RESUMEN
Neurons are polarized cells of extreme scale and compartmentalization. To fulfill their role in electrochemical signaling, axons must maintain a specific complement of membrane proteins. Despite being the subject of considerable attention, the trafficking pathway of axonal membrane proteins is not well understood. Two pathways, direct delivery and transcytosis, have been proposed. Previous studies reached contradictory conclusions about which of these mediates delivery of axonal membrane proteins to their destination, in part because they evaluated long-term distribution changes and not vesicle transport. We developed a novel strategy to selectively label vesicles in different trafficking pathways and determined the trafficking of two canonical axonal membrane proteins, neuron-glia cell adhesion molecule and vesicle-associated membrane protein-2. Results from detailed quantitative analyses of transporting vesicles differed substantially from previous studies and found that axonal membrane proteins overwhelmingly undergo direct delivery. Transcytosis plays only a minor role in axonal delivery of these proteins. In addition, we identified a novel pathway by which wayward axonal proteins that reach the dendritic plasma membrane are targeted to lysosomes. These results redefine how axonal proteins achieve their polarized distribution, a crucial requirement for elucidating the underlying molecular mechanisms.
Asunto(s)
Axones/metabolismo , Moléculas de Adhesión Celular Neurona-Glia/metabolismo , Transporte de Proteínas/fisiología , Proteína 2 de Membrana Asociada a Vesículas/metabolismo , Animales , Transporte Biológico , Moléculas de Adhesión Celular Neurona-Glia/fisiología , Polaridad Celular , Dendritas/metabolismo , Endocitosis/fisiología , Endosomas/metabolismo , Hipocampo/metabolismo , Potenciales de la Membrana/fisiología , Neuronas/metabolismo , Cultivo Primario de Células/métodos , Ratas , Transducción de Señal , Transcitosis/fisiología , Vesículas Transportadoras/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/fisiologíaRESUMEN
The primary hallmark of Parkinson's disease (PD) is the generation of Lewy bodies of which major component is α-synuclein (α-Syn). Because of increasing evidence of the fundamental roles of α-Syn oligomers in disease progression, α-Syn oligomers have become potential targets for therapeutic interventions for PD. One of the potential toxicities of α-Syn oligomers is their inhibition of SNARE-mediated vesicle fusion by specifically interacting with vesicle-SNARE protein synaptobrevin-2 (Syb2), which hampers dopamine release. Here, we show that α-Syn monomers and oligomers cooperatively inhibit neuronal SNARE-mediated vesicle fusion. α-Syn monomers at submicromolar concentrations increase the fusion inhibition by α-Syn oligomers. This cooperative pathological effect stems from the synergically enhanced vesicle clustering. Based on this cooperative inhibition mechanism, we reverse the fusion inhibitory effect of α-Syn oligomers using small peptide fragments. The small peptide fragments, derivatives of α-Syn, block the binding of α-Syn oligomers to Syb2 and dramatically reverse the toxicity of α-Syn oligomers in vesicle fusion. Our findings demonstrate a new strategy for therapeutic intervention in PD and related diseases based on this specific interaction of α-Syn.
Asunto(s)
Fusión de Membrana/efectos de los fármacos , Proteínas SNARE/antagonistas & inhibidores , alfa-Sinucleína/farmacología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Dopamina/metabolismo , Dopamina/farmacología , Evaluación Preclínica de Medicamentos , Liposomas , Lípidos de la Membrana/metabolismo , Modelos Moleculares , Mutación Missense , Fragmentos de Péptidos/farmacología , Mutación Puntual , Unión Proteica , Multimerización de Proteína , Proteolípidos/química , Proteínas Recombinantes de Fusión/farmacología , Proteínas SNARE/fisiología , Proteína 2 de Membrana Asociada a Vesículas/antagonistas & inhibidores , Proteína 2 de Membrana Asociada a Vesículas/fisiología , alfa-Sinucleína/química , alfa-Sinucleína/genética , alfa-Sinucleína/toxicidadRESUMEN
Vesicle fusion is mediated by assembly of SNARE proteins between opposing membranes. While previous work suggested an active role of SNARE transmembrane domains (TMDs) in promoting membrane merger (Dhara et al., 2016), the underlying mechanism remained elusive. Here, we show that naturally-occurring v-SNARE TMD variants differentially regulate fusion pore dynamics in mouse chromaffin cells, indicating TMD flexibility as a mechanistic determinant that facilitates transmitter release from differentially-sized vesicles. Membrane curvature-promoting phospholipids like lysophosphatidylcholine or oleic acid profoundly alter pore expansion and fully rescue the decelerated fusion kinetics of TMD-rigidifying VAMP2 mutants. Thus, v-SNARE TMDs and phospholipids cooperate in supporting membrane curvature at the fusion pore neck. Oppositely, slowing of pore kinetics by the SNARE-regulator complexin-2 withstands the curvature-driven speeding of fusion, indicating that pore evolution is tightly coupled to progressive SNARE complex formation. Collectively, TMD-mediated support of membrane curvature and SNARE force-generated membrane bending promote fusion pore formation and expansion.
