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Synaptic dysfunction is an early mechanism in Alzheimer's disease that involves progressively larger areas of the brain over time. However, how it starts and propagates is unknown. Here we show that amyloid-ß released by microglia in association with large extracellular vesicles (Aß-EVs) alters dendritic spine morphology in vitro, at the site of neuron interaction, and impairs synaptic plasticity both in vitro and in vivo in the entorhinal cortex-dentate gyrus circuitry. One hour after Aß-EV injection into the mouse entorhinal cortex, long-term potentiation was impaired in the entorhinal cortex but not in the dentate gyrus, its main target region, while 24â h later it was also impaired in the dentate gyrus, revealing a spreading of long-term potentiation deficit between the two regions. Similar results were obtained upon injection of extracellular vesicles carrying Aß naturally secreted by CHO7PA2 cells, while neither Aß42 alone nor inflammatory extracellular vesicles devoid of Aß were able to propagate long-term potentiation impairment. Using optical tweezers combined to time-lapse imaging to study Aß-EV-neuron interaction, we show that Aß-EVs move anterogradely at the axon surface and that their motion can be blocked through annexin-V coating. Importantly, when Aß-EV motility was inhibited, no propagation of long-term potentiation deficit occurred along the entorhinal-hippocampal circuit, implicating large extracellular vesicle motion at the neuron surface in the spreading of long-term potentiation impairment. Our data indicate the involvement of large microglial extracellular vesicles in the rise and propagation of early synaptic dysfunction in Alzheimer's disease and suggest a new mechanism controlling the diffusion of large extracellular vesicles and their pathogenic signals in the brain parenchyma, paving the way for novel therapeutic strategies to delay the disease.
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Enfermedad de Alzheimer , Vesículas Extracelulares , Péptidos beta-Amiloides , Animales , Hipocampo , Potenciación a Largo Plazo , Ratones , MicroglíaRESUMEN
Recent evidence indicates synaptic dysfunction as an early mechanism affected in neuroinflammatory diseases, such as multiple sclerosis, which are characterized by chronic microglia activation. However, the mode(s) of action of reactive microglia in causing synaptic defects are not fully understood. In this study, we show that inflammatory microglia produce extracellular vesicles (EVs) which are enriched in a set of miRNAs that regulate the expression of key synaptic proteins. Among them, miR-146a-5p, a microglia-specific miRNA not present in hippocampal neurons, controls the expression of presynaptic synaptotagmin1 (Syt1) and postsynaptic neuroligin1 (Nlg1), an adhesion protein which play a crucial role in dendritic spine formation and synaptic stability. Using a Renilla-based sensor, we provide formal proof that inflammatory EVs transfer their miR-146a-5p cargo to neuron. By western blot and immunofluorescence analysis we show that vesicular miR-146a-5p suppresses Syt1 and Nlg1 expression in receiving neurons. Microglia-to-neuron miR-146a-5p transfer and Syt1 and Nlg1 downregulation do not occur when EV-neuron contact is inhibited by cloaking vesicular phosphatidylserine residues and when neurons are exposed to EVs either depleted of miR-146a-5p, produced by pro-regenerative microglia, or storing inactive miR-146a-5p, produced by cells transfected with an anti-miR-146a-5p. Morphological analysis reveals that prolonged exposure to inflammatory EVs leads to significant decrease in dendritic spine density in hippocampal neurons in vivo and in primary culture, which is rescued in vitro by transfection of a miR-insensitive Nlg1 form. Dendritic spine loss is accompanied by a decrease in the density and strength of excitatory synapses, as indicated by reduced mEPSC frequency and amplitude. These findings link inflammatory microglia and enhanced EV production to loss of excitatory synapses, uncovering a previously unrecognized role for microglia-enriched miRNAs, released in association to EVs, in silencing of key synaptic genes.
