RESUMEN
RTN4-binding proteins were widely studied as "NoGo" receptors, but their physiological interactors and roles remain elusive. Similarly, BAI adhesion-GPCRs were associated with numerous activities, but their ligands and functions remain unclear. Using unbiased approaches, we observed an unexpected convergence: RTN4 receptors are high-affinity ligands for BAI adhesion-GPCRs. A single thrombospondin type 1-repeat (TSR) domain of BAIs binds to the leucine-rich repeat domain of all three RTN4-receptor isoforms with nanomolar affinity. In the 1.65 Å crystal structure of the BAI1/RTN4-receptor complex, C-mannosylation of tryptophan and O-fucosylation of threonine in the BAI TSR-domains creates a RTN4-receptor/BAI interface shaped by unusual glycoconjugates that enables high-affinity interactions. In human neurons, RTN4 receptors regulate dendritic arborization, axonal elongation, and synapse formation by differential binding to glial versus neuronal BAIs, thereby controlling neural network activity. Thus, BAI binding to RTN4/NoGo receptors represents a receptor-ligand axis that, enabled by rare post-translational modifications, controls development of synaptic circuits.
Asunto(s)
Inhibidores de la Angiogénesis/metabolismo , Encéfalo/metabolismo , Neurogénesis , Neuronas/metabolismo , Proteínas Nogo/metabolismo , Receptores Nogo/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Adipoquinas/metabolismo , Secuencia de Aminoácidos , Animales , Axones/metabolismo , Adhesión Celular , Moléculas de Adhesión Celular Neuronal/metabolismo , Complemento C1q/metabolismo , Dendritas/metabolismo , Glicosilación , Células HEK293 , Células Madre Embrionarias Humanas/metabolismo , Humanos , Ligandos , Ratones Endogámicos C57BL , Red Nerviosa/metabolismo , Polisacáridos/metabolismo , Unión Proteica , Dominios Proteicos , Eliminación de Secuencia , Sinapsis/metabolismo , Transmisión Sináptica/fisiologíaRESUMEN
Neuromodulators bind to pre- and postsynaptic G protein-coupled receptors (GPCRs), are able to quickly change intracellular cyclic AMP (cAMP) and Ca2+ levels, and are thought to play important roles in neuropsychiatric and neurodegenerative diseases. Here, we discovered in human neurons an unanticipated presynaptic mechanism that acutely changes synaptic ultrastructure and regulates synaptic communication. Activation of neuromodulator receptors bidirectionally controlled synaptic vesicle numbers within nerve terminals. This control correlated with changes in the levels of cAMP-dependent protein kinase A-mediated phosphorylation of synapsin-1. Using a conditional deletion approach, we reveal that the neuromodulator-induced control of synaptic vesicle numbers was largely dependent on synapsin-1. We propose a mechanism whereby non-phosphorylated synapsin-1 "latches" synaptic vesicles to presynaptic clusters at the active zone. cAMP-dependent phosphorylation of synapsin-1 then removes the vesicles. cAMP-independent dephosphorylation of synapsin-1 in turn recruits vesicles. Synapsin-1 thereby bidirectionally regulates synaptic vesicle numbers and modifies presynaptic neurotransmitter release as an effector of neuromodulator signaling in human neurons.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Terminales Presinápticos/metabolismo , Sinapsinas/metabolismo , Transmisión Sináptica , Vesículas Sinápticas/metabolismo , Animales , Células Cultivadas , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Neurotransmisores/metabolismo , Receptores de Neurotransmisores/metabolismo , Transducción de SeñalRESUMEN
Teneurins (TENs) are cell-surface adhesion proteins with critical roles in tissue development and axon guidance. Here, we report the 3.1-Å cryoelectron microscopy structure of the human TEN2 extracellular region (ECR), revealing a striking similarity to bacterial Tc-toxins. The ECR includes a large ß barrel that partially encapsulates a C-terminal domain, which emerges to the solvent through an opening in the mid-barrel region. An immunoglobulin (Ig)-like domain seals the bottom of the barrel while a ß propeller is attached in a perpendicular orientation. We further show that an alternatively spliced region within the ß propeller acts as a switch to regulate trans-cellular adhesion of TEN2 to latrophilin (LPHN), a transmembrane receptor known to mediate critical functions in the central nervous system. One splice variant activates trans-cellular signaling in a LPHN-dependent manner, whereas the other induces inhibitory postsynaptic differentiation. These results highlight the unusual structural organization of TENs giving rise to their multifarious functions.