Asunto(s)
Exocitosis , Fusión de Membrana , Complejos Multiproteicos/fisiología , Neurotransmisores/fisiología , Fosfolípidos/metabolismo , Proteínas SNARE/fisiología , Proteína 2 de Membrana Asociada a Vesículas/fisiología , Animales , Calcio/fisiología , Membrana Celular/metabolismo , Células Cultivadas , Células Cromafines , Cinética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Mutantes/fisiología , Unión Proteica , Dominios Proteicos , Vesículas Secretoras/fisiologíaRESUMEN
VAMP2 encodes the vesicular SNARE protein VAMP2 (also called synaptobrevin-2). Together with its partners syntaxin-1A and synaptosomal-associated protein 25 (SNAP25), VAMP2 mediates fusion of synaptic vesicles to release neurotransmitters. VAMP2 is essential for vesicular exocytosis and activity-dependent neurotransmitter release. Here, we report five heterozygous de novo mutations in VAMP2 in unrelated individuals presenting with a neurodevelopmental disorder characterized by axial hypotonia (which had been present since birth), intellectual disability, and autistic features. In total, we identified two single-amino-acid deletions and three non-synonymous variants affecting conserved residues within the C terminus of the VAMP2 SNARE motif. Affected individuals carrying de novo non-synonymous variants involving the C-terminal region presented a more severe phenotype with additional neurological features, including central visual impairment, hyperkinetic movement disorder, and epilepsy or electroencephalography abnormalities. Reconstituted fusion involving a lipid-mixing assay indicated impairment in vesicle fusion as one of the possible associated disease mechanisms. The genetic synaptopathy caused by VAMP2 de novo mutations highlights the key roles of this gene in human brain development and function.
Asunto(s)
Discapacidad Intelectual/genética , Hipotonía Muscular/genética , Trastornos del Neurodesarrollo/genética , Neuronas/metabolismo , Sinapsis/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/genética , Adolescente , Trastorno Autístico/genética , Trastorno Autístico/metabolismo , Encéfalo/diagnóstico por imagen , Niño , Preescolar , Epilepsia/metabolismo , Exocitosis , Femenino , Heterocigoto , Humanos , Lípidos/química , Imagen por Resonancia Magnética , Masculino , Fusión de Membrana , Trastornos del Movimiento/genética , Mutación , Trastornos del Neurodesarrollo/metabolismo , Neurotransmisores/metabolismo , Fenotipo , Dominios Proteicos , Proteínas R-SNARE/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/fisiologíaRESUMEN
Lateral inhibition in the vertebrate retina depends on a negative feedback synapse between horizontal cells (HCs) and rod and cone photoreceptors. A change in pH is thought to be the signal for negative feedback, but its spatial profile in the synaptic cleft is unknown. Here we use three different membrane proteins, each fused to the same genetically-encoded pH-sensitive Green Fluorescent Protein (GFP) (pHluorin), to probe synaptic pH in retina from transgenic zebrafish (Danio rerio) of either sex. We used the cone transducin promoter to express SynaptopHluorin (pHluorin on vesicle-associated membrane protein (VAMP2)) or CalipHluorin (pHluorin on an L-type Ca2+ channel) and the HC-specific connexin-55.5 promoter to express AMPApHluorin (pHluorin on an AMPA receptor). Stimulus light led to increased fluorescence of all three probes, consistent with alkalinization of the synaptic cleft. The receptive field size, sensitivity to surround illumination, and response to activation of an alien receptor expressed exclusively in HCs, are consistent with lateral inhibition as the trigger for alkalinization. However, SynaptopHluorin and AMPApHluorin, which are displaced farther from cone synaptic ribbons than CalipHluorin, reported a smaller pH change. Hence, unlike feedforward glutamatergic transmission, which spills over to allow cross talk between terminals in the cone network, the pH change underlying HC feedback is compartmentalized to individual synaptic invaginations within a cone terminal, consistent with private line communication.SIGNIFICANCE STATEMENT Lateral inhibition (LI) is a fundamental feature of information processing in sensory systems, enhancing contrast sensitivity and enabling edge discrimination. Horizontal cells (HCs) are the first cellular substrate of LI in the vertebrate retina, but the synaptic mechanisms underlying LI are not completely understood, despite decades of study. This paper makes a significant contribution to our understanding of LI, by showing that each HC-cone synapse is a "private-line" that operates independently from other HC-cone connections. Using transgenic zebrafish expressing pHluorin, a pH-sensitive GFP variant spliced onto three different protein platforms expressed either in cones or HCs we show that the feedback pH signal is constrained to individual cone terminals, and more stringently, to individual synaptic contact sites within each terminal.