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Vesículas Extracelulares/inmunología , Inflamación/metabolismo , MicroARNs/metabolismo , Neuroglía/inmunología , Neuronas/inmunología , Sinapsis/inmunología , Animales , Células Cultivadas , Líquido Cefalorraquídeo/metabolismo , Técnicas de Cocultivo , Vesículas Extracelulares/patología , Femenino , Hipocampo/inmunología , Hipocampo/patología , Humanos , Inflamación/patología , Masculino , Ratones Endogámicos C57BL , Neuroglía/patología , Plasticidad Neuronal/fisiología , Neuronas/patología , Cultivo Primario de Células , Ratas Sprague-Dawley , Sinapsis/patologíaRESUMEN
Growing evidence indicates that sphingosine-1-P (S1P) upregulates glutamate secretion in hippocampal neurons. However, the molecular mechanisms through which S1P enhances excitatory activity remain largely undefined. The aim of this study was to identify presynaptic targets of S1P action controlling exocytosis. Confocal analysis of rat hippocampal neurons showed that S1P applied at nanomolar concentration alters the distribution of Synapsin I (SynI), a presynaptic phosphoprotein that controls the availability of synaptic vesicles for exocytosis. S1P induced SynI relocation to extrasynaptic regions of mature neurons, as well as SynI dispersion from synaptic vesicle clusters present at axonal growth cones of developing neurons. S1P-induced SynI relocation occurred in a Ca(2+)-independent but ERK-dependent manner, likely through the activation of S1P3 receptors, as it was prevented by the S1P3 receptor selective antagonist CAY1044 and in neurons in which S1P3 receptor was silenced. Our recent evidence indicates that microvesicles (MVs) released by microglia enhance the metabolism of endogenous sphingolipids in neurons and stimulate excitatory transmission. We therefore investigated whether MVs affect SynI distribution and whether endogenous S1P could be involved in the process. Analysis of SynI immunoreactivity showed that exposure to microglial MVs induces SynI mobilization at presynaptic sites and growth cones, whereas the use of inhibitors of sphingolipid cascade identified S1P as the sphingolipid mediating SynI redistribution. Our data represent the first demonstration that S1P induces SynI mobilization from synapses, thereby indicating the phosphoprotein as a novel target through which S1P controls exocytosis. SIGNIFICANCE STATEMENT: Growing evidence indicates that the bioactive lipid sphingosine and its metabolite sphingosine-1-P (S1P) stimulate excitatory transmission. While it has been recently clarified that sphingosine influences directly the exocytotic machinery by activating the synaptic vesicle protein VAMP2 to form SNARE fusion complexes, the molecular mechanism by which S1P promotes neurotransmission remained largely undefined. In this study, we identify Synapsin I, a presynaptic phosphoprotein involved in the control of availability of synaptic vesicles for exocytosis, as the key target of S1P action. In addition, we provide evidence that S1P can be produced at mature axon terminals as well as at immature growth cones in response to microglia-derived signals, which may be important to stabilize nascent synapses and to restore or potentiate transmission.
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Lisofosfolípidos/fisiología , Terminales Presinápticos/metabolismo , Esfingosina/análogos & derivados , Sinapsis/metabolismo , Sinapsinas/biosíntesis , Animales , Células Cultivadas , Femenino , Hipocampo/química , Hipocampo/citología , Hipocampo/metabolismo , Lisofosfolípidos/análisis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Terminales Presinápticos/química , Ratas , Ratas Sprague-Dawley , Esfingosina/análisis , Esfingosina/fisiología , Sinapsis/química , Sinapsinas/análisisRESUMEN
Endocannabinoids primarily influence neuronal synaptic communication within the nervous system. To exert their function, endocannabinoids need to travel across the intercellular space. However, how hydrophobic endocannabinoids cross cell membranes and move extracellularly remains an unresolved problem. Here, we show that endocannabinoids are secreted through extracellular membrane vesicles produced by microglial cells. We demonstrate that microglial extracellular vesicles carry on their surface N-arachidonoylethanolamine (AEA), which is able to stimulate type-1 cannabinoid receptors (CB1), and inhibit presynaptic transmission, in target GABAergic neurons. This is the first demonstration of a functional role of extracellular vesicular transport of endocannabinoids.
