and obstructive sleep apnea (OSA), among others, can provoke hypoxia. Untreated OSA patients showed a decrease in BK-ß1 subunit mRNA levels and high arterial tension. Treatment with continuous positive airway pressure (CPAP) upregulated ß1 subunit mRNA level, decreased arterial pressures, and improved endothelial function coupled with a reduction in morbidity and mortality associated with OSA. These reports suggest that the BK channel has a role in the response involved in hypoxia-associated hypertension derived from OSA. Thus, this review aims to describe the mechanisms involved in the BK channel activation after a hypoxic stimulus and their relationship with disorders like OSA. A deep understanding of the molecular mechanism involved in hypoxic response may help in the therapeutic approaches to treat the pathological processes associated with diseases involving cellular hypoxia.

Dynamin-2 R465W mutation induces long range perturbation in highly ordered oligomeric structures.

High order oligomers are crucial for normal cell physiology, and protein function perturbed by missense mutations underlies several autosomal dominant diseases. Dynamin-2 is one of such protein forming helical oligomers that catalyze membrane fission. Mutations in this protein, where R465W is the most frequent, cause dominant centronuclear myopathy, but the molecular mechanisms underpinning the functional modifications remain to be investigated. To unveil the structural impact of this mutation in dynamin-2, we used full-atom molecular dynamics simulations and coarse-grained models and built dimers and helices of wild-type (WT) monomers, mutant monomers, or both WT and mutant monomers combined. Our results show that the mutation R465W causes changes in the interactions with neighbor amino acids that propagate through the oligomer. These new interactions perturb the contact between monomers and favor an extended conformation of the bundle signaling element (BSE), a dynamin region that transmits the conformational changes from the GTPase domain to the rest of the protein. This extended configuration of the BSE that is only relevant in the helices illustrates how a small change in the microenvironment surrounding a single residue can propagate through the oligomer structures of dynamin explaining how dominance emerges in large protein complexes.

Dynamin-2 mutations linked to Centronuclear Myopathy impair actin-dependent trafficking in muscle cells.

Dynamin-2 is a ubiquitously expressed GTP-ase that mediates membrane remodeling. Recent findings indicate that dynamin-2 also regulates actin dynamics. Mutations in dynamin-2 cause dominant centronuclear myopathy (CNM), a congenital myopathy characterized by progressive weakness and atrophy of skeletal muscles. However, the muscle-specific roles of dynamin-2 affected by these mutations remain elusive. Here we show that, in muscle cells, the GTP-ase activity of dynamin-2 is involved in de novo actin polymerization as well as in actin-mediated trafficking of the glucose transporter GLUT4. Expression of dynamin-2 constructs carrying CNM-linked mutations disrupted the formation of new actin filaments as well as the stimulus-induced translocation of GLUT4 to the plasma membrane. Similarly, mature muscle fibers isolated from heterozygous knock-in mice that harbor the dynamin-2 mutation p.R465W, an animal model of CNM, exhibited altered actin organization, reduced actin polymerization and impaired insulin-induced translocation of GLUT4 to the sarcolemma. Moreover, GLUT4 displayed aberrant perinuclear accumulation in biopsies from CNM patients carrying dynamin-2 mutations, further suggesting trafficking defects. These results suggest that dynamin-2 is a key regulator of actin dynamics and GLUT4 trafficking in muscle cells. Our findings also support a model in which impairment of actin-dependent trafficking contributes to the pathological mechanism in dynamin-2-associated CNM.

Cell Junction Pathology of Neural Stem Cells Is Associated With Ventricular Zone Disruption, Hydrocephalus, and Abnormal Neurogenesis.

Fetal-onset hydrocephalus affects 1 to 3 per 1,000 live births. It is not only a disorder of cerebrospinal fluid dynamics but also a brain disorder that corrective surgery does not ameliorate. We hypothesized that cell junction abnormalities of neural stem cells (NSCs) lead to the inseparable phenomena of fetal-onset hydrocephalus and abnormal neurogenesis. We used bromodeoxyuridine labeling, immunocytochemistry, electron microscopy, and cell culture to study the telencephalon of hydrocephalic HTx rats and correlated our findings with those in human hydrocephalic and nonhydrocephalic human fetal brains (n = 12 each). Our results suggest that abnormal expression of the intercellular junction proteins N-cadherin and connexin-43 in NSC leads to 1) disruption of the ventricular and subventricular zones, loss of NSCs and neural progenitor cells; and 2) abnormalities in neurogenesis such as periventricular heterotopias and abnormal neuroblast migration. In HTx rats, the disrupted NSC and progenitor cells are shed into the cerebrospinal fluid and can be grown into neurospheres that display intercellular junction abnormalities similar to those of NSC of the disrupted ventricular zone; nevertheless, they maintain their potential for differentiating into neurons and glia. These NSCs can be used to investigate cellular and molecular mechanisms underlying this condition, thereby opening the avenue for stem cell therapy.