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The synaptic vesicle cluster (SVC) is an essential component of chemical synapses, which provides neurotransmitter-loaded vesicles during synaptic activity, at the same time as also controlling the local concentrations of numerous exo- and endocytosis cofactors. In addition, the SVC hosts molecules that participate in other aspects of synaptic function, from cytoskeletal components to adhesion proteins, and affects the location and function of organelles such as mitochondria and the endoplasmic reticulum. We argue here that these features extend the functional involvement of the SVC in synapse formation, signalling and plasticity, as well as synapse stabilization and metabolism. We also propose that changes in the size of the SVC coalesce with changes in the postsynaptic compartment, supporting the interplay between pre- and postsynaptic dynamics. Thereby, the SVC could be seen as an 'all-in-one' regulator of synaptic structure and function, which should be investigated in more detail, to reveal molecular mechanisms that control synaptic function and heterogeneity.
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Communication between cells is largely orchestrated by proteins on the cell surface, which allow information transfer across the cell membrane. Super-resolution and single-molecule visualization of these proteins can be achieved by genetically grafting HTP (HaloTag Protein) into the protein of interest followed by brief incubation of cells with a dye-HTL (dye-linked HaloTag Ligand). This approach allows for use of cutting-edge fluorophores optimized for specific optical techniques or a cell-impermeable dye-HTL to selectively label surface proteins without labeling intracellular copies. However, these two goals often conflict, as many high-performing dyes exhibit membrane permeability. Traditional methods to eliminate cell permeability face synthetic bottlenecks and risk altering photophysical properties. Here we report that dye-HTL reagents can be made cell-impermeable by inserting a charged sulfonate directly into the HTL, leaving the dye moiety unperturbed. This simple, one-step method requires no purification and is compatible with both the original HTL and second-generation HTL.2, the latter offering accelerated labeling. We validate such compounds, termed dye-SHTL ('dye shuttle') conjugates, in live cells via widefield microscopy, demonstrating exclusive membrane staining of extracellular HTP fusion proteins. In transduced primary hippocampal neurons, we label mGluR2, a neuromodulatory G protein-coupled receptor (GPCR), with dyes optimized for stimulated emission by depletion (STED) super-resolution microscopy, allowing unprecedented accuracy in distinguishing surface and receptors from those in internal compartments of the presynaptic terminal, important in neural communication. This approach offers broad utility for surface-specific protein labelling.
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The presynaptic SNARE-complex regulator complexin (Cplx) enhances the fusogenicity of primed synaptic vesicles (SVs). Consequently, Cplx deletion impairs action potential-evoked transmitter release. Conversely, though, Cplx loss enhances spontaneous and delayed asynchronous release at certain synapse types. Using electrophysiology and kinetic modeling, we show that such seemingly contradictory transmitter release phenotypes seen upon Cplx deletion can be explained by an additional of Cplx in the control of SV priming, where its ablation facilitates the generation of a "faulty" SV fusion apparatus. Supporting this notion, a sequential two-step priming scheme, featuring reduced vesicle fusogenicity and increased transition rates into the faulty primed state, reproduces all aberrations of transmitter release modes and short-term synaptic plasticity seen upon Cplx loss. Accordingly, we propose a dual presynaptic function for the SNARE-complex interactor Cplx, one as a "checkpoint" protein that guarantees the proper assembly of the fusion machinery during vesicle priming, and one in boosting vesicle fusogenicity.