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Endocannabinoides/metabolismo , Microglía/metabolismo , Animales , Ácidos Araquidónicos , Células Cultivadas , Exosomas/metabolismo , Alcamidas Poliinsaturadas , Terminales Presinápticos/metabolismo , Ratas , Receptores de Cannabinoides/metabolismo , Transmisión Sináptica/fisiologíaRESUMEN
Exosomes and ectosomes, extracellular vesicles of two types generated by all cells at multivesicular bodies and the plasma membrane, respectively, play critical roles in physiology and pathology. A key mechanism of their function, analogous for both types of vesicles, is the fusion of their membrane to the plasma membrane of specific target cells, followed by discharge to the cytoplasm of their luminal cargo containing proteins, RNAs, and DNA. Here we summarize the present knowledge about the interactions, binding and fusions of vesicles with the cell plasma membrane. The sequence initiates with dynamic interactions, during which vesicles roll over the plasma membrane, followed by the binding of specific membrane proteins to their cell receptors. Membrane binding is then converted rapidly into fusion by mechanisms analogous to those of retroviruses. Specifically, proteins of the extracellular vesicle membranes are structurally rearranged, and their hydrophobic sequences insert into the target cell plasma membrane which undergoes lipid reorganization, protein restructuring and membrane dimpling. Single fusions are not the only process of vesicle/cell interactions. Upon intracellular reassembly of their luminal cargoes, vesicles can be regenerated, released and fused horizontally to other target cells. Fusions of extracellular vesicles are relevant also for specific therapy processes, now intensely investigated.
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Membrana Celular/metabolismo , Animales , Micropartículas Derivadas de Células/metabolismo , Exosomas/metabolismo , HumanosRESUMEN
Emerging evidence indicates that activation of microglia, the immune cells of the brain, is strictly associated to both secretion of soluble molecules and release of extracellular membrane vesicles (EMVs) into the pericellular space. Through these processes, microglia heavily influence brain cell functions, either propagating inflammation and causing damage to neurons or playing a supportive, neuroprotective role. In this review, we highlight the emerging concepts related to vesicular mechanisms of secretion operating in microglial cells, with the aim of dissecting how microglia communicate with other cell types within the brain microenvironment in health and disease.
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Encéfalo/citología , Micropartículas Derivadas de Células/metabolismo , Vesículas Citoplasmáticas/fisiología , Microglía/citología , Vías Secretoras/fisiología , Animales , Encéfalo/inmunología , Micropartículas Derivadas de Células/inmunología , Interleucina-1beta/metabolismoRESUMEN
We have uncovered a novel role for astrocytes-derived extracellular vesicles (EVs) in controlling intraneuronal Ca2+ concentration ([Ca2+]i) and identified transglutaminase-2 (TG2) as a surface-cargo of astrocytes-derived EVs. Incubation of hippocampal neurons with primed astrocyte-derived EVs have led to an increase in [Ca2+]i, unlike EVs from TG2-knockout astrocytes. Exposure of neurons or brain slices to extracellular TG2 promoted a [Ca2+]i rise, which was reversible upon TG2 removal and was dependent on Ca2+ influx through the plasma membrane. Patch-clamp and calcium imaging recordings revealed TG2-dependent neuronal membrane depolarization and activation of inward currents, due to the Na+/Ca2+-exchanger (NCX) operating in the reverse mode and indirect activation of L-type VOCCs, as indicated by VOCCs/NCX pharmacological inhibitors. A subunit of Na+/K+-ATPase was selected by comparative proteomics and identified as being functionally inhibited by extracellular TG2, implicating Na+/K+-ATPase inhibition in NCX reverse mode-switching leading to Ca2+ influx and higher basal [Ca2+]i. These data suggest that reactive astrocytes control intraneuronal [Ca2+]i through release of EVs with TG2 as responsible cargo, which could have a significant impact on synaptic activity in brain inflammation.