Asunto(s)
Retroalimentación Fisiológica/fisiología , Células Fotorreceptoras Retinianas Conos/fisiología , Células Horizontales de la Retina/fisiología , Sinapsis/fisiología , Animales , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/fisiología , Conexinas/metabolismo , Femenino , Glutamatos/fisiología , Concentración de Iones de Hidrógeno , Masculino , Protones , Receptores AMPA/metabolismo , Células Fotorreceptoras Retinianas Conos/ultraestructura , Células Horizontales de la Retina/ultraestructura , Sinapsis/ultraestructura , Transmisión Sináptica/fisiología , Proteína 2 de Membrana Asociada a Vesículas/genética , Proteína 2 de Membrana Asociada a Vesículas/fisiología , Pez CebraRESUMEN
The soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins synaptobrevin (Syb), syntaxin, and SNAP-25 function in Ca2+-triggered exocytosis in both endocrine cells and neurons. The transmembrane domains (TMDs) of Syb and syntaxin span the vesicle and plasma membrane, respectively, and influence flux through fusion pores in endocrine cells as well as fusion pores formed during SNARE-mediated fusion of reconstituted membranes. These results support a model for exocytosis in which SNARE TMDs form the initial fusion pore. The present study sought to test this model in synaptic terminals. Patch-clamp recordings of miniature EPSCs (mEPSCs) were used to probe fusion pore properties in cultured hippocampal neurons from mice of both sexes. Mutants harboring tryptophan at four different sites in the Syb TMD reduced the rate-of-rise of mEPSCs. A computer model that simulates glutamate diffusion and receptor activation kinetics could account for this reduction in mEPSC rise rate by slowing the flux of glutamate through synaptic fusion pores. TMD mutations introducing positive charge also reduced the mEPSC rise rate, but negatively charged residues and glycine, which should have done the opposite, had no effect. The sensitivity of mEPSCs to pharmacological blockade of receptor desensitization was enhanced by a mutation that slowed the mEPSC rate-of-rise, suggesting that the mutation prolonged the residence of glutamate in the synaptic cleft. The same four Syb TMD residues found here to influence synaptic release were found previously to influence endocrine release, leading us to propose that a similar TMD-lined fusion pore functions widely in Ca2+-triggered exocytosis in mammalian cells.SIGNIFICANCE STATEMENT SNARE proteins function broadly in biological membrane fusion. Evidence from non-neuronal systems suggests that SNARE proteins initiate fusion by forming a fusion pore lined by transmembrane domains, but this model has not yet been tested in synapses. The present study addressed this question by testing mutations in the synaptic vesicle SNARE synaptobrevin for an influence on the rise rate of miniature synaptic currents. These results indicate that synaptobrevin's transmembrane domain interacts with glutamate as it passes through the fusion pore. The sites in synaptobrevin that influence this flux are identical to those shown previously to influence flux through endocrine fusion pores. Thus, SNARE transmembrane domains may function in the fusion pores of Ca2+-triggered exocytosis of both neurotransmitters and hormones.
