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Volume reconstruction from electron microscopy datasets is a tool increasingly used to study the ultrastructure of the synapse in the broader context of neuronal network and brain organization. Fine modifications of synapse structure, such as activity-dependent dendritic spine enlargement and changes in the size and shape of the postsynaptic density, occur upon maturation and plasticity. The lack of structural plasticity or the inability to stabilize potentiated synapses are associated with synaptic and neuronal functional impairment. Mapping these rearrangements with the high resolution of electron microscopy proved to be essential in order to establish precise correlations between the geometry of synapses and their functional states. In this review we discuss recent discoveries on the substructure of the postsynaptic compartment of central excitatory synapses and how those are correlated with functional states of the neuronal network. The added value of volume electron microscopy analyses with respect to conventional transmission electron microscopy studies is highlighted considering that some limitations of volume-based methods imposed several adjustments to describe the geometry of this synaptic compartment and new parameters-that are good indicators of synapses strength and activity-have been introduced. [ABSTRACT FROM AUTHOR]

Rho GTPases are a class of G-proteins involved in several aspects of cellular biology, including the regulation of actin cytoskeleton. The most studied members of this family are RHOA and RAC1 that act in concert to regulate actin dynamics. Recently, Rho GTPases gained much attention as synaptic regulators in the mammalian central nervous system (CNS). In this context, ARHGAP22 protein has been previously shown to specifically inhibit RAC1 activity thus standing as critical cytoskeleton regulator in cancer cell models; however, whether this function is maintained in neurons in the CNS is unknown. Here, we generated a knockout animal model for arhgap22 and provided evidence of its role in the hippocampus. Specifically, we found that ARHGAP22 absence leads to RAC1 hyperactivity and to an increase in dendritic spine density with defects in synaptic structure, molecular composition, and plasticity. Furthermore, arhgap22 silencing causes impairment in cognition and a reduction in anxiety-like behavior in mice. We also found that inhibiting RAC1 restored synaptic plasticity in ARHGAP22 KO mice. All together, these results shed light on the specific role of ARHGAP22 in hippocampal excitatory synapse formation and function as well as in learning and memory behaviors. [ABSTRACT FROM AUTHOR]

Fragile X syndrome (FXS) is the most frequent form of inherited intellectual disability and the best-described monogenic cause of autism. CGG-repeat expansion in the FMR1 gene leads to FMR1 silencing, loss-of-expression of the Fragile X Mental Retardation Protein (FMRP), and is a common cause of FXS. Missense mutations in the FMR1 gene were also identified in FXS patients, including the recurrent FMRP-R138Q mutation. To investigate the mechanisms underlying FXS caused by this mutation, we generated a knock-in mouse model (Fmr1R138Q) expressing the FMRP-R138Q protein. We demonstrate that, in the hippocampus of the Fmr1R138Q mice, neurons show an increased spine density associated with synaptic ultrastructural defects and increased AMPA receptor-surface expression. Combining biochemical assays, high-resolution imaging, electrophysiological recordings, and behavioural testing, we also show that the R138Q mutation results in impaired hippocampal long-term potentiation and socio-cognitive deficits in mice. These findings reveal the functional impact of the FMRP-R138Q mutation in a mouse model of FXS. The R138Q mutation in the Fragile X Mental Retardation 1 (FMR1) gene has been associated with Fragile X syndrome (FXS). Here, the authors present a Fmr1R138Q Knock-In mouse model and show that R138Q mutation results in impaired long-term potentiation and socio-cognitive performance in these mice. [ABSTRACT FROM AUTHOR]

Key points: The present study demonstrates, by in vitro and in vivo analyses, the novel concept that signal transmission between sympathetic neurons and the heart, underlying the physiological regulation of cardiac function, operates in a quasi‐synaptic fashion. This is a result of the direct coupling between neurotransmitter releasing sites and effector cardiomyocyte membranes. Abstract: Cardiac sympathetic neurons (SNs) finely tune the rate and strength of heart contractions to match blood demand, both at rest and during acute stress, through the release of noradrenaline (NE). Junctional sites at the interface between the two cell types have been observed, although whether direct neurocardiac coupling has a role in heart physiology has not been clearly demonstrated to date. We investigated the dynamics of SN/cardiomyocyte intercellular signalling, both by fluorescence resonance energy transfer‐based imaging of cAMP in co‐cultures, as a readout of cardiac β‐adrenergic receptor activation, and in vivo, using optogenetics in transgenic mice with SN‐specific expression of Channelrhodopsin‐2. We demonstrate that SNs and cardiomyocytes interact at specific sites in the human and rodent heart, as well as in co‐cultures. Accordingly, neuronal activation elicited intracellular cAMP increases only in directly contacted myocytes and cell–cell coupling utilized a junctional extracellular signalling domain with an elevated NE concentration. In the living mouse, optogenetic activation of cardiac SNs innervating the sino‐atrial node resulted in an instantaneous chronotropic effect, which shortened the heartbeat interval with single beat precision. Remarkably, inhibition of the optogenetically elicited chronotropic responses required a high dose of propranolol (20–50 mg kg–1), suggesting that sympathetic neurotransmission in the heart occurs at a locally elevated NE concentration. Our in vitro and in vivo data suggest that the control of cardiac function by SNs occurs via direct intercellular coupling as a result of the establishment of a specific junctional site. [ABSTRACT FROM AUTHOR]