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Astrocitos , Vesículas Extracelulares , Adenosina Trifosfatasas , Astrocitos/metabolismo , Calcio/metabolismo , Vesículas Extracelulares/metabolismo , Homeostasis , Humanos , Neuronas/metabolismo , Proteína Glutamina Gamma Glutamiltransferasa 2 , Intercambiador de Sodio-Calcio/metabolismoRESUMEN
Expression of neurosecretion by nerve cells requires the levels of the transcription repressor element-1 silencing transcription factor (REST) to be very low. However, when high-REST clones of PC12 cells, defective of neurosecretion, were fused to other high-REST, non-neurosecretory cells, some neurosecretion was recovered. To clarify the mechanism of this recovery, we fused defective PC12 cells with human lymphocytes. A cytogenetic analysis revealed all hybrid clones that recovered neurosecretion to contain a fragment of chromosome 11 including the gene encoding BHC80, a protein of one of the complexes that mediate REST repression. In these clones, REST levels were as high as in defective PC12, whereas BHC80, localized in the nucleus, was 4- to 5-fold higher. Transient transfection of defective PC12 with various amounts of BHC80 cDNA induced (1) in defective PC12, the reexpression of only neurosecretion mRNAs; (2) in defective PC12 cotransfected with the REST negative construct DNA-binding domain (to attenuate gene repression), the recovery of a weak, but complete neurosecretory phenotype, including dense-core granules and their regulated exocytosis. Chromatin immunoprecipitation and immunodepletion analyses revealed the extensive BHC80 association with REST at the genes of two neurosecretion proteins, chromograninB and SNAP25, however only in the low-REST PC12, whereas in high-REST defective PC12 no association was appreciable. In defective PC12 transfected with BHC80 some association was reestablished. Therefore, the recovery of neurosecretion observed after fusion/transfection of defective PC12 depends on the reciprocal level of BHC80 and REST, with BHC80 working as a negative modulator of REST repression. This role appears of possible cell physiological and pathological importance.
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Histona Desacetilasas/metabolismo , Neurosecreción/fisiología , Proteínas Represoras/metabolismo , Animales , Línea Celular , Núcleo Celular/metabolismo , Cromogranina B/metabolismo , ADN Complementario/metabolismo , Histona Desacetilasas/genética , Humanos , Linfocitos , Células PC12 , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Proteína 25 Asociada a Sinaptosomas/metabolismo , TransfecciónRESUMEN
BACKGROUND: To estimate the prevalence of caries and to study oral health habits (oral hygiene, toothbrushing frequency, cariogenic or no cariogenic diet, visits to dentist) in children aged 6 to 12 with social exclusion risk. MATERIAL AND METHODS: A cross sectional study was conducted in a sample of 160 children aged form 6 to 12 years belonging to Colegio Madre Petra in Torrent and Colegio Nuestra Señora de los Desamparados in Nazaret (Valencia). Among other variables DMFT and Greene and Vermilion simplified plaque index were analyzed. RESULTS: The prevalence of caries observed was 81.87% and the global DMFT was 4.481. The mean plaque index observed was 1.12. No statistically significant differences were found between global DMFT and sex, global DMFT and age, global DMFT and diet, global DMFT and visits to the dentist and global DMFT and plaque index. A significant association was seen between global DMFT and ethnicity and global DMFT and brushing frequency. Statistically significant association was found also between plaque index and visits to the dentist and plaque index and diet. CONCLUSIONS: It was observed that children at risk of social exclusion had a very high global DMFT and a regular plaque index. So, it can be concluded that social exclusion constitute an underlying factor that increase caries prevalence and global DMFT and a marker of poor oral hygiene habits. Key words:Dental caries, caries prevalence, oral hygiene habits, plaque index, toothbrushing frequency, social exclusion.
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BACKGROUND: The embryonic root groove is an anatomical abnormality that starts in the cingulum and extends longitudinally down the long axis root towards the apex. This developmental anomaly is more frequently reported in maxillary lateral incisors. Gu YC in 2011 established three types of radicular grooves depending on its severity. According to this classification, type III presents a greater diagnostic and therapeutic complexity. The prevalence of palatogingival grooves in maxillary lateral incisors ranges from 1.9 to 14%. This case report provides valuable information about the diagnosis and treatment plan of palatogingival grooves with Cone-beam computed tomography (CBCT) scan. CASE REPORT: The patient was referred to the University Dental Clinic of European University of Valencia, with recurrent abscesses at the upper right lateral incisor region for the last two years. Palpation and percussion tests were positive for tooth 1.2. There was no clinical history of caries or previous trauma. Periapical radiography showed periapical radiolucent lesions located, not only in the apical area of tooth 1.2, but also in tooth 1.3. Both teeth had previously been endodontically treated. Periodontal probing showed normal values. CBCT scan was perfomed in order to establish a definitive diagnosis and appropriate treatment plan. DISCUSSION: The complex anatomy of the palatal root groove requires detailed knowledge of the internal root morphology for endodontic treatment success. This complementary tool allows a more accurate image of hard tissue structures, such as palatal grooves and/or accessory roots, in comparison to conventional periapical radiography. The treatment plan of this primary periodontal lesion with secondary endodontic involvement was as follows: periapical surgery combined with root amputation and sealing with MTA, and guided bone regeneration. Key words:Palatal radicular groove, palatogingival groove, maxillary lateral incisor, cone-beam computed tomography, endodontic-periodontal lesion, guided bone regeneration.