Asunto(s)
Exocitosis/fisiología , Ácido Glutámico/metabolismo , Potenciales Postsinápticos Miniatura/fisiología , Neuronas/fisiología , Proteína 2 de Membrana Asociada a Vesículas/fisiología , Sustitución de Aminoácidos , Animales , Transporte Biológico , Calcio/fisiología , Simulación por Computador , Difusión , Femenino , Técnicas de Inactivación de Genes , Hipocampo/citología , Cinética , Masculino , Fusión de Membrana , Ratones , Modelos Biológicos , Técnicas de Placa-Clamp , Dominios Proteicos , Proteínas SNARE/fisiología , Triptófano/análisis , Proteína 2 de Membrana Asociada a Vesículas/química , Proteína 2 de Membrana Asociada a Vesículas/genéticaRESUMEN
Synaptic soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) couple their stepwise folding to fusion of synaptic vesicles with plasma membranes. In this process, three SNAREs assemble into a stable four-helix bundle. Arguably, the first and rate-limiting step of SNARE assembly is the formation of an activated binary target (t)-SNARE complex on the target plasma membrane, which then zippers with the vesicle (v)-SNARE on the vesicle to drive membrane fusion. However, the t-SNARE complex readily misfolds, and its structure, stability, and dynamics are elusive. Using single-molecule force spectroscopy, we modeled the synaptic t-SNARE complex as a parallel three-helix bundle with a small frayed C terminus. The helical bundle sequentially folded in an N-terminal domain (NTD) and a C-terminal domain (CTD) separated by a central ionic layer, with total unfolding energy of â¼17 kBT, where kB is the Boltzmann constant and T is 300 K. Peptide binding to the CTD activated the t-SNARE complex to initiate NTD zippering with the v-SNARE, a mechanism likely shared by the mammalian uncoordinated-18-1 protein (Munc18-1). The NTD zippering then dramatically stabilized the CTD, facilitating further SNARE zippering. The subtle bidirectional t-SNARE conformational switch was mediated by the ionic layer. Thus, the t-SNARE complex acted as a switch to enable fast and controlled SNARE zippering required for synaptic vesicle fusion and neurotransmission.
Asunto(s)
Proteínas SNARE/química , Secuencia de Aminoácidos , Animales , Fusión de Membrana , Ratones , Microscopía de Fuerza Atómica , Simulación de Dinámica Molecular , Proteínas Munc18/química , Proteínas Munc18/fisiología , Pinzas Ópticas , Conformación Proteica , Dominios Proteicos , Pliegue de Proteína , Estabilidad Proteica , Proteínas Qa-SNARE/química , Proteínas Qa-SNARE/fisiología , Proteínas SNARE/genética , Proteínas SNARE/fisiología , Transmisión Sináptica/fisiología , Proteína 25 Asociada a Sinaptosomas/química , Proteína 25 Asociada a Sinaptosomas/fisiología , Proteína 2 de Membrana Asociada a Vesículas/química , Proteína 2 de Membrana Asociada a Vesículas/fisiologíaRESUMEN
Synaptic vesicle fusion during neurotransmitter release is mediated by assembly of SNARE- and SM-protein complexes composed of syntaxin-1, SNAP-25, synaptobrevin-2/VAMP2, and Munc18-1. Current models suggest that SNARE-complex assembly catalyzes membrane fusion by pulling the transmembrane regions (TMRs) of SNARE proteins together, thus allowing their TMRs to form a fusion pore. These models are consistent with the requirement for TMRs in viral fusion proteins. However, the role of the SNARE TMRs in synaptic vesicle fusion has not yet been tested physiologically. Here, we examined whether synaptic SNAREs require TMRs for catalysis of synaptic vesicle fusion, which was monitored electrophysiologically at millisecond time resolution. Surprisingly, we find that both lipid-anchored syntaxin-1 and lipid-anchored synaptobrevin-2 lacking TMRs efficiently promoted spontaneous and Ca(2+)-triggered membrane fusion. Our data suggest that SNARE proteins function during fusion primarily as force generators, consistent with the notion that forcing lipid membranes close together suffices to induce membrane fusion.
Asunto(s)
Fusión de Membrana/fisiología , Lípidos de la Membrana/metabolismo , Neurotransmisores/metabolismo , Proteínas SNARE/metabolismo , Transmisión Sináptica/fisiología , Vesículas Sinápticas/metabolismo , Animales , Células Cultivadas , Potenciales Postsinápticos Inhibidores/fisiología , Ratones , Ratones Noqueados , Ratones Transgénicos , Neuronas/fisiología , Proteínas SNARE/química , Proteínas SNARE/genética , Sintaxina 1/genética , Sintaxina 1/fisiología , Proteína 2 de Membrana Asociada a Vesículas/genética , Proteína 2 de Membrana Asociada a Vesículas/fisiologíaRESUMEN
Parkinson disease and dementia with Lewy bodies are featured with the formation of Lewy bodies composed mostly of α-synuclein (α-Syn) in the brain. Although evidence indicates that the large oligomeric or protofibril forms of α-Syn are neurotoxic agents, the detailed mechanisms of the toxic functions of the oligomers remain unclear. Here, we show that large α-Syn oligomers efficiently inhibit neuronal SNARE-mediated vesicle lipid mixing. Large α-Syn oligomers preferentially bind to the N-terminal domain of a vesicular SNARE protein, synaptobrevin-2, which blocks SNARE-mediated lipid mixing by preventing SNARE complex formation. In sharp contrast, the α-Syn monomer has a negligible effect on lipid mixing even with a 30-fold excess compared with the case of large α-Syn oligomers. Thus, the results suggest that large α-Syn oligomers function as inhibitors of dopamine release, which thus provides a clue, at the molecular level, to their neurotoxicity.