Neuromuscular junction assembly and plasticity during embryonic, postnatal, and adult life are tightly regulated by the continuous cross-talk among motor nerve endings, muscle fibers, and glial cells. Altered communications among these components is thought to be responsible for the physiological age-related changes at this synapse and possibly for its destruction in pathological states. Neuromuscular junction dismantling plays a crucial role in the onset of Amyotrophic Lateral Sclerosis (ALS). ALS is characterized by the degeneration and death of motor neurons leading to skeletal muscle denervation, atrophy and, most often, death of the patient within five years from diagnosis. ALS is a non-cell autonomous disease as, besides motor neuron degeneration, glial cells, and possibly muscle fibers, play a role in its onset and progression. Here, we will review the recent literature regarding the mechanisms leading to neuromuscular junction disassembly and muscle denervation focusing on the role of the three players of this peripheral tripartite synapse. [ABSTRACT FROM AUTHOR]

Aims Caveolinopathies are a family of genetic disorders arising from alterations of the caveolin-3 (cav-3) gene. The T78M cav-3 variant has been associated with both skeletal and cardiac muscle pathologies but its functional contribution, especially to cardiac diseases, is still controversial. Here, we evaluated the effect of the T78M cav-3 variant on cardiac ion channel function and membrane excitability. Methods and results We transfected either the wild type (WT) or T78M cav-3 in caveolin-1 knock-out mouse embryonic fibroblasts and found by immunofluorescence and electron microscopy that both are expressed at the plasma membrane and form caveolae. Two ion channels known to interact and co-immunoprecipitate with the cav-3, hKv1.5 and hHCN4, interact also with T78M cav-3 and reside in lipid rafts. Electrophysiological analysis showed that the T78M cav-3 causes hKv1.5 channels to activate and inactivate at more hyperpolarized potentials and the hHCN4 channels to activate at more depolarized potentials, in a dominant way. In spontaneously beating neonatal cardiomyocytes, the expression of the T78M cav-3 significantly increased action potential peak-to-peak variability without altering neither the mean rate nor the maximum diastolic potential. We also found that in a small cohort of patients with supraventricular arrhythmias, the T78M cav-3 variant is more frequent than in the general population. Finally, in silico analysis of both sinoatrial and atrial cell models confirmed that the T78M-dependent changes are compatible with a pro-arrhythmic effect. Conclusion This study demonstrates that the T78M cav-3 induces complex modifications in ion channel function that ultimately alter membrane excitability. The presence of the T78M cav-3 can thus generate a susceptible substrate that, in concert with other structural alterations and/or genetic mutations, may become arrhythmogenic. [ABSTRACT FROM AUTHOR]

Myosin IXa (Myo9a) is a motor protein that is highly expressed in the brain. However, the role of Myo9a in neurons remains unknown. Here, we investigated Myo9a function in hippocampal synapses. In rat hippocampal neurons, Myo9a localizes to the postsynaptic density (PSD) and binds the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) GluA2 subunit. Myo9a+/- mice displayed a thicker PSD and increased levels of PSD95 and surface AMPAR expression. Furthermore, synaptic transmission, long-term potentiation (LTP) and cognitive functions were impaired in Myo9a+/- mice. Together, these results support a key role for Myo9a in controlling the molecular structure and function of hippocampal synapses. [ABSTRACT FROM AUTHOR]

ABSTRACT Confocal microscopy imaging of cells allows to visualize the presence of specific antigens by using fluorescent tags or fluorescent proteins, with resolution of few hundreds of nanometers, providing their localization in a large field-of-view and the understanding of their cellular function. Conversely, in scanning electron microscopy (SEM), the surface morphology of cells is imaged down to nanometer scale using secondary electrons. Combining both imaging techniques have brought to the correlative light and electron microscopy, contributing to investigate the existing relationships between biological surface structures and functions. Furthermore, in SEM, backscattered electrons (BSE) can image local compositional differences, like those due to nanosized gold particles labeling cellular surface antigens. To perform SEM imaging of cells, they could be grown on conducting substrates, but obtaining images of limited quality. Alternatively, they could be rendered electrically conductive, coating them with a thin metal layer. However, when BSE are collected to detect gold-labeled surface antigens, heavy metals cannot be used as coating material, as they would mask the BSE signal produced by the markers. Cell surface could be then coated with a thin layer of chromium, but this results in a loss of conductivity due to the fast chromium oxidation, if the samples come in contact with air. In order to overcome these major limitations, a thin layer of indium-tin-oxide was deposited by ion-sputtering on gold-decorated HeLa cells and neurons. Indium-tin-oxide was able to provide stable electrical conductivity and preservation of the BSE signal coming from the gold-conjugated markers. Microsc. Res. Tech. 78:433-443, 2015. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Correlative light electron microscopy (CLEM) combines the advantages of light and electron microscopy, thus making it possible to follow dynamic events in living cells at nanometre resolution. Various CLEM approaches and devices have been developed, each of which has its own advantages and technical challenges. We here describe our customized patterned glass substrates, which improve the feasibility of correlative fluorescence/confocal and scanning electron microscopy. [ABSTRACT FROM AUTHOR]