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BACKGROUND: The success of endodontic treatment derives from the complete elimination of microorganisms capable of causing an intraradicular or extraradicular infection. To achieve a more effective eradication of these microorganisms, endodontic instrumentation must always be implemented with abundant irrigation, which has to achieve chemical, mechanical and biological effects. The irrigators most used today are NaOCl, CHX and EDTA, released into the ducts through different techniques such as syringe, manual agitation, positive or negative apical pressure, sonic or ultrasonic activation, PIPS and PDT. The objective of this review is to update the different irrigating solutions and intracanal disinfection drugs, as well as to establish an irrigation protocol in the endodontic treatment. MATERIAL AND METHODS: Systematic search of scientific articles in the databases PubMed, Medline and Google Scholar, with the following keywords Endodontic, Infection, Failure, Irrigation, Retreatment and Irrigation protocol. The exclusion criteria were "case report" articles and articles with a publication date prior to 2000. RESULTS: 48 articles that met the inclusion criteria were analyzed. Comparing the different articles it can be seen that the NaOCl is the "gold standard" in terms of immediate antimicrobial efficacy, followed by the CHX that has a long-term antibacterial effect. As an intra-conductive drug it is advisable to use the combination of Ca(OH)2 with CPMC. CONCLUSIONS: The most adequate irrigation protocol consists of using 2.5% NaOCl activated with ultrasound followed by a final wash with 7% MA or 0.2% CTR combined with 2% CHX. Key words:Endodontic failure, endodontic infection, enterococcus faecalis, endodontic retreatment, irrigation, sodium hipoclorite, irrigation protocol.
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Paracrine and endocrine roles have increasingly been ascribed to extracellular vesicles (EVs) generated by multicellular organisms. Central to the biogenesis, content, and function of EVs are their delimiting lipid bilayer membranes. To evaluate research progress on membranes and EVs, the International Society for Extracellular Vesicles (ISEV) conducted a workshop in March 2018 in Baltimore, Maryland, USA, bringing together key opinion leaders and hands-on researchers who were selected on the basis of submitted applications. The workshop was accompanied by two scientific surveys and covered four broad topics: EV biogenesis and release; EV uptake and fusion; technologies and strategies used to study EV membranes; and EV transfer and functional assays. In this ISEV position paper, we synthesize the results of the workshop and the related surveys to outline important outstanding questions about EV membranes and describe areas of consensus. The workshop discussions and survey responses reveal that while much progress has been made in the field, there are still several concepts that divide opinion. Good consensus exists in some areas, including particular aspects of EV biogenesis, uptake and downstream signalling. Areas with little to no consensus include EV storage and stability, as well as whether and how EVs fuse with target cells. Further research is needed in these key areas, as a better understanding of membrane biology will contribute substantially towards advancing the field of extracellular vesicles.
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Studies carried out by immunofluorescence, patch-clamping and FM dye fluorescence consistently showed that the Ca(2+)-induced exocytosis of enlargeosomes, specific vesicles expressed by many cell types, is strongly reinforced by pre-treatment of the cells with genistein, a wide spectrum blocker of tyrosine kinases, which also induces many additional effects. Various other blockers of tyrosine kinases, however, were ineffective, and the same occurred with drugs mimicking most of the rapid, non-tyrosine kinase-dependent effects of genistein. The reinforcement of enlargeosome-regulated exocytosis, therefore, is a new effect of genistein and a peculiar property of the enlargeosome exocytosis, not shared by analogous processes.