Asunto(s)
Neuronas/fisiología , Proteínas SNARE/fisiología , alfa-Sinucleína/química , alfa-Sinucleína/fisiología , Animales , Exocitosis/efectos de los fármacos , Exocitosis/fisiología , Metabolismo de los Lípidos/efectos de los fármacos , Modelos Neurológicos , Neuronas/efectos de los fármacos , Neurotoxinas/química , Neurotoxinas/toxicidad , Células PC12 , Unión Proteica , Estructura Cuaternaria de Proteína , Proteolípidos/metabolismo , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/toxicidad , Vesículas Secretoras/efectos de los fármacos , Vesículas Secretoras/fisiología , Transducción Genética , Proteína 2 de Membrana Asociada a Vesículas/fisiología , alfa-Sinucleína/genética , alfa-Sinucleína/toxicidadRESUMEN
In this study we used live-cell immunocytochemistry and confocal microscopy to study the release from a single vesicle in a simplified system called membrane lawns. The lawns were prepared by exposing differentiated pituitary prolactin (PRL)-secreting cells to a hypoosmotic shear stress. The density of the immunolabeled ternary soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) complexes that bind complexin was approximately 10 times lower than the PRL-positive, lawn-resident vesicles; this indicates that some but not all vesicles are associated with ternary SNARE complexes. However, lawn-resident PRL vesicles colocalized relatively well with particular SNARE proteins: synaptobrevin 2 (35%), syntaxin 1 (22%), and 25-kDa synaptosome associated protein (6%). To study vesicle discharge, we prepared lawn-resident vesicles, derived from atrial natriuretic peptide tagged with emerald fluorescent protein (ANP.emd)-transfected cells, which label vesicles. These maintained the structural passage to the exterior because approximately 40% of ANP.emd-loaded vesicles were labeled by extracellular PRL antibodies. Cargo release from the lawn-resident vesicles, monitored by the decline in the ANP.emd fluorescence intensity, was similar to that in intact cells. It is likely that SNARE proteins are required for calcium-dependent release from these vesicles. This is because the expression of the dominant-negative SNARE peptide, which interferes with SNARE complex formation, reduced the number of PRL-positive spots per cell (PRL antibodies placed extracellularly) significantly, from 58 ± 9 to 4 ± 2. In dominant-negative SNARE-treated cells, the PRL-positive area was reduced from 0.259 ± 0.013 to 0.123 ± 0.014 µm(2), which is consistent with a hindered vesicle luminal access for extracellular PRL antibodies. These results indicate that vesicle discharge is regulated by SNARE-mediated fusion pore widening.
Asunto(s)
Hipófisis/metabolismo , Prolactina/metabolismo , Proteínas SNARE/fisiología , Vesículas Secretoras/metabolismo , Animales , Calcio/metabolismo , Diferenciación Celular , Inmunohistoquímica , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Fusión de Membrana/fisiología , Microscopía Confocal , Hipófisis/citología , Ratas , Ratas Wistar , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vesículas Secretoras/ultraestructura , Sinaptosomas/fisiología , Sintaxina 1/fisiología , Proteína 2 de Membrana Asociada a Vesículas/fisiologíaAsunto(s)
Axones/fisiología , Membrana Celular/fisiología , Animales , Transporte Biológico , Movimiento Celular , Citoesqueleto/fisiología , Citoesqueleto/ultraestructura , Endocitosis , Conos de Crecimiento/fisiología , Conos de Crecimiento/ultraestructura , Ratones , Modelos Biológicos , Factores de Crecimiento Nervioso/fisiología , Sistema Nervioso/embriología , Netrina-1 , Proteínas SNARE/fisiología , Semaforina-3A/fisiología , Proteína 25 Asociada a Sinaptosomas/fisiología , Sintaxina 1/fisiología , Proteínas Supresoras de Tumor/fisiología , Proteína 2 de Membrana Asociada a Vesículas/fisiologíaRESUMEN
Loss of sleep causes an increase in sleep drive and deficits in hippocampal-dependent memory. Both of these responses are thought to require activation of adenosine A1 receptors (adorA1Rs) and release of transmitter molecules including ATP, which is rapidly converted to adenosine in the extracellular space, from astrocytes in a process termed gliotransmission. Although it is increasingly clear that astrocyte-derived adenosine plays an important role in driving the homeostatic sleep response and the effects of sleep loss on memory (Halassa et al., 2009; Florian et al., 2011), previous studies have not determined whether the concentration of this signaling molecule increases in response to wakefulness. Here, we show that the level of adorA1R activation increases in response to wakefulness in mice (Mus musculus). We found that this increase affected synaptic transmission in the hippocampus and modulated network activity in the cortex. Direct biosensor-based measurement of adenosine showed that the net extracellular concentration of this transmitter increased in response to normal wakefulness and sleep deprivation. Genetic inhibition of gliotransmission prevented this increase and attenuated the wakefulness-dependent changes in synaptic and network regulation by adorA1R. Consequently, we conclude that wakefulness increases the level of extracellular adenosine in the hippocampus and that this increase requires the release of transmitters from astroctyes.