Asunto(s)
Toxinas Bacterianas/química , Proteínas de la Membrana/química , Proteínas del Tejido Nervioso/química , Sinapsis/metabolismo , Empalme Alternativo , Secuencias de Aminoácidos , Animales , Axones , Adhesión Celular , Línea Celular , AMP Cíclico/metabolismo , Femenino , Células HEK293 , Hormonas/química , Humanos , Insectos , Proteínas de la Membrana/metabolismo , Ratones , Conformación Molecular , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Neuropéptidos/química , Unión Proteica , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/química , Transducción de SeñalRESUMEN
Synapses are specialized junctions between neurons in brain that transmit and compute information, thereby connecting neurons into millions of overlapping and interdigitated neural circuits. Here, we posit that the establishment, properties, and dynamics of synapses are governed by a molecular logic that is controlled by diverse trans-synaptic signaling molecules. Neurexins, expressed in thousands of alternatively spliced isoforms, are central components of this dynamic code. Presynaptic neurexins regulate synapse properties via differential binding to multifarious postsynaptic ligands, such as neuroligins, cerebellin/GluD complexes, and latrophilins, thereby shaping the input/output relations of their resident neural circuits. Mutations in genes encoding neurexins and their ligands are associated with diverse neuropsychiatric disorders, especially schizophrenia, autism, and Tourette syndrome. Thus, neurexins nucleate an overall trans-synaptic signaling network that controls synapse properties, which thereby determines the precise responses of synapses to spike patterns in a neuron and circuit and which is vulnerable to impairments in neuropsychiatric disorders.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Vías Nerviosas , Sinapsis , Empalme Alternativo , Animales , Trastorno Autístico/metabolismo , Trastorno Autístico/patología , Moléculas de Adhesión Celular Neuronal/química , Moléculas de Adhesión Celular Neuronal/genética , Humanos , Glicoproteínas de Membrana/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Esquizofrenia/metabolismo , Esquizofrenia/patología , Transducción de Señal , Síndrome de Tourette/metabolismo , Síndrome de Tourette/patologíaRESUMEN
Human apolipoprotein E (ApoE) apolipoprotein is primarily expressed in three isoforms (ApoE2, ApoE3, and ApoE4) that differ only by two residues. ApoE4 constitutes the most important genetic risk factor for Alzheimer's disease (AD), ApoE3 is neutral, and ApoE2 is protective. How ApoE isoforms influence AD pathogenesis, however, remains unclear. Using ES-cell-derived human neurons, we show that ApoE secreted by glia stimulates neuronal Aß production with an ApoE4 > ApoE3 > ApoE2 potency rank order. We demonstrate that ApoE binding to ApoE receptors activates dual leucine-zipper kinase (DLK), a MAP-kinase kinase kinase that then activates MKK7 and ERK1/2 MAP kinases. Activated ERK1/2 induces cFos phosphorylation, stimulating the transcription factor AP-1, which in turn enhances transcription of amyloid-ß precursor protein (APP) and thereby increases amyloid-ß levels. This molecular mechanism also regulates APP transcription in mice in vivo. Our data describe a novel signal transduction pathway in neurons whereby ApoE activates a non-canonical MAP kinase cascade that enhances APP transcription and amyloid-ß synthesis.