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Exocitosis/efectos de los fármacos , Genisteína/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Vesículas Secretoras/efectos de los fármacos , Animales , Calcio/metabolismo , Ionomicina/farmacología , Ionóforos/farmacología , Células PC12 , Técnicas de Placa-Clamp , Proteínas Tirosina Quinasas/antagonistas & inhibidores , RatasRESUMEN
Trehalose is a nonreducing disaccharide that has recently attracted much attention because of its ability to inhibit protein aggregation, induce autophagy, and protect against dissections and strokes. In vertebrates, the biosynthesis of trehalose was long considered absent due to the lack of annotated genes involved in this process. In contrast, trehalase (TreH), which is an enzyme required for the cleavage of trehalose, is known to be conserved and expressed. Here, we show that trehalose is present as an endogenous metabolite in the rodent hippocampus. We found that primary astrocytes were able to synthesize trehalose and release it into the extracellular space. Notably, the TreH enzyme was observed only in the soma of neurons, which are the exclusive users of this substrate. A statistical analysis of the metabolome during different stages of maturation indicated that this metabolite is implicated in neuronal maturation. A morphological analysis of primary neurons confirmed that trehalose is required for neuronal arborization.
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Astrocitos/enzimología , Hipocampo/enzimología , Plasticidad Neuronal/fisiología , Neuronas/enzimología , Trehalosa/biosíntesis , Animales , Astrocitos/citología , Células Cultivadas , Cerebelo/citología , Cerebelo/enzimología , Corteza Cerebral/citología , Corteza Cerebral/enzimología , Proteína Ácida Fibrilar de la Glía/metabolismo , Hipocampo/citología , Masculino , Metaboloma , Metabolómica , Ratones Endogámicos C57BL , Microglía/citología , Microglía/enzimología , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas/citología , Bulbo Olfatorio/citología , Bulbo Olfatorio/enzimología , Ratas Sprague-Dawley , Ratas WistarRESUMEN
Extracellular ATP is among molecules promoting microglia activation and inducing the release of extracellular vesicles (EVs), which are potent mediators of intercellular communication between microglia and the microenvironment. We previously showed that EVs produced under ATP stimulation (ATP-EVs) propagate a robust inflammatory reaction among astrocytes and microglia in vitro and in mice with subclinical neuroinflammation (Verderio et al., 2012). However, the proteome of EVs released upon ATP stimulation has not yet been elucidated. In this study we applied a label free proteomic approach to characterize the proteome of EVs released constitutively and during microglia activation with ATP. We show that ATP drives sorting in EVs of a set of proteins implicated in cell adhesion/extracellular matrix organization, autophagy-lysosomal pathway and cellular metabolism, that may influence the response of recipient astrocytes to EVs. These data provide new clues to molecular mechanisms involved in microglia response to ATP and in microglia signaling to the environment via EVs.
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In the substantia nigra of human brain, neuromelanin (NM) released by degenerating neurons can activate microglia with consequent neurodegeneration, typical of Parkinson's disease (PD). Synthetic analogues of NM were prepared to develop a PD model reproducing the neuropathological conditions of the disease. Soluble melanin-protein conjugates were obtained by melanization of fibrillated ß-lactoglobulin (fLG). The melanic portion of the conjugates contains either eumelanic (EufLG) or mixed eumelanic/pheomelanic composition (PheofLG), the latter better simulating natural NMs. In addition, the conjugates can be loaded with controlled amounts of iron. Upon melanization, PheofLG-Fe conjugates maintain the amyloid cross-ß protein core as the only structurally organized element, similarly to human NMs. The similarity in composition and structural organization with the natural pigment is reflected by the ability of synthetic NMs to activate microglia, showing potential of the novel conjugates to model NM induced neuroinflammation. Thus, synthetic NM/microglia constitute a new model to develop anti-Parkinson drugs.
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Melaninas/análisis , Melaninas/síntesis química , Melaninas/farmacología , Microglía/efectos de los fármacos , Enfermedad de Parkinson/patología , Amiloide/farmacocinética , Animales , Animales Recién Nacidos , Arginasa/genética , Arginasa/metabolismo , Células Cultivadas , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Dopamina/metabolismo , Proteínas Filagrina , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Hierro/metabolismo , Lactoglobulinas/farmacocinética , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Receptor de Manosa , Lectinas de Unión a Manosa/genética , Lectinas de Unión a Manosa/metabolismo , Melaninas/química , Microglía/ultraestructura , Modelos Biológicos , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismoRESUMEN
Extracellular vesicles (EVs) are spherical membrane structures released by most cells. These highly conserved mediators of intercellular communication carry proteins, lipids, and nucleic acids, and transfer these cellular components between cells by different mechanisms, such as endocytosis, macropinocytosis, or fusion. However, the temporal and spatial dynamics of vesicle-cell interactions still remain largely unexplored. Here we used optical tweezers to drive single EVs produced by microglial cells onto the surface of astrocytes or microglia in primary culture. By visualizing single EV-cell contacts, we observed that microglial vesicles displayed different motilities on the surface of astrocytes compared with microglia. After contact, EVs positioned on astrocytes displayed some minor oscillatory motion around the point of adhesion, while vesicles dragged to microglia displayed quite regular directional movement on the plasma membrane. Both the adhesion and motion of vesicles on glial cells were strongly reduced by cloaking phosphatidylserine (PS) residues, which are externalized on the vesicle membrane and act as determinants for vesicle recognition by target cells. These data identify optical manipulation as a powerful tool to monitor in vitro vesicle-cell dynamics with high temporal and spatial resolution and to determine in a quantitative manner the contribution of surface receptors/extracellular protein ligands to the contact.
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Adhesión Celular/genética , Vesículas Extracelulares/metabolismo , Pinzas Ópticas , Animales , Astrocitos/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Vesículas Extracelulares/genética , Microglía/metabolismo , Cultivo Primario de Células , RatasRESUMEN
The Repressor Element-1 (RE-1) Silencing Transcription (REST) factor, which is highly expressed in stem cells and non-neural cells, with low expression in neurons and other neural cells, orchestrates neural differentiation and preserves the unique neural phenotype. REST also plays a role in proliferation, although its effect differs depending on the cell type. It acts as an oncogene in neural cells and tumors (medulloblastomas, neuroblastomas, glioblastomas) and as a tumor suppressor in carcinomas of the lung, breast, and colon. The mechanisms underlying this duality have started to emerge recently and new therapeutic approaches based on these findings are being developed. Here, we present the mechanisms proposed to account for the oncogenic and antioncogenic roles of REST and discuss the therapeutic perspective of recent advances, particularly for small-cell lung cancer.
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Oncogenes , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteínas Supresoras de Tumor/genética , Carcinoma/genética , Carcinoma/metabolismo , Diferenciación Celular , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Meduloblastoma/genética , Meduloblastoma/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuronas/metabolismo , Células Madre/metabolismoRESUMEN
Astrocytes are the brain nonnerve cells that are competent for gliosecretion, i.e., for expression and regulated exocytosis of clear and dense-core vesicles (DCVs). We investigated whether expression of astrocyte DCVs is governed by RE-1-silencing transcription factor (REST)/neuron-restrictive silencer factor (NRSF), the transcription repressor that orchestrates nerve cell differentiation. Rat astrocyte cultures exhibited high levels of REST and expressed neither DCVs nor their markers (granins, peptides, and membrane proteins). Transfection of a dominant-negative construct of REST induced the appearance of DCVs filled with secretogranin 2 and neuropeptide Y (NPY) and distinct from other organelles. Total internal reflection fluorescence analysis revealed NPY-monomeric red fluorescent protein-labeled DCVs to undergo Ca(2+)-dependent exocytosis, which was largely prevented by botulinum toxin B. In the I-II layers of the human temporal brain cortex, all neurons and microglia exhibited the expected inappreciable and high levels of REST, respectively. In contrast, astrocyte REST was variable, going from inappreciable to high, and accompanied by a variable expression of DCVs. In conclusion, astrocyte DCV expression and gliosecretion are governed by REST. The variable in situ REST levels may contribute to the well-known structural/functional heterogeneity of astrocytes.