Asunto(s)
Adenosina/metabolismo , Astrocitos/metabolismo , Líquido Extracelular/metabolismo , Transmisión Sináptica/fisiología , Vigilia/fisiología , Adenosina/fisiología , Animales , Corteza Cerebral/fisiología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptor de Adenosina A1/efectos de los fármacos , Receptor de Adenosina A1/fisiología , Privación de Sueño/metabolismo , Transmisión Sináptica/efectos de los fármacos , Teofilina/análogos & derivados , Teofilina/farmacología , Proteína 2 de Membrana Asociada a Vesículas/genética , Proteína 2 de Membrana Asociada a Vesículas/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/fisiologíaRESUMEN
Attractive and repulsive molecules such as Semaphorins (Sema) trigger rapid responses that control the navigation of axonal growth cones. The role of vesicular traffic in axonal guidance is still largely unknown. The exocytic vesicular soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor (SNARE) Synaptobrevin 2 (Syb2) is known for mediating neurotransmitter release in mature neurons, but its potential role in axonal guidance remains elusive. Here we show that Syb2 is required for Sema3A-dependent repulsion but not Sema3C-dependent attraction in cultured neurons and in the mouse brain. Syb2 associated with Neuropilin 1 and Plexin A1, two essential components of the Sema3A receptor, via its juxtatransmembrane domain. Sema3A receptor and Syb2 colocalize in endosomal membranes. Moreover, upon Sema3A treatment, Syb2-deficient neurons failed to collapse and transport Plexin A1 to cell bodies. Reconstitution of Sema3A receptor in nonneuronal cells revealed that Sema3A further inhibited the exocytosis of Syb2. Therefore, Sema3A-mediated signaling and axonal repulsion require Syb2-dependent vesicular traffic.
Asunto(s)
Axones/fisiología , Proteínas R-SNARE/fisiología , Semaforina-3A/fisiología , Proteína 2 de Membrana Asociada a Vesículas/fisiología , Animales , Células COS , Chlorocebus aethiops , Cuerpo Calloso/anatomía & histología , Exocitosis/fisiología , Conos de Crecimiento/fisiología , Ratones , Ratones Noqueados , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Semaforina-3A/metabolismo , Transducción de Señal , Proteína 2 de Membrana Asociada a Vesículas/genética , Proteína 2 de Membrana Asociada a Vesículas/metabolismoRESUMEN
Our previous work has correlated permanent alterations in the rat neurosecretory machinery with epileptogenesis. Such findings highlighted the need for a greater understanding of the molecular mechanisms underlying epilepsy so that novel therapeutic regimens can be designed. To this end, we examined kindling in transgenic mice with a defined reduction of a key element of the neurosecretory machinery: the v-SNARE (vesicle-bound SNAP [soluble NSF attachment protein] receptor), synaptobrevin/vesicle-associated membrane protein 2 (VAMP2). Initial analysis of biochemical markers, which previously displayed kindling-dependent alterations in rat hippocampal synaptosomes, showed similar trends in both wild-type and VAMP2(+/-) mice, demonstrating that kindled rat and mouse models are comparable. This report focuses on the effects that a ~50% reduction of synaptosomal VAMP2 has on the progression of electrical kindling and on glutamate release in hippocampal subregions. Our studies show that epileptogenesis is dramatically attenuated in VAMP2(+/-) mice, requiring both higher current and more stimulations to reach a fully kindled state (two successive Racine stage 5 seizures). Progression through the five identifiable Racine stages was slower and more variable in the VAMP2(+/-) animals compared with the almost linear progression seen in wild-type littermates. Consistent with the expected effects of reducing a major neuronal v-SNARE, glutamate-selective, microelectrode array (MEA) measurements in specific hippocampal subregions of VAMP2(+/-) mice showed significant reductions in potassium-evoked glutamate release. Taken together these studies demonstrate that manipulating the levels of the neurosecretory machinery not only affects neurotransmitter release but also mitigates kindling-induced epileptogenesis.
Asunto(s)
Epilepsia/fisiopatología , Excitación Neurológica/genética , Excitación Neurológica/fisiología , Proteína 2 de Membrana Asociada a Vesículas/biosíntesis , Proteína 2 de Membrana Asociada a Vesículas/fisiología , Animales , Región CA1 Hipocampal/efectos de los fármacos , Región CA1 Hipocampal/metabolismo , Región CA2 Hipocampal/efectos de los fármacos , Región CA2 Hipocampal/metabolismo , Interpretación Estadística de Datos , Giro Dentado/efectos de los fármacos , Giro Dentado/metabolismo , Estimulación Eléctrica , Ácido Glutámico/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microelectrodos , Proteínas SNARE/fisiología , Proteína 2 de Membrana Asociada a Vesículas/genéticaRESUMEN
BACKGROUND: The aim of this study was to assess the distribution of key SNARE proteins in glutamatergic and GABAergic synapses of the adult rat cerebellar cortex using light microscopy immunohistochemical techniques. Analysis was made of co-localizations of vGluT-1 and vGluT-2, vesicular transporters of glutamate and markers of glutamatergic synapses, or GAD, the GABA synthetic enzyme and marker of GABAergic synapses, with VAMP-2, SNAP-25A/B and syntaxin-1. RESULTS: The examined SNARE proteins were found to be diffusely expressed in glutamatergic synapses, whereas they were rarely observed in GABAergic synapses. However, among glutamatergic synapses, subpopulations which did not contain VAMP-2, SNAP-25A/B and syntaxin-1 were detected. They included virtually all the synapses established by terminals of climbing fibres (immunoreactive for vGluT-2) and some synapses established by terminals of parallel and mossy fibres (immunoreactive for vGluT-1, and for vGluT-1 and 2, respectively). The only GABA synapses expressing the SNARE proteins studied were the synapses established by axon terminals of basket neurons. CONCLUSION: The present study supplies a detailed morphological description of VAMP-2, SNAP-25A/B and syntaxin-1 in the different types of glutamatergic and GABAergic synapses of the rat cerebellar cortex. The examined SNARE proteins characterize most of glutamatergic synapses and only one type of GABAergic synapses. In the subpopulations of glutamatergic and GABAergic synapses lacking the SNARE protein isoforms examined, alternative mechanisms for regulating trafficking of synaptic vesicles may be hypothesized, possibly mediated by different isoforms or homologous proteins.
Asunto(s)
Corteza Cerebelosa/fisiología , Ácido Glutámico/fisiología , Sinapsis/metabolismo , Proteína 25 Asociada a Sinaptosomas/fisiología , Sintaxina 1/fisiología , Proteína 2 de Membrana Asociada a Vesículas/fisiología , Ácido gamma-Aminobutírico/fisiología , Animales , Transporte Axonal/fisiología , Corteza Cerebelosa/citología , Corteza Cerebelosa/metabolismo , Terminales Presinápticos/metabolismo , Terminales Presinápticos/fisiología , Ratas , Ratas Wistar , Vesículas Sinápticas/fisiologíaRESUMEN
The crystal structure of complexin bound to a prefusion SNAREpin mimetic shows that the accessory helix extends away from the SNAREpin in an 'open' conformation, binding another SNAREpin and inhibiting its assembly, to clamp fusion. In contrast, the accessory helix in the postfusion complex parallels the SNARE complex in a 'closed' conformation. Here we use targeted mutations, FRET spectroscopy and a functional assay that reconstitutes Ca(2+)-triggered exocytosis to show that the conformational switch from open to closed in complexin is needed for synaptotagmin-Ca(2+) to trigger fusion. Triggering fusion requires the zippering of three crucial aspartate residues in the switch region (residues 64-68) of v-SNARE. Conformational switching in complexin is integral to clamp release and is probably triggered when its accessory helix is released from its trans-binding to the neighboring SNAREpin, allowing the v-SNARE to complete zippering and open a fusion pore.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/química , Proteínas del Tejido Nervioso/química , Sinaptotagminas/fisiología , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/fisiología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , Humanos , Fusión de Membrana/fisiología , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/fisiología , Estructura Terciaria de Proteína , Ratas , Proteína 25 Asociada a Sinaptosomas/química , Proteína 25 Asociada a Sinaptosomas/metabolismo , Sinaptotagminas/metabolismo , Sintaxina 1/química , Sintaxina 1/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/química , Proteína 2 de Membrana Asociada a Vesículas/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/fisiologíaRESUMEN
The core mechanism of intracellular vesicle fusion consists of SNAREpin zippering between vesicular and target membranes. Recent studies indicate that the same SNARE-binding protein, complexin (CPX), can act either as a facilitator or as an inhibitor of membrane fusion, constituting a controversial dilemma. Here we take energetic measurements with the surface force apparatus that reveal that CPX acts sequentially on assembling SNAREpins, first facilitating zippering by nearly doubling the distance at which v- and t-SNAREs can engage and then clamping them into a half-zippered fusion-incompetent state. Specifically, we find that the central helix of CPX allows SNAREs to form this intermediate energetic state at 9-15 nm but not when the bilayers are closer than 9 nm. Stabilizing the activated-clamped state at separations of less than 9 nm requires the accessory helix of CPX, which prevents membrane-proximal assembly of SNAREpins.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Proteínas SNARE/química , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/química , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/fisiología , Animales , Sitios de Unión , Humanos , Membrana Dobles de Lípidos/metabolismo , Fusión de Membrana/fisiología , Ratones , Modelos Moleculares , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/fisiología , Estructura Terciaria de Proteína , Ratas , Proteínas SNARE/metabolismo , Proteínas SNARE/fisiología , Proteína 25 Asociada a Sinaptosomas/química , Proteína 25 Asociada a Sinaptosomas/metabolismo , Proteína 25 Asociada a Sinaptosomas/fisiología , Sintaxina 1/química , Sintaxina 1/metabolismo , Sintaxina 1/fisiología , Proteína 2 de Membrana Asociada a Vesículas/química , Proteína 2 de Membrana Asociada a Vesículas/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/fisiologíaRESUMEN
Complexin prevents SNAREs from releasing neurotransmitters until an action potential arrives at the synapse. To understand the mechanism for this inhibition, we determined the structure of complexin bound to a mimetic of a prefusion SNAREpin lacking the portion of the v-SNARE that zippers last to trigger fusion. The 'central helix' of complexin is anchored to one SNARE complex, while its 'accessory helix' extends away at ~45° and bridges to a second complex, occupying the vacant v-SNARE binding site to inhibit fusion. We expected the accessory helix to compete with the v-SNARE for t-SNARE binding but found instead that the interaction occurs intermolecularly. Thus, complexin organizes the SNAREs into a zigzag topology that, when interposed between the vesicle and plasma membranes, is incompatible with fusion.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/química , Proteínas del Tejido Nervioso/química , Proteína 2 de Membrana Asociada a Vesículas/química , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/fisiología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , Humanos , Fusión de Membrana/fisiología , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/fisiología , Estructura Terciaria de Proteína , Ratas , Sintaxina 1/química , Sintaxina 1/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/fisiologíaAsunto(s)
Proteínas SNARE/fisiología , Transmisión Sináptica/fisiología , Adenosina Trifosfato/fisiología , Animales , Exocitosis , Humanos , Fusión de Membrana , Modelos Neurológicos , Neurotransmisores/metabolismo , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Subunidades de Proteína , Protones , Vesículas Sinápticas/enzimología , Vesículas Sinápticas/metabolismo , ATPasas de Translocación de Protón Vacuolares , Proteína 2 de Membrana Asociada a Vesículas/fisiologíaRESUMEN
Neurotransmitter release is mediated by the SNARE proteins synaptobrevin II (sybII, also known as VAMP2), syntaxin, and SNAP-25, generating a force transfer to the membranes and inducing fusion pore formation. However, the molecular mechanism by which this force leads to opening of a fusion pore remains elusive. Here we show that the ability of sybII to support exocytosis is inhibited by addition of one or two residues to the sybII C terminus depending on their energy of transfer from water to the membrane interface, following a Boltzmann distribution. These results suggest that following stimulation, the SNARE complex pulls the C terminus of sybII deeper into the vesicle membrane. We propose that this movement disrupts the vesicular membrane continuity leading to fusion pore formation. In contrast to current models, the experiments suggest that fusion pore formation begins with molecular rearrangements at the intravesicular membrane leaflet and not between the apposed cytoplasmic leaflets.