Asunto(s)
Precursor de Proteína beta-Amiloide/genética , Apolipoproteínas E/metabolismo , Sistema de Señalización de MAP Quinasas , Enfermedad de Alzheimer/metabolismo , Animales , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Fibroblastos/metabolismo , Humanos , Ratones , Neuronas/metabolismo , Isoformas de Proteínas/metabolismoRESUMEN
α- and ß-neurexins are presynaptic cell-adhesion molecules implicated in autism and schizophrenia. We find that, although ß-neurexins are expressed at much lower levels than α-neurexins, conditional knockout of ß-neurexins with continued expression of α-neurexins dramatically decreased neurotransmitter release at excitatory synapses in cultured cortical neurons. The ß-neurexin knockout phenotype was attenuated by CB1-receptor inhibition, which blocks presynaptic endocannabinoid signaling, or by 2-arachidonoylglycerol synthesis inhibition, which impairs postsynaptic endocannabinoid release. In synapses formed by CA1-region pyramidal neurons onto burst-firing subiculum neurons, presynaptic in vivo knockout of ß-neurexins aggravated endocannabinoid-mediated inhibition of synaptic transmission and blocked LTP; presynaptic CB1-receptor antagonists or postsynaptic 2-arachidonoylglycerol synthesis inhibition again reversed this block. Moreover, conditional knockout of ß-neurexins in CA1-region neurons impaired contextual fear memories. Thus, our data suggest that presynaptic ß-neurexins control synaptic strength in excitatory synapses by regulating postsynaptic 2-arachidonoylglycerol synthesis, revealing an unexpected role for ß-neurexins in the endocannabinoid-dependent regulation of neural circuits.
Asunto(s)
Endocannabinoides/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Vías Nerviosas/metabolismo , Sinapsis/metabolismo , Animales , Ácidos Araquidónicos/biosíntesis , Calcio/metabolismo , Proteínas de Unión al Calcio , Endocannabinoides/biosíntesis , Glicéridos/biosíntesis , Hipocampo/citología , Hipocampo/metabolismo , Ratones , Ratones Noqueados , Moléculas de Adhesión de Célula Nerviosa/genética , Neuronas/metabolismo , Neurotransmisores/metabolismo , Transducción de SeñalRESUMEN
Social communication guides decision-making, which is essential for survival. Social transmission of food preference (STFP) is an ecologically relevant memory paradigm in which an animal learns a desirable food odour from another animal in a social context, creating a long-term memory1,2. How food-preference memory is acquired, consolidated and stored is unclear. Here we show that the posteromedial nucleus of the cortical amygdala (COApm) serves as a computational centre in long-term STFP memory consolidation by integrating social and sensory olfactory inputs. Blocking synaptic signalling by the COApm-based circuit selectively abolished STFP memory consolidation without impairing memory acquisition, storage or recall. COApm-mediated STFP memory consolidation depends on synaptic inputs from the accessory olfactory bulb and on synaptic outputs to the anterior olfactory nucleus. STFP memory consolidation requires protein synthesis, suggesting a gene-expression mechanism. Deep single-cell and spatially resolved transcriptomics revealed robust but distinct gene-expression signatures induced by STFP memory formation in the COApm that are consistent with synapse restructuring. Our data thus define a neural circuit for the consolidation of a socially communicated long-term memory, thereby mechanistically distinguishing protein-synthesis-dependent memory consolidation from memory acquisition, storage or retrieval.
Asunto(s)
Amígdala del Cerebelo , Preferencias Alimentarias , Consolidación de la Memoria , Memoria a Largo Plazo , Conducta Social , Animales , Masculino , Ratones , Amígdala del Cerebelo/fisiología , Amígdala del Cerebelo/citología , Consolidación de la Memoria/fisiología , Memoria a Largo Plazo/fisiología , Ratones Endogámicos C57BL , Odorantes/análisis , Bulbo Olfatorio/fisiología , Bulbo Olfatorio/citología , Análisis de la Célula Individual , Sinapsis/metabolismo , Transcriptoma , Preferencias Alimentarias/fisiología , Preferencias Alimentarias/psicologíaRESUMEN
The assembly and specification of synapses in the brain is incompletely understood1-3. Latrophilin-3 (encoded by Adgrl3, also known as Lphn3)-a postsynaptic adhesion G-protein-coupled receptor-mediates synapse formation in the hippocampus4 but the mechanisms involved remain unclear. Here we show in mice that LPHN3 organizes synapses through a convergent dual-pathway mechanism: activation of Gαs signalling and recruitment of phase-separated postsynaptic protein scaffolds. We found that cell-type-specific alternative splicing of Lphn3 controls the LPHN3 G-protein-coupling mode, resulting in LPHN3 variants that predominantly signal through Gαs or Gα12/13. CRISPR-mediated manipulation of Lphn3 alternative splicing that shifts LPHN3 from a Gαs- to a Gα12/13-coupled mode impaired synaptic connectivity as severely as the overall deletion of Lphn3, suggesting that Gαs signalling by LPHN3 splice variants mediates synapse formation. Notably, Gαs-coupled, but not Gα12/13-coupled, splice variants of LPHN3 also recruit phase-transitioned postsynaptic protein scaffold condensates, such that these condensates are clustered by binding of presynaptic teneurin and FLRT ligands to LPHN3. Moreover, neuronal activity promotes alternative splicing of the synaptogenic Gαs-coupled variant of LPHN3. Together, these data suggest that activity-dependent alternative splicing of a key synaptic adhesion molecule controls synapse formation by parallel activation of two convergent pathways: Gαs signalling and clustered phase separation of postsynaptic protein scaffolds.