To optimise the efficiency of cell machinery, cells can use the same protein (often called a hub protein) to participate in different cell functions by simply changing its target molecules. There are large data sets describing protein-protein interactions (“interactome”) but they frequently fail to consider the functional significance of the interactions themselves. We studied the interaction between two potential hub proteins, ICln and 4.1R (in the form of its two splicing variants 4.1R80 and 4.1R135), which are involved in such crucial cell functions as proliferation, RNA processing, cytoskeleton organisation and volume regulation. The sub-cellular localisation and role of native and chimeric 4.1R over-expressed proteins in human embryonic kidney (HEK) 293 cells were examined. ICln interacts with both 4.1R80 and 4.1R135 and its over-expression displaces 4.1R from the membrane regions, thus affecting 4.1R interaction with ß-actin. It was found that 4.1R80 and 4.1R135 are differently involved in regulating the swelling activated anion current (ICl,swell) upon hypotonic shock, a condition under which both isoforms are dislocated from the membrane region and thus contribute to ICl,swell current regulation. Both 4.1R isoforms are also differently involved in regulating cell morphology, and ICln counteracts their effects. The findings of this study confirm that 4.1R plays a role in cell volume regulation and cell morphology and indicate that ICln is a new negative regulator of 4.1R functions. [ABSTRACT FROM AUTHOR]

Actin-based remodelling underlies spine structural changes occurring during synaptic plasticity, the process that constantly reshapes the circuitry of the adult brain in response to external stimuli, leading to learning and memory formation. A positive correlation exists between spine shape and synaptic strength and, consistently, abnormalities in spine number and morphology have been described in a number of neurological disorders. In the present study, we demonstrate that the actin-regulating protein, Eps8, is recruited to the spine head during chemically induced long-term potentiation in culture and that inhibition of its actin-capping activity impairs spine enlargement and plasticity. Accordingly, mice lacking Eps8 display immature spines, which are unable to undergo potentiation, and are impaired in cognitive functions. Additionally, we found that reduction in the levels of Eps8 occurs in brains of patients affected by autism compared to controls. Our data reveal the key role of Eps8 actin-capping activity in spine morphogenesis and plasticity and indicate that reductions in actin-capping proteins may characterize forms of intellectual disabilities associated with spine defects. [ABSTRACT FROM AUTHOR]

Objective: Microvesicles (MVs) have been indicated as important mediators of intercellular communication and are emerging as new biomarkers of tissue damage. Our previous data indicate that reactive microglia/macrophages release MVs in vitro. The aim of the study was to evaluate whether MVs are released by microglia/macrophages in vivo and whether their number varies in brain inflammatory conditions, such as multiple sclerosis (MS). Methods: Electron and fluorescence microscopy and flow cytometry were used to detect myeloid MVs in the cerebrospinal fluid (CSF) of healthy controls, MS patients, and rodents affected by experimental autoimmune encephalomyelitis (EAE), the animal model of MS. Results: Myeloid MVs were detected in CSF of healthy controls. In relapsing and remitting EAE mice, the concentration of myeloid MVs in the CSF was significantly increased and closely associated with disease course. Analysis of MVs in the CSF of 28 relapsing patients and 28 patients with clinical isolated syndrome from 2 independent cohorts revealed higher levels of myeloid MVs than in 13 age-matched controls, indicating a clinical value of MVs as a companion tool to capture disease activity. Myeloid MVs were found to spread inflammatory signals both in vitro and in vivo at the site of administration; mice impaired in MV shedding were protected from EAE, suggesting a pathogenic role for MVs in the disease. Finally, FTY720, the first approved oral MS drug, significantly reduced the amount of MVs in the CSF of EAE-treated mice. Interpretation: These findings identify myeloid MVs as a marker and therapeutic target of brain inflammation. ANN NEUROL 2012;72:610-624 [ABSTRACT FROM AUTHOR]

VAPB (vesicle-associated membrane protein-associated protein B) is a ubiquitously expressed, ER-resident tail-anchored protein that functions as adaptor for lipid-exchange proteins. Its mutant form, P56S-VAPB, is linked to a dominantly inherited form of amyotrophic lateral sclerosis (ALS8). P56S-VAPB forms intracellular inclusions, whose role in ALS pathogenesis has not yet been elucidated. We recently demonstrated that these inclusions are formed by profoundly remodelled stacked ER cisternae. Here, we used stable HeLa-TetOff cell lines inducibly expressing wild-type VAPB and P56S-VAPB, as well as microinjection protocols in non-transfected cells, to investigate the dynamics of inclusion generation and degradation. Shortly after synthesis, the mutant protein forms small, polyubiquitinated clusters, which then congregate in the juxtanuclear region independently of the integrity of the microtubule cytoskeleton. The rate of degradation of the aggregated mutant is higher than that of the wild-type protein, so that the inclusions are cleared only a few hours after cessation of P56SVAPB synthesis. At variance with other inclusion bodies linked to neurodegenerative diseases, clearance of P56S-VAPB inclusions involves the proteasome, with no apparent participation of macro-autophagy. Transfection of a dominant-negative form of the AAA ATPase p97/VCP stabilizes mutant VAPB, suggesting a role for this ATPase in extracting the aggregated protein from the inclusions. Our results demonstrate that the structures induced by P56S-VAPB stand apart from other inclusion bodies, both in the mechanism of their genesis and of their clearance from the cell, with possible implications for the pathogenic mechanism of the mutant protein. [ABSTRACT FROM AUTHOR]

Several fluorescent proteins ( FPs) are prone to forming low-affinity oligomers. This undesirable tendency is exacerbated when FPs are confined to membranes or when fused to naturally oligomeric proteins. Oligomerization of FPs limits their suitability for creating fusions with proteins of interest. Unfortunately, no standardized method evaluates the biologically relevant oligomeric state of FPs. Here, we describe a quantitative visual assay for assessing whether FPs are sufficiently monomeric under physiologic conditions. Membrane-associated FP-fusion proteins, by virtue of their constrained planar geometry, achieve high effective concentrations. We exploited this propensity to develop an assay to measure FP tendencies to oligomerize in cells. FPs were fused on the cytoplasmic end of an endoplasmic reticulum ( ER) signal-anchor membrane protein ( CytERM) and expressed in cells. Cells were scored based on the ability of CytERM to homo-oligomerize with proteins on apposing membranes and restructure the ER from a tubular network into organized smooth ER ( OSER) whorl structures. The ratio of nuclear envelope and OSER structures mean fluorescent intensities for cells expressing enhanced green fluorescent protein ( EGFP) or monomeric green fluorescent protein ( mGFP) CytERM established standards for comparison of uncharacterized FPs. We tested three FPs and identified two as sufficiently monomeric, while a third previously reported as monomeric was found to strongly oligomerize. [ABSTRACT FROM AUTHOR]

During the initial stage of neuromuscular junction (NMJ) formation, nerve-derived agrin cooperates with muscle-autonomous mechanisms in the organization and stabilization of a plaque-like postsynaptic specialization at the site of nerve-muscle contact. Subsequent NMJ maturation to the characteristic pretzel-like appearance requires extensive structural reorganization. We found that the progress of plaque-to-pretzel maturation is regulated by agrin. Excessive cleavage of agrin via transgenic overexpression of an agrin-cleaving protease, neurotrypsin, in motoneurons resulted in excessive reorganizational activity of the NMJs, leading to rapid dispersal of the synaptic specialization. By contrast, expression of cleavage-resistant agrin in motoneurons slowed down NMJ remodeling and delayed NMJ maturation. Neurotrypsin, which is the sole agrin-cleaving protease in the CNS, was excluded as the physiological agrin-cleaving protease at the NMJ, because NMJ maturation was normal in neurotrypsin-deficient mice. Together, our analyses characterize agrin cleavage at its proteolytic α- and β-sites by an as-yet-unspecified protease as a regulatory access for relieving the agrin-dependent constraint on endplate reorganization during NMJ maturation. [ABSTRACT FROM AUTHOR]

VAPB (vesicle-associated membrane protein-associated protein B) is an endoplasmic reticulum (ER)-resident tail-anchored adaptor protein involved in lipid transport. A dominantly inherited mutant, P56S-VAPB, causes a familial form of amyotrophic lateral sclerosis (ALS) and forms poorly characterized inclusion bodies in cultured cells. To provide a cell biological basis for the understanding of mutant VAPB pathogenicity, we investigated its biogenesis and the inclusions that it generates. Translocation assays in cell-free systems and in cultured mammalian cells were used to investigate P56S-VAPB membrane insertion, and the inclusions were characterized by confocal imaging and electron microscopy. We found that mutant VAPB inserts post-translationally into ER membranes in a manner indistinguishable from the wild-type protein but that it rapidly clusters to form inclusions that remain continuous with the rest of the ER. Inclusions were induced by the mutant also when it was expressed at levels comparable to the endogenous wild-type protein. Ultrastructural analysis revealed that the inclusions represent a novel form of organized smooth ER (OSER) consisting in a limited number of parallel cisternae (usually 2 or 3) interleaved by a ~30 nm-thick electron-dense cytosolic layer. Our results demonstrate that the ALS-linked VAPB mutant causes dramatic ER restructuring that may underlie its pathogenicity in motoneurons. [ABSTRACT FROM AUTHOR]

Granins are major constituents of dense-core secretory granules in neuroendocrine cells, but their function is still a matter of debate. Work in cell lines has suggested that the most abundant and ubiquitously expressed granins, chromogranin A and B (CgA and CgB), are involved in granulogenesis and protein sorting. Here we report the generation and characterization of mice lacking chromogranin B (CgB-ko), which were viable and fertile. Unlike neuroendocrine tissues, pancreatic islets of these animals lacked compensatory changes in other granins and were therefore analyzed in detail. Stimulated secretion of insulin, glucagon and somatostatin was reduced in CgB-ko islets, in parallel with somewhat impaired glucose clearance and reduced insulin release, but normal insulin sensitivity in vivo. CgB-ko islets lacked specifically the rapid initial phase of stimulated secretion, had elevated basal insulin release, and stored and released twice as much proinsulin as wildtype (wt) islets. Stimulated release of glucagon and somatostatin was reduced as well. Surprisingly, biogenesis, morphology and function of insulin granules were normal, and no differences were found with regard to β-cell stimulus-secretion coupling. We conclude that CgB is not required for normal insulin granule biogenesis or maintenance in vivo, but is essential for adequate secretion of islet hormones. Consequentially CgB-ko animals display some, but not all, hallmarks of human type-2 diabetes. However, the molecular mechanisms underlying this defect remain to be determined. [ABSTRACT FROM AUTHOR]

We have earlier shown that microglia, the immune cells of the CNS, release microparticles from cell plasma membrane after ATP stimulation. These vesicles contain and release IL-1β, a crucial cytokine in CNS inflammatory events. In this study, we show that microparticles are also released by astrocytes and we get insights into the mechanism of their shedding. We show that, on activation of the ATP receptor P2X7, microparticle shedding is associated with rapid activation of acid sphingomyelinase, which moves to plasma membrane outer leaflet. ATP-induced shedding and IL-1β release are markedly reduced by the inhibition of acid sphingomyelinase, and completely blocked in glial cultures from acid sphingomyelinase knockout mice. We also show that p38 MAPK cascade is relevant for the whole process, as specific kinase inhibitors strongly reduce acid sphingomyelinase activation, microparticle shedding and IL-1β release. Our results represent the first demonstration that activation of acid sphingomyelinase is necessary and sufficient for microparticle release from glial cells and define key molecular effectors of microparticle formation and IL-1β release, thus, opening new strategies for the treatment of neuroinflammatory diseases. [ABSTRACT FROM AUTHOR]

In this study, we examined the role of the L27 [(LIN2-LIN7) domain] and PDZ domain (domain previously found in PSD95-DlgA-ZO-1) for protein–protein interaction of the scaffold protein LIN7 in tight junction (TJ) assembly in Madin–Darby canine kidney (MDCK) cells and found that the stable expression of a LIN7 mutant lacking the L27 domain (ΔL27 mutant) acts as a dominant interfering protein by inhibiting TJ localization of endogenous LIN7. The loss of LIN7 did not alter the localization of the PALS1 (protein associated with LIN7) partner of the L27 domain but prevented TJ localization of the insulin receptor substrate p53 (IRSp53), a partner of the PDZ domain of LIN7. The function of both L27 and PDZ domains of LIN7 in IRSp53 localization to TJs has been further demonstrated by reducing the expression of LIN7 (LIN7 small hairpin RNA experiments) and by expression of IRSp53 deleted of its motif for PDZ interaction (IRSp53Δ5) or fused to the L27 domain of LIN7 (L27-IRSp53Δ5). Cell lines with decreased localization of LIN7 and IRSp53 to TJs showed defects during assembly of TJs and cyst polarization and failed to activate Rac1, a member of the Rho guanosine triphosphatases family crucially involved in actin organization and orientation of apicobasal polarity. These data therefore indicate that LIN7–IRSp53 association plays a role during assembly of functional TJs and surface polarization in epithelial cells. [ABSTRACT FROM AUTHOR]

Mitochondrial damage induced by superoxide dismutase (SOD1) mutants has been proposed to have a causative role in the selective degeneration of motoneurons in amyotrophic lateral sclerosis (ALS). In order to investigate the basis of the tissue specificity of mutant SOD1 we compared the effect of the continuous expression of wild-type or mutant (G93A) human SOD1 on mitochondrial morphology in the NSC-34 motoneuronal-like, the N18TG2 neuroblastoma and the non-neuronal Madin–Darby Canine Kidney (MDCK) cell lines. Morphological alterations of mitochondria were observed in NSC-34 expressing the G93A mutant (NSC-G93A) but not the wild-type SOD1, whereas a ten-fold greater level of total expression of the mutant had no effect on mitochondria of non-motoneuronal cell lines. Fragmented network, swelling and cristae remodelling but not vacuolization of mitochondria or other intracellular organelles were observed only in NSC-G93A cells. The mitochondrial alterations were not explained by a preferential localization of the mutant within NSC-G93A mitochondria, as a higher amount of the mutant SOD1 was found in mitochondria of MDCK-G93A cells. Our results suggest that mitochondrial vulnerability of motoneurons to G93ASOD1 is recapitulated in NSC-34 cells, and that peculiar features in network dynamics may account for the selective alterations of motoneuronal mitochondria. [ABSTRACT FROM AUTHOR]

The SNARE-dependent exocytosis of glutamate-containing vesicles in astrocytes is increasingly viewed as an important signal at the basis of the astrocyte-to-neurone communication system in the brain. Here we provide further insights into the molecular features and dynamics of vesicles in cultured astrocytes. We found that immunoisolated synaptobrevin2 vesicles are clear vesicles quite heterogenous in size and contain the vesicular glutamate transporter v-Glut-2. Moreover, they are immunopositive for synaptotagmin IV, for AMPA receptor subunits GluR2,3 and, to a lesser extent, for GluR1. We also provide direct evidence for the functional expression of v-Glut-2 in astrocytes and demonstrate that synaptobrevin2-positive vesicles can specifically take up (3H)l-glutamate via a bafilomycin-sensitive mechanism. Finally, by time lapse confocal microscopy, we show that a subpopulation of vesicles (tagged with a synaptobrevin2–EGFP chimera) is highly mobile and can fuse with the plasma membrane, preferentially at the level of the astrocyte processes, in a Ca2+-dependent manner. These latter observations, together with the evidence reported here for the expression of functional v-Glut-2 in synaptobrevin2-positive vesicles, provide a molecular basis for regulated exocytosis in astrocyte. [ABSTRACT FROM AUTHOR]

C-tail-anchored (TA) proteins often require a transmembrane domain of moderate hydrophobicity to maintain their endoplasmic reticulum residence, but the suggested role of protein-lipid interactions in this phenomenon has not been established. Here, we studied the interaction of TA proteins with lipids by differential scanning calorimetry by using a model system consisting of liposomes embedding either of two forms of cytochrome b5: the endoplasmic reticulum-resident wild-type (b5wt) and a mutant thereof (b5ext), that contains five extra nonpolar amino acids in its transmembrane domain and, therefore, reaches the plasma membrane. The proteins were incorporated into liposomes of palmitoyloleyl-phosphatidyicholine (POPC) or POPC mixed with either distearoyl-phosphatidylseri ne (DSPS), palmitoyl-oleyl-phosphatidylserine (POPS), distearoyl-phosphatidylcholine (DSPC), or C16- ceramide (CER). POPC liposomes displayed a single thermotropic transition centered at -3.4°C. When present the second lipid formed a domain within the POPC bilayer, as indicated by the appearance of an additional peak. This peak was centered at temperatures close to 0°C in the case of liposomes containing 10% CER, DSPS, and POPS and at 23°C in the case of DSPC, likely reflecting a higher degree of molecular packing for DSPC domains. In DSPS/POPC, POPS/POPC, or CER/POPC, but not in DSPC/POPC liposomes, the insertion of b5wt increased, whereas b5ext decreased, the relative contribution to the total enthalpy of the higher temperature, phase-separated component. These results were confirmed with fluorescence measurements by using pyrene-labeled phospholipids. The dissimilar interaction with lipids of these two differently localized TA proteins could have implications for their intracellular sorting. [ABSTRACT FROM AUTHOR]

We recently found that long-term exposure to nitric oxide (NO) triggers mitochondrial biogenesis in mammalian cells and tissues by activation of guanylate cyclase and generation of cGMP. Here, we report that the NO/cGMP-dependent mitochondrial biogenesis is associated with enhanced coupled respiration and content of ATP in U937, L6, and PC12 cells. The observed increase in ATP content depended entirely on oxidative phosphorylation, because ATP formation by glycolysis was unchanged. Brain, kidney, liver, heart, and gastrocnemius muscle from endothelial NO synthase null mutant mice displayed markedly reduced mitochondrial content associated with significantly lower oxygen consumption and ATP content. In these tissues, ultrastructural analyses revealed significantly smaller mitochondria. Furthermore, a significant reduction !n the number of mitochondria was observed in the subsarcolemmal region of the gastrocnemius muscle. We conclude that NO/cGMP stimulates mitochondrial biogenesis, both in vitro and in vivo, and that this stimulation is associated with increased mitochondrial function, resulting in enhanced formation of ATP. [ABSTRACT FROM AUTHOR]

We have recently shown that oxytocin inhibits cell proliferation when the vast majority of oxytocin receptors are excluded from caveolin-1-enriched microdomains, and that, on the contrary, it has a mitogenic effect when the receptors are targeted to these plasma membrane domains. In this study, we investigated whether the receptors located inside and outside caveolar microdomains initiate different signalling pathways and how this may lead to opposite effects on cell proliferation. Our data indicate that, depending on their localization, oxytocin receptors transactivate EGFR and activate ERK1/2 using different signalling intermediates. The final outcome is a different temporal pattern of EGFR and ERK1/2 phosphorylation, which is more persistent when the receptors are located outside caveolar microdomains and inhibit cell growth, and very transient when they are located in caveolar microdomains and stimulate cell growth. Finally, only the activation of receptors located outside caveolar microdomains correlates with the activation of the cell cycle inhibitor p21WAF1/CIP1, thus suggesting that the antiproliferative OTR effects may, in this case, be achieved by a sustained activation of EGFR and MAPK leading to the induction of this cell cycle regulator.Oncogene (2003) 22, 6054-6060. doi:10.1038/sj.onc.1206612 [ABSTRACT FROM AUTHOR]

Presents a study that investigated the functional role of the localization of human oxytocin receptor in caveolin-1 enriched membrane domains. Functions of caveolae; List of signaling molecules; Impact of G protein-coupled receptors (GPRC) on caveolae; Analysis of mitogenic effects mediated by some GPRC.

Penetration of the gut mucosa by pathogens expressing invasion genes is believed to occur mainly through specialized epithelial cells, called M cells, that are located in Peyer's patches. However, Salmonella typhimurium that are deficient in invasion genes encoded by Salmonella pathogenicity island 1 (SPI1) are still able to reach the spleen after oral administration. This suggests the existence of an alternative route for bacterial invasion, one that is independent of M cells. We report here a new mechanism for bacterial uptake in the mucosa tissues that is mediated by dendritic cells (DCs). DCs open the tight junctions between epithelial cells, send dendrites outside the epithelium and directly sample bacteria. In addition, because DCs express tight-junction proteins such as occludin, claudin 1 and zonula occludens 1, the integrity of the epithelial barrier is preserved. [ABSTRACT FROM AUTHOR]

The effects of hexarelin, a growth hormone (GH) secretagogue, and human GH on the mechanical and metabolic changes measured in isolated rat hearts submitted to 5 min of Ca deprivation followed by reperfusion with Ca-containing medium, the so-called calcium paradox phenomenon, were studied. Hexarelin (80 μg/kg bid, subcutaneously) administered for 7 d to male rats effectively antagonized the sudden increase in resting tension measured in vitro on Ca repletion. Moreover, during Ca repletion the release of creatine kinase activity (an index of cell damage) in the perfusate of these hearts was reduced up to 40% compared with controls. By contrast, administration of hexarelin for 3 d or GH (400 μg/kg bid, subcutaneously) for 7 d did not affect the mechanical and metabolic alterations induced by the calcium paradox. To assess its direct and acute cardiac effects, hexarelin (8 μg/mL) was perfused in vitro in recirculating conditions for 60 min through the hearts of normal rats. In this case, hexarelin did not stimulate heart contractility and failed to prevent ventricular contracture upon Ca readmission, whereas diltiazem, a Ca channel blocker, effectively antagonized the calcium paradox phenomenon. We conclude that short-term in vivo exposure to hexarelin, but not GH, enables cardiac myocyites to prevent cytoplasmatic electrolytic unbalance and to control intracellular Ca gain, two functions largely impaired during the calcium paradox phenomenon. Moreover, because the effect of hexarelin is not acute but dependent on the length of in vivo treatment, we suggest that it requires modifications of myocardiocyte physiology. [ABSTRACT FROM AUTHOR]

The effects of antifungal agents, with different mechanisms of action, on the morphogenetic transformation by synchronised yeast-phase Candida albicans cells in vitro and their respective anti-Candida activities are described. MIC data demonstrated that the azoles, amphotericin B and echinocandin were the most active agents against four C. albicans strains. Morphogenetic transformation experiments demonstrated that amphotericin B was significantly better at preventing the transformation, under a variety of test conditions, than the azoles and flucytosine: amphotericin B abolished the transformation at low concentrations while the azoles only prevented the morphogenetic transformation at much higher concentrations (>100 × MIC). [ABSTRACT FROM PUBLISHER]

The N-type voltage-operated calcium channel has been characterized over the years as a high-threshold channel, with variable inactivation kinetics, and a unique ability to bind with high affinity and specificity ω-conotoxin GVIA and related toxins. This channel is particularly expressed in some neurons and endocrine cells, where it participates in several calcium-dependent processes, including secretion. ω-conotoxin GVIA was instrumental not only for the biophysical and pharmacological characterization of N-type channels but also for the development of in vitro assays for studying N-type VOCC subcellular localization, biosynthesis, turnover, as well as short-and long-term regulation of its expression. We here summarize our studies on N-type VOCC expression in neurosecretory cells, with a major emphasis on recent data demonstrating the presence of N-type channels in intracellular secretory organelles and their recruitment to the cell surface during regulated exocytosis. [ABSTRACT FROM AUTHOR]

Three-dimensional (3D) reconstruction from electron microscopy (EM) datasets is a widely used tool that has improved our knowledge of synapse ultrastructure and organization in the brain. Rearrangements of synapse structure following maturation and in synaptic plasticity have been broadly described and, in many cases, the defective architecture of the synapse has been associated to functional impairments. It is therefore important, when studying brain connectivity, to map these rearrangements with the highest accuracy possible, considering the affordability of the different EM approaches to provide solid and reliable data about the structure of such a small complex. The aim of this work is to compare quantitative data from two dimensional (2D) and 3D EM of mouse hippocampal CA1 (apical dendrites), to define whether the results from the two approaches are consistent. We examined asymmetric excitatory synapses focusing on post synaptic density and dendritic spine area and volume as well as spine density, and we compared the results obtained with the two methods. The consistency between the 2D and 3D results questions the need—for many applications—of using volumetric datasets (costly and time consuming in terms of both acquisition and analysis), with respect to the more accessible measurements from 2D EM projections. [ABSTRACT FROM AUTHOR]

Although acetylcholine is the major neurotransmitter operating at the skeletal neuromuscular junction of many invertebrates and of vertebrates, glutamate participates in modulating cholinergic transmission and plastic changes in the last. Presynaptic terminals of neuromuscular junctions contain and release glutamate that contribute to the regulation of synaptic neurotransmission through its interaction with pre- and post-synaptic receptors activating downstream signaling pathways that tune synaptic efficacy and plasticity. During vertebrate development, the chemical nature of the neurotransmitter at the vertebrate neuromuscular junction can be experimentally shifted from acetylcholine to other mediators (including glutamate) through the modulation of calcium dynamics in motoneurons and, when the neurotransmitter changes, the muscle fiber expresses and assembles new receptors to match the nature of the new mediator. Finally, in adult rodents, by diverting descending spinal glutamatergic axons to a denervated muscle, a functional reinnervation can be achieved with the formation of new neuromuscular junctions that use glutamate as neurotransmitter and express ionotropic glutamate receptors and other markers of central glutamatergic synapses. Here, we summarize the past and recent experimental evidences in support of a role of glutamate as a mediator at the synapse between the motor nerve ending and the skeletal muscle fiber, focusing on the molecules and signaling pathways that are present and activated by glutamate at the vertebrate neuromuscular junction. [ABSTRACT FROM AUTHOR]

Shrm4, a protein expressed only in polarized tissues, is encoded by the KIAA1202 gene, whose mutations have been linked to epilepsy and intellectual disability. However, a physiological role for Shrm4 in the brain is yet to be established. Here, we report that Shrm4 is localized to synapses where it regulates dendritic spine morphology and interacts with the C terminus of GABAB receptors (GABABRs) to control their cell surface expression and intracellular trafficking via a dynein-dependent mechanism. Knockdown of Shrm4 in rat severely impairs GABABR activity causing increased anxiety-like behaviour and susceptibility to seizures. Moreover, Shrm4 influences hippocampal excitability by modulating tonic inhibition in dentate gyrus granule cells, in a process involving crosstalk between GABABRs and extrasynaptic δ-subunit-containing GABAARs. Our data highlights a role for Shrm4 in synaptogenesis and in maintaining GABABR-mediated inhibition, perturbation of which may be responsible for the involvement of Shrm4 in cognitive disorders and epilepsy. [ABSTRACT FROM AUTHOR]

Background information ATP is the main transmitter stored and released from astrocytes under physiological and pathological conditions. Morphological and functional evidence suggest that besides secretory granules, secretory lysosomes release ATP. However, the molecular mechanisms involved in astrocytic lysosome fusion remain still unknown. Results In the present study, we identify tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP, also called VAMP7) as the vesicular SNARE which mediates secretory lysosome exocytosis, contributing to release of both ATP and cathepsin B from glial cells. We also demonstrate that fusion of secretory lysosomes is triggered by slow and locally restricted calcium elevations, distinct from calcium spikes which induce the fusion of glutamate-containing clear vesicles. Downregulation of TI-VAMP/VAMP7 expression inhibited the fusion of ATP-storing vesicles and ATP-mediated calcium wave propagation. TI-VAMP/VAMP7 downregulation also significantly reduced secretion of cathepsin B from glioma. Conclusions Given that sustained ATP release from glia upon injury greatly contributes to secondary brain damage and cathepsin B plays a critical role in glioma dissemination, TI-VAMP silencing can represent a novel strategy to control lysosome fusion in pathological conditions. [ABSTRACT FROM AUTHOR]

Cholesterol and sphingolipids are abundant in neuronal membranes, where they help the organisation of the membrane microdomains involved in major roles such as axonal and dendritic growth, and synapse and spine stability. The aim of this study was to analyse their roles in presynaptic physiology. We first confirmed the presence of proteins of the exocytic machinery (SNARES and Cav2.1 channels) in the lipid microdomains of cultured neurons, and then incubated the neurons with fumonisin B (an inhibitor of sphingolipid synthesis), or with mevastatin or zaragozic acid (two compounds that affect the synthesis of cholesterol by inhibiting HMG-CoA reductase or squalene synthase). The results demonstrate that fumonisin B and zaragozic acid efficiently decrease sphingolipid and cholesterol levels without greatly affecting the viability of neurons or the expression of synaptic proteins. Electron microscopy showed that the morphology and number of synaptic vesicles in the presynaptic boutons of cholesterol-depleted neurons were similar to those observed in control neurons. Zaragozic acid (but not fumonisin B) treatment impaired synaptic vesicle uptake of the lipophilic dye FM1-43 and an antibody directed against the luminal epitope of synaptotagmin-1, effects that depended on the reduction in cholesterol because they were reversed by cholesterol reloading. The time-lapse confocal imaging of neurons transfected with ecliptic SynaptopHluorin showed that cholesterol depletion affects the post-depolarisation increase in fluorescence intensity. Taken together, these findings show that reduced cholesterol levels impair synaptic vesicle exocytosis in cultured neurons. [ABSTRACT FROM AUTHOR]

Correction to: The EMBO Journal (2009) 28, 1043–1054. doi:10.1038/emboj.2009.45 [ABSTRACT FROM AUTHOR]

In many cells, the endoplasmic reticulum (ER) contains segregated smooth and rough domains, but the mechanism of this segregation is unclear. Here, we used a HeLa cell line, inducibly expressing a GFP fusion protein [GFP-b(5)tail] anchored to the ER membrane, as a tool to investigate factors influencing ER organisation. Induction of GFP-b(5)tail expression caused proliferation of the ER, but its normal branching polygonal meshwork architecture was maintained. Experiments designed to test the effects of drugs that alter ceramide levels revealed that treatment of these cells with Phenyl-2-decanoyl-amino-3-morpholino-1-propanol-hydrocholride (PDMP) generated patches of segregated smooth ER, organised as a random tubular network, which rapidly dispersed after removal of the drug. The effect of PDMP was independent of its activity as sphingolipid synthesis inhibitor, but could be partially reversed by a membrane-permeant Ca2+ chelator. Although the smooth ER patches maintained connectivity with the remaining ER, they appeared to represent distinct domains differing in protein and lipid composition from the remaining ER. PDMP did not cause detachment of membrane-bound ribosomes, indicating that smooth ER patch generation was due to a reorganisation of pre-existing ribosome-free areas. Our results demonstrate a dynamic relationship between smooth and rough ER and have implications for the mechanisms regulating ER architecture. [ABSTRACT FROM AUTHOR]