Assuntos
Sinapses , Vesículas Sinápticas , Sinapses/metabolismo , Vesículas Sinápticas/metabolismo , Potenciais de Ação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Transmissão Sináptica/fisiologiaRESUMO
Munc13 proteins are priming factors for SNARE-dependent exocytosis, which are activated by diacylglycerol (DAG)-binding to their C1-domain. Several Munc13 paralogs exist, but their differential roles are not well understood. We studied the interdependence of phorbolesters (DAG mimics) with Munc13-1 and ubMunc13-2 in mouse adrenal chromaffin cells. Although expression of either Munc13-1 or ubMunc13-2 stimulated secretion, phorbolester was only stimulatory for secretion when ubMunc13-2 expression dominated, but inhibitory when Munc13-1 dominated. Accordingly, phorbolester stimulated secretion in wildtype cells, or cells overexpressing ubMunc13-2, but inhibited secretion in Munc13-2/Unc13b knockout (KO) cells or in cells overexpressing Munc13-1. Phorbolester was more stimulatory in the Munc13-1/Unc13a KO than in WT littermates, showing that endogenous Munc13-1 limits the effects of phorbolester. Imaging showed that ubMunc13-2 traffics to the plasma membrane with a time-course matching Ca2+-dependent secretion, and trafficking is independent of Synaptotagmin-7 (Syt7). However, in the absence of Syt7, phorbolester became inhibitory for both Munc13-1 and ubMunc13-2-driven secretion, indicating that stimulatory phorbolester x Munc13-2 interaction depends on functional pairing with Syt7. Overall, DAG/phorbolester, ubMunc13-2 and Syt7 form a stimulatory triad for dense-core vesicle priming.
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Diglicerídeos , Ésteres de Forbol , Animais , Camundongos , Vesículas de Núcleo Denso , Exocitose , Proteínas SNARE/metabolismo , SinaptotagminasRESUMO
Despite the importance of dopamine for striatal circuit function, mechanistic understanding of dopamine transmission remains incomplete. We recently showed that dopamine secretion relies on the presynaptic scaffolding protein RIM, indicating that it occurs at active zone-like sites similar to classical synaptic vesicle exocytosis. Here, we establish using a systematic gene knockout approach that Munc13 and Liprin-α, active zone proteins for vesicle priming and release site organization, are important for dopamine secretion. Furthermore, RIM zinc finger and C2B domains, which bind to Munc13 and Liprin-α, respectively, are needed to restore dopamine release after RIM ablation. In contrast, and different from typical synapses, the active zone scaffolds RIM-BP and ELKS, and RIM domains that bind to them, are expendable. Hence, dopamine release necessitates priming and release site scaffolding by RIM, Munc13, and Liprin-α, but other active zone proteins are dispensable. Our work establishes that efficient release site architecture mediates fast dopamine exocytosis.
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Dopamina , Transmissão Sináptica , Corpo Estriado , Dopamina/metabolismo , Exocitose , Sinapses/metabolismoRESUMO
During ongoing presynaptic action potential (AP) firing, transmitter release is limited by the availability of release-ready synaptic vesicles (SVs). The rate of SV recruitment (SVR) to release sites is strongly upregulated at high AP frequencies to balance SV consumption. We show that Munc13-1-an essential SV priming protein-regulates SVR via a Ca2+-phospholipid-dependent mechanism. Using knockin mouse lines with point mutations in the Ca2+-phospholipid-binding C2B domain of Munc13-1, we demonstrate that abolishing Ca2+-phospholipid binding increases synaptic depression, slows recovery of synaptic strength after SV pool depletion, and reduces temporal fidelity of synaptic transmission, while increased Ca2+-phospholipid binding has the opposite effects. Thus, Ca2+-phospholipid binding to the Munc13-1-C2B domain accelerates SVR, reduces short-term synaptic depression, and increases the endurance and temporal fidelity of neurotransmission, demonstrating that Munc13-1 is a core vesicle priming hub that adjusts SV re-supply to demand.
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Fosfolipídeos , Transmissão Sináptica , Potenciais de Ação , Animais , Cálcio/metabolismo , Camundongos , Plasticidade Neuronal/fisiologia , Fosfolipídeos/metabolismo , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismoRESUMO
Exploring the interactions between the Ca2+ binding protein calmodulin (CaM) and its target proteins remains a challenging task. Members of the Munc13 protein family play an essential role in short-term synaptic plasticity, modulated via the interaction with CaM at the presynaptic compartment. In this study, we focus on the bMunc13-2 isoform expressed in the brain, as strong changes in synaptic transmission were observed upon its mutagenesis or deletion. The CaMâbMunc13-2 interaction was previously characterized at the molecular level using short bMunc13-2-derived peptides only, revealing a classical 1â5â10 CaM binding motif. Using larger protein constructs, we have now identified for the first time a novel and unique CaM binding site in bMunc13-2 that contains an N-terminal extension of a classical 1â5â10 CaM binding motif. We characterize this motif using a range of biochemical and biophysical methods and highlight its importance for the CaMâbMunc13-2 interaction.
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Calmodulina/metabolismo , Reagentes de Ligações Cruzadas/metabolismo , Espectrometria de Massas , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Calmodulina/química , Bovinos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Ratos , SuínosRESUMO
Synapses are fundamental information-processing units of the brain, and synaptic dysregulation is central to many brain disorders ("synaptopathies"). However, systematic annotation of synaptic genes and ontology of synaptic processes are currently lacking. We established SynGO, an interactive knowledge base that accumulates available research about synapse biology using Gene Ontology (GO) annotations to novel ontology terms: 87 synaptic locations and 179 synaptic processes. SynGO annotations are exclusively based on published, expert-curated evidence. Using 2,922 annotations for 1,112 genes, we show that synaptic genes are exceptionally well conserved and less tolerant to mutations than other genes. Many SynGO terms are significantly overrepresented among gene variations associated with intelligence, educational attainment, ADHD, autism, and bipolar disorder and among de novo variants associated with neurodevelopmental disorders, including schizophrenia. SynGO is a public, universal reference for synapse research and an online analysis platform for interpretation of large-scale -omics data (https://syngoportal.org and http://geneontology.org).
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Encéfalo/citologia , Ontologia Genética , Proteômica , Software , Sinapses/fisiologia , Animais , Encéfalo/fisiologia , Bases de Dados Genéticas , Humanos , Bases de Conhecimento , Potenciais Sinápticos/fisiologia , SinaptossomosRESUMO
The aim of this report is to present a tentative clinical and pathophysiological approach to diseases affecting the neuronal presynaptic terminal, with a major focus on synaptic vesicles (SVs). Diseases are classified depending on which step of the neurobiology of the SV is predominantly affected: (1) biogenesis of vesicle precursors in the neuronal soma; (2) transport along the axon; (3) vesicle cycle at the presynaptic terminal (exocytosis-endocytosis cycle, with the main purpose of neurotransmitter release). Given that SVs have been defined as individual organelles, we highlight the link between the biological processes disturbed by genetic mutations and the clinical presentation of these disorders. The great majority of diseases may present as epileptic encephalopathies, intellectual disability (syndromic or nonsyndromic) with/without autism spectrum disorder (and other neuropsychiatric symptoms), and movement disorders. These symptoms may overlap and present in patients as a combination of clinical signs that results in the spectrum of the synaptopathies. A small number of diseases may also exhibit neuromuscular signs. In general, SV disorders tend to be severe, early encephalopathies that interfere with neurodevelopment. As a consequence, developmental delay and intellectual disability are constant in almost all the defects described. Considering that some of these diseases might mimic other neurometabolic conditions (and in particular treatable disorders), an initial extensive metabolic workup should always be considered. Further knowledge into pathophysiological mechanisms and biomarkers, as well as descriptions of new presynaptic disorders, will probably take place in the near future.
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Encefalopatias Metabólicas Congênitas/complicações , Terminações Pré-Sinápticas/patologia , Transmissão Sináptica , Vesículas Sinápticas/patologia , Transtorno do Espectro Autista/etiologia , Endocitose , Epilepsia/etiologia , Exocitose , Humanos , Deficiência Intelectual/etiologia , Transtornos dos Movimentos/etiologia , Doenças Neurodegenerativas , Doenças Neuromusculares/etiologiaRESUMO
KEY POINTS: Synaptic transmission relies on the recruitment of neurotransmitter-filled vesicles to presynaptic release sites. Increased intracellular calcium buffering slows the recovery from synaptic depression, suggesting that vesicle recruitment is a calcium-dependent process. However, the molecular mechanisms of vesicle recruitment have only been investigated at some synapses. We investigate the role of calcium in vesicle recruitment at the cerebellar mossy fibre to granule cell synapse. We find that increased intracellular calcium buffering slows the recovery from depression following physiological stimulation. However, the recovery is largely resistant to perturbation of the molecular pathways previously shown to mediate calcium-dependent vesicle recruitment. Furthermore, we find two pools of vesicles with different recruitment speeds and show that models incorporating two pools of vesicles with different calcium-independent recruitment rates can explain our data. In this framework, increased calcium buffering prevents the release of intrinsically fast-recruited vesicles but does not change the vesicle recruitment rates themselves. ABSTRACT: During sustained synaptic transmission, recruitment of new transmitter-filled vesicles to the release site counteracts vesicle depletion and thus synaptic depression. An elevated intracellular Ca2+ concentration has been proposed to accelerate the rate of vesicle recruitment at many synapses. This conclusion is often based on the finding that increased intracellular Ca2+ buffering slows the recovery from synaptic depression. However, the molecular mechanisms of the activity-dependent acceleration of vesicle recruitment have only been analysed at some synapses. Using physiological stimulation patterns in postsynaptic recordings and step depolarizations in presynaptic bouton recordings, we investigate vesicle recruitment at cerebellar mossy fibre boutons. We show that increased intracellular Ca2+ buffering slows recovery from depression dramatically. However, pharmacological and genetic interference with calmodulin or the calmodulin-Munc13 pathway, which has been proposed to mediate Ca2+ -dependence of vesicle recruitment, barely affects vesicle recovery from depression. Furthermore, we show that cerebellar mossy fibre boutons have two pools of vesicles: rapidly fusing vesicles that recover slowly and slowly fusing vesicles that recover rapidly. Finally, models adopting such two pools of vesicles with Ca2+ -independent recruitment rates can explain the slowed recovery from depression upon increased Ca2+ buffering. Our data do not rule out the involvement of the calmodulin-Munc13 pathway during stronger stimuli or other molecular pathways mediating Ca2+ -dependent vesicle recruitment at cerebellar mossy fibre boutons. However, we show that well-established two-pool models predict an apparent Ca2+ -dependence of vesicle recruitment. Thus, previous conclusions of Ca2+ -dependent vesicle recruitment based solely on increased intracellular Ca2+ buffering should be considered with caution.
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Potenciais de Ação , Cálcio/metabolismo , Córtex Cerebelar/fisiologia , Terminações Pré-Sinápticas/fisiologia , Sinapses/fisiologia , Transmissão Sináptica , Vesículas Sinápticas/fisiologia , Animais , Calmodulina/metabolismo , Córtex Cerebelar/citologia , Potenciais Pós-Sinápticos Excitadores , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibras Nervosas/fisiologiaRESUMO
INTRODUCTION: Calmodulin (CaM) is a highly conserved Ca2+-binding protein that is exceptionally abundant in the brain. In the presynaptic compartment of neurons, CaM transduces changes in Ca2+ concentration into the regulation of synaptic transmission dynamics. Areas covered: We review selected literature including published CaM interactor screens and outline established and candidate presynaptic CaM targets. We present a workflow of biochemical and structural proteomic methods that were used to identify and characterize the interactions between CaM and Munc13 proteins. Finally, we outline the potential of ion mobility-mass spectrometry (IM-MS) for conformational screening and of protein-protein cross-linking for the structural characterization of CaM complexes. Expert commentary: Cross-linking/MS and native MS can be applied with considerable throughput to protein mixtures under near-physiological conditions, and thus effectively complement high-resolution structural biology techniques. Experimental distance constraints are applicable best when obtained by combining different cross-linking strategies, i.e. by using cross-linkers with different spacer length and reactivity, and by using the incorporation of unnatural photo-reactive amino acids. Insights from structural proteomics can be used to generate CaM-insensitive mutants of CaM targets for functional studies in vitro or ideally in vivo.
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Calmodulina/genética , Proteômica , Transmissão Sináptica/genética , Sequência de Aminoácidos/genética , Cálcio/metabolismo , Humanos , Espectrometria de Massas , Neurônios/metabolismo , Terminações Pré-Sinápticas/metabolismo , Ligação ProteicaRESUMO
Munc13 proteins are essential regulators of neurotransmitter release at nerve cell synapses. They mediate the priming step that renders synaptic vesicles fusion-competent, and their genetic elimination causes a complete block of synaptic transmission. Here we have described a patient displaying a disorder characterized by a dyskinetic movement disorder, developmental delay, and autism. Using whole-exome sequencing, we have shown that this condition is associated with a rare, de novo Pro814Leu variant in the major human Munc13 paralog UNC13A (also known as Munc13-1). Electrophysiological studies in murine neuronal cultures and functional analyses in Caenorhabditis elegans revealed that the UNC13A variant causes a distinct dominant gain of function that is characterized by increased fusion propensity of synaptic vesicles, which leads to increased initial synaptic vesicle release probability and abnormal short-term synaptic plasticity. Our study underscores the critical importance of fine-tuned presynaptic control in normal brain function. Further, it adds the neuronal Munc13 proteins and the synaptic vesicle priming process that they control to the known etiological mechanisms of psychiatric and neurological synaptopathies.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Transtornos Motores/metabolismo , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/metabolismo , Transmissão Sináptica , Vesículas Sinápticas/metabolismo , Substituição de Aminoácidos , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Linhagem Celular , Feminino , Humanos , Lactente , Masculino , Transtornos Motores/genética , Proteínas do Tecido Nervoso/genética , Plasticidade Neuronal , Neurônios/metabolismo , Vesículas Sinápticas/genéticaRESUMO
Increased levels of the second messenger lipid diacylglycerol (DAG) induce downstream signaling events including the translocation of C1-domain-containing proteins toward the plasma membrane. Here, we introduce three light-sensitive DAGs, termed PhoDAGs, which feature a photoswitchable acyl chain. The PhoDAGs are inactive in the dark and promote the translocation of proteins that feature C1 domains toward the plasma membrane upon a flash of UV-A light. This effect is quickly reversed after the termination of photostimulation or by irradiation with blue light, permitting the generation of oscillation patterns. Both protein kinase C and Munc13 can thus be put under optical control. PhoDAGs control vesicle release in excitable cells, such as mouse pancreatic islets and hippocampal neurons, and modulate synaptic transmission in Caenorhabditis elegans. As such, the PhoDAGs afford an unprecedented degree of spatiotemporal control and are broadly applicable tools to study DAG signaling.
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Diglicerídeos/metabolismo , Diglicerídeos/efeitos da radiação , Processos Fotoquímicos/efeitos da radiação , Proteína Quinase C/metabolismo , Proteína Quinase C/efeitos da radiação , Raios Ultravioleta , Animais , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/efeitos da radiação , Diglicerídeos/química , Camundongos , Fenômenos Ópticos , Proteína Quinase C/química , Transdução de Sinais/efeitos da radiaçãoRESUMO
Neurotransmitter is released at synapses by fusion of synaptic vesicles with the plasma membrane. To sustain synaptic transmission, compensatory retrieval of membranes and vesicular proteins is essential. We combined capacitance measurements and pH-imaging via pH-sensitive vesicular protein marker (anti-synaptotagmin2-cypHer5E), and compared the retrieval kinetics of membranes and vesicular proteins at the calyx of Held synapse. Membrane and Syt2 were retrieved with a similar time course when slow endocytosis was elicited. When fast endocytosis was elicited, Syt2 was still retrieved together with the membrane, but endocytosed organelle re-acidification was slowed down, which provides strong evidence for two distinct endocytotic pathways. Strikingly, CaM inhibitors or the inhibition of the Ca(2+)-calmodulin-Munc13-1 signaling pathway only impaired the uptake of Syt2 while leaving membrane retrieval intact, indicating different recycling mechanisms for membranes and vesicle proteins. Our data identify a novel mechanism of stimulus- and Ca(2+)-dependent regulation of coordinated endocytosis of synaptic membranes and vesicle proteins.
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Endocitose/genética , Transmissão Sináptica/genética , Sinaptotagmina II/genética , Sinaptotagmina II/metabolismo , Animais , Calmodulina/antagonistas & inibidores , Membrana Celular/genética , Membrana Celular/metabolismo , Exocitose/genética , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Neurotransmissores/metabolismo , Terminações Pré-Sinápticas/metabolismo , Ratos , Transdução de Sinais , Sinapses/metabolismo , Vesículas Sinápticas/metabolismoRESUMO
Munc13 proteins are essential presynaptic regulators that mediate synaptic vesicle priming and play a role in the regulation of neuronal short-term synaptic plasticity. All four Munc13 isoforms share a common domain structure, including a calmodulin (CaM) binding site in their otherwise divergent N-termini. Here, we summarize recent results on the investigation of the CaM/Munc13 interaction. By combining chemical cross-linking, photoaffinity labeling, and mass spectrometry, we showed that all neuronal Munc13 isoforms exhibit similar CaM binding modes. Moreover, we demonstrated that the 1-5-8-26 CaM binding motif discovered in Munc13-1 cannot be induced in the classical CaM target skMLCK, indicating unique features of the Munc13 CaM binding motif.
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Calmodulina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Calmodulina/química , Humanos , Modelos Moleculares , Proteínas do Tecido Nervoso/química , Ligação Proteica , Conformação ProteicaRESUMO
Short-term synaptic plasticity, the dynamic alteration of synaptic strength during high-frequency activity, is a fundamental characteristic of all synapses. At the calyx of Held, repetitive activity eventually results in short-term synaptic depression, which is in part due to the gradual exhaustion of releasable synaptic vesicles. This is counterbalanced by Ca(2+)-dependent vesicle replenishment, but the molecular mechanisms of this replenishment are largely unknown. We studied calyces of Held in knockin mice that express a Ca(2+)-Calmodulin insensitive Munc13-1(W464R) variant of the synaptic vesicle priming protein Munc13-1. Calyces of these mice exhibit a slower rate of synaptic vesicle replenishment, aberrant short-term depression and reduced recovery from synaptic depression after high-frequency stimulation. Our data establish Munc13-1 as a major presynaptic target of Ca(2+)-Calmodulin signaling and show that the Ca(2+)-Calmodulin-Munc13-1 complex is a pivotal component of the molecular machinery that determines short-term synaptic plasticity characteristics.
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Cálcio/metabolismo , Calmodulina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal/fisiologia , Vesículas Sinápticas/metabolismo , Animais , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Transdução de Sinais/fisiologia , Sinapses/metabolismo , Transmissão Sináptica/fisiologiaRESUMO
Accumulated genetic evidence suggests that attenuation of the ratio between cerebral amyloid-ß Aß40 and Aß42 isoforms is central to familial Alzheimer's disease (FAD) pathogenesis. However, FAD mutations account for only 1-2% of Alzheimer's disease cases, leaving the experience-dependent mechanisms regulating Aß40/42 an enigma. Here we explored regulation of Aß40/42 ratio by temporal spiking patterns in the rodent hippocampus. Spike bursts boosted Aß40/42 through a conformational change in presenilin1 (PS1), the catalytic subunit of γ-secretase, and subsequent increase in Aß40 production. Conversely, single spikes did not alter basal PS1 conformation and Aß40/42. Burst-induced PS1 conformational shift was mediated by means of Ca(2+)-dependent synaptic vesicle exocytosis. Presynaptic inhibition in vitro and visual deprivation in vivo augmented synaptic and Aß40/42 facilitation by bursts in the hippocampus. Thus, burst probability and transfer properties of synapses represent fundamental features regulating Aß40/42 by experience and may contribute to the initiation of the common, sporadic Alzheimer's disease.
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Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Presenilina-1/metabolismo , Animais , Animais Recém-Nascidos , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Adaptação à Escuridão/fisiologia , Relação Dose-Resposta a Droga , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Hipocampo/citologia , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurotransmissores/farmacologia , Presenilina-1/química , Presenilina-1/genética , Conformação Proteica , Ratos , Ratos Wistar , Córtex Visual/citologiaRESUMO
Munc13s are presynaptic proteins that mediate synaptic vesicle priming and thereby control the size of the readily releasable pool of vesicles. During high synaptic activity, Munc13-1 and its closely related homolog, ubMunc13-2, bind Ca(2+)/calmodulin, resulting in enhanced priming activity and in changes of short-term synaptic plasticity characteristics. Here, we studied whether bMunc13-2 and Munc13-3, two remote isoforms of Munc13-1 with a neuronal subtype-specific expression pattern, mediate synaptic vesicle priming and regulate short-term synaptic plasticity in a Ca(2+)/calmodulin-dependent manner. We identified a single functional Ca(2+)/calmodulin binding site in these isoforms and provide structural evidence that all Munc13s employ a common mode of interaction with calmodulin despite the lack of sequence homology between their Ca(2+)/calmodulin binding sites. Electrophysiological analysis showed that, during high-frequency activity, Ca(2+)/calmodulin binding positively regulates the priming activity of bMunc13-2 and Munc13-3, resulting in an increase in the size of the readily releasable pool of vesicles and subsequently in strong short-term synaptic enhancement of neurotransmission. We conclude that Ca(2+)/calmodulin-dependent regulation of priming activity is structurally and functionally conserved in all Munc13 proteins, and that the composition of Munc13 isoforms in a neuron differentially controls its short-term synaptic plasticity characteristics.
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Cálcio/metabolismo , Calmodulina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal/genética , Neurônios/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Calmodulina/genética , Expressão Gênica , Hipocampo/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Mutação , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Técnicas de Patch-Clamp , Plasmídeos , Cultura Primária de Células , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transmissão Sináptica/fisiologia , TransfecçãoRESUMO
Munc13 proteins are essential regulators of exocytosis. In hippocampal glutamatergic neurons, the genetic deletion of Munc13s results in the complete loss of primed synaptic vesicles (SVs) in direct contact with the presynaptic active zone membrane, and in a total block of neurotransmitter release. Similarly drastic consequences of Munc13 loss are detectable in hippocampal and striatal GABAergic neurons. We show here that, in the adult mouse retina, the two Munc13-2 splice variants bMunc13-2 and ubMunc13-2 are selectively localized to conventional and ribbon synapses, respectively, and that ubMunc13-2 is the only Munc13 isoform in mature photoreceptor ribbon synapses. Strikingly, the genetic deletion of ubMunc13-2 has little effect on synaptic signaling by photoreceptor ribbon synapses and does not prevent membrane attachment of synaptic vesicles at the photoreceptor ribbon synaptic site. Thus, photoreceptor ribbon synapses and conventional synapses differ fundamentally with regard to their dependence on SV priming proteins of the Munc13 family. Their function is only moderately affected by Munc13 loss, which leads to slight perturbations of signal integration in the retina.