Asunto(s)
Empalme Alternativo , Receptores Acoplados a Proteínas G , Receptores de Péptidos , Sinapsis , Animales , Ratones , Empalme Alternativo/genética , Subunidades alfa de la Proteína de Unión al GTP G12-G13 , Subunidades alfa de la Proteína de Unión al GTP Gs , Ligandos , Receptores Acoplados a Proteínas G/deficiencia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/deficiencia , Receptores de Péptidos/genética , Receptores de Péptidos/metabolismo , Sinapsis/metabolismo , Transducción de SeñalRESUMEN
Memory encodes past experiences, thereby enabling future plans. The basolateral amygdala is a centre of salience networks that underlie emotional experiences and thus has a key role in long-term fear memory formation1. Here we used spatial and single-cell transcriptomics to illuminate the cellular and molecular architecture of the role of the basolateral amygdala in long-term memory. We identified transcriptional signatures in subpopulations of neurons and astrocytes that were memory-specific and persisted for weeks. These transcriptional signatures implicate neuropeptide and BDNF signalling, MAPK and CREB activation, ubiquitination pathways, and synaptic connectivity as key components of long-term memory. Notably, upon long-term memory formation, a neuronal subpopulation defined by increased Penk and decreased Tac expression constituted the most prominent component of the memory engram of the basolateral amygdala. These transcriptional changes were observed both with single-cell RNA sequencing and with single-molecule spatial transcriptomics in intact slices, thereby providing a rich spatial map of a memory engram. The spatial data enabled us to determine that this neuronal subpopulation interacts with adjacent astrocytes, and functional experiments show that neurons require interactions with astrocytes to encode long-term memory.
Asunto(s)
Astrocitos , Comunicación Celular , Perfilación de la Expresión Génica , Memoria a Largo Plazo , Neuronas , Astrocitos/citología , Astrocitos/metabolismo , Astrocitos/fisiología , Complejo Nuclear Basolateral/citología , Complejo Nuclear Basolateral/metabolismo , Complejo Nuclear Basolateral/fisiología , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Memoria a Largo Plazo/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuronas/citología , Neuronas/metabolismo , Neuronas/fisiología , Análisis de Secuencia de ARN , Imagen Individual de Molécula , Análisis de Expresión Génica de una Sola Célula , UbiquitinaciónRESUMEN
In humans, neuroligin-3 mutations are associated with autism, whereas in mice, the corresponding mutations produce robust synaptic and behavioral changes. However, different neuroligin-3 mutations cause largely distinct phenotypes in mice, and no causal relationship links a specific synaptic dysfunction to a behavioral change. Using rotarod motor learning as a proxy for acquired repetitive behaviors in mice, we found that different neuroligin-3 mutations uniformly enhanced formation of repetitive motor routines. Surprisingly, neuroligin-3 mutations caused this phenotype not via changes in the cerebellum or dorsal striatum but via a selective synaptic impairment in the nucleus accumbens/ventral striatum. Here, neuroligin-3 mutations increased rotarod learning by specifically impeding synaptic inhibition onto D1-dopamine receptor-expressing but not D2-dopamine receptor-expressing medium spiny neurons. Our data thus suggest that different autism-associated neuroligin-3 mutations cause a common increase in acquired repetitive behaviors by impairing a specific striatal synapse and thereby provide a plausible circuit substrate for autism pathophysiology.
Asunto(s)
Trastorno Autístico/genética , Trastorno Autístico/fisiopatología , Moléculas de Adhesión Celular Neuronal/genética , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Animales , Trastorno Autístico/metabolismo , Ganglios Basales/metabolismo , Ganglios Basales/fisiopatología , Moléculas de Adhesión Celular Neuronal/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Mutación , Proteínas del Tejido Nervioso/metabolismo , Núcleo Accumbens/metabolismo , Prueba de Desempeño de Rotación con Aceleración ConstanteRESUMEN
Neurexins are essential presynaptic cell adhesion molecules that are linked to schizophrenia and autism and are subject to extensive alternative splicing. Here, we used a genetic approach to test the physiological significance of neurexin alternative splicing. We generated knockin mice in which alternatively spliced sequence #4 (SS4) of neuexin-3 is constitutively included but can be selectively excised by cre-recombination. SS4 of neurexin-3 was chosen because it is highly regulated and controls neurexin binding to neuroligins, LRRTMs, and other ligands. Unexpectedly, constitutive inclusion of SS4 in presynaptic neurexin-3 decreased postsynaptic AMPA, but not NMDA receptor levels, and enhanced postsynaptic AMPA receptor endocytosis. Moreover, constitutive inclusion of SS4 in presynaptic neurexin-3 abrogated postsynaptic AMPA receptor recruitment during NMDA receptor-dependent LTP. These phenotypes were fully rescued by constitutive excision of SS4 in neurexin-3. Thus, alternative splicing of presynaptic neurexin-3 controls postsynaptic AMPA receptor trafficking, revealing an unanticipated alternative splicing mechanism for trans-synaptic regulation of synaptic strength and long-term plasticity.
Asunto(s)
Empalme Alternativo , Neuronas/metabolismo , Receptores AMPA/metabolismo , Animales , Endocitosis , Técnicas de Sustitución del Gen , Hipocampo/metabolismo , Potenciación a Largo Plazo , Ratones , Proteínas del Tejido Nervioso/genética , SinapsisRESUMEN
Direct lineage reprogramming is a promising approach for human disease modeling and regenerative medicine, with poorly understood mechanisms. Here, we reveal a hierarchical mechanism in the direct conversion of fibroblasts into induced neuronal (iN) cells mediated by the transcription factors Ascl1, Brn2, and Myt1l. Ascl1 acts as an "on-target" pioneer factor by immediately occupying most cognate genomic sites in fibroblasts. In contrast, Brn2 and Myt1l do not access fibroblast chromatin productively on their own; instead, Ascl1 recruits Brn2 to Ascl1 sites genome wide. A unique trivalent chromatin signature in the host cells predicts the permissiveness for Ascl1 pioneering activity among different cell types. Finally, we identified Zfp238 as a key Ascl1 target gene that can partially substitute for Ascl1 during iN cell reprogramming. Thus, a precise match between pioneer factors and the chromatin context at key target genes is determinative for transdifferentiation to neurons and likely other cell types.
Asunto(s)
Reprogramación Celular , Embrión de Mamíferos/citología , Fibroblastos/citología , Redes Reguladoras de Genes , Neuronas/citología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular , Cromatina/metabolismo , Fibroblastos/metabolismo , Estudio de Asociación del Genoma Completo , Humanos , Ratones , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Factores del Dominio POU/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Neurons are highly polarized cells that face the fundamental challenge of compartmentalizing a vast and diverse repertoire of proteins in order to function properly1. The axon initial segment (AIS) is a specialized domain that separates a neuron's morphologically, biochemically and functionally distinct axon and dendrite compartments2,3. How the AIS maintains polarity between these compartments is not fully understood. Here we find that in Caenorhabditis elegans, mouse, rat and human neurons, dendritically and axonally polarized transmembrane proteins are recognized by endocytic machinery in the AIS, robustly endocytosed and targeted to late endosomes for degradation. Forcing receptor interaction with the AIS master organizer, ankyrinG, antagonizes receptor endocytosis in the AIS, causes receptor accumulation in the AIS, and leads to polarity deficits with subsequent morphological and behavioural defects. Therefore, endocytic removal of polarized receptors that diffuse into the AIS serves as a membrane-clearance mechanism that is likely to work in conjunction with the known AIS diffusion-barrier mechanism to maintain neuronal polarity on the plasma membrane. Our results reveal a conserved endocytic clearance mechanism in the AIS to maintain neuronal polarity by reinforcing axonal and dendritic compartment membrane boundaries.
Asunto(s)
Segmento Inicial del Axón , Polaridad Celular , Endocitosis , Animales , Segmento Inicial del Axón/metabolismo , Caenorhabditis elegans , Membrana Celular/metabolismo , Dendritas/metabolismo , Difusión , Endosomas/metabolismo , Humanos , Ratones , Transporte de Proteínas , Proteolisis , Ratas , Receptores de Superficie Celular/metabolismoRESUMEN
Synaptic adhesion molecules (SAMs) are evolutionarily conserved proteins that play an important role in the form and function of neuronal synapses. Teneurins (Tenms) and latrophilins (Lphns) are well-known cell adhesion molecules that form a transsynaptic complex. Recent studies suggest that Tenm3 and Lphn2 (gene symbol Adgrl2) are involved in hippocampal circuit assembly via their topographical expression. However, it is not known whether other teneurins and latrophilins display similar topographically restricted expression patterns during embryonic and postnatal development. Here, we reveal the cartography of all teneurin (Tenm1-4) and latrophilin (Lphn1-3 [Adgrl1-3]) paralog expression in the mouse hippocampus across prenatal and postnatal development as monitored by large-scale single-molecule RNA in situ hybridization mapping. Our results identify a striking heterogeneity in teneurin and latrophilin expression along the spatiotemporal axis of the hippocampus. Tenm2 and Tenm4 expression levels peak at the neonatal stage when compared to Tenm1 and Tenm3, while Tenm1 expression is restricted to the postnatal pyramidal cell layer. Tenm4 expression in the dentate gyrus (DG) exhibits an opposing topographical expression pattern in the embryonic and neonatal hippocampus. Our findings were validated by analyses of multiple RNA-seq datasets at bulk, single-cell, and spatial levels. Thus, our study presents a comprehensive spatiotemporal map of Tenm and Lphn expression in the hippocampus, showcasing their diverse expression patterns across developmental stages in distinct spatial axes.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Hipocampo , Proteínas del Tejido Nervioso , Receptores de Péptidos , Animales , Femenino , Masculino , Ratones , Hipocampo/metabolismo , Hipocampo/embriología , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Receptores Acoplados a Proteínas G , Receptores de Péptidos/metabolismo , Receptores de Péptidos/genética , TenascinaRESUMEN
Ionotropic glutamate delta receptors 1 (GluD1) and 2 (GluD2) exhibit the molecular architecture of postsynaptic ionotropic glutamate receptors, but assemble into trans-synaptic adhesion complexes by binding to secreted cerebellins that in turn interact with presynaptic neurexins1-4. It is unclear whether neurexin-cerebellin-GluD1/2 assemblies serve an adhesive synapse-formation function or mediate trans-synaptic signalling. Here we show in hippocampal synapses, that binding of presynaptic neurexin-cerebellin complexes to postsynaptic GluD1 controls glutamate receptor activity without affecting synapse numbers. Specifically, neurexin-1-cerebellin-2 and neurexin-3-cerebellin-2 complexes differentially regulate NMDA (N-methyl-D-aspartate) receptors and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors by activating distinct postsynaptic GluD1 effector signals. Of note, minimal GluD1 and GluD2 constructs containing only their N-terminal cerebellin-binding and C-terminal cytoplasmic domains, joined by an unrelated transmembrane region, fully control the levels of NMDA and AMPA receptors. The distinct signalling specificity of presynaptic neurexin-1 and neurexin-35,6 is encoded by their alternatively spliced splice site 4 sequences, whereas the regulatory functions of postsynaptic GluD1 are mediated by conserved cytoplasmic sequence motifs spanning 5-13 residues. Thus, GluDs are signalling molecules that regulate NMDA and AMPA receptors by an unexpected transduction mechanism that bypasses their ionotropic receptor architecture and directly converts extracellular neurexin-cerebellin signals into postsynaptic receptor responses.
Asunto(s)
Glutamato Deshidrogenasa/metabolismo , Receptores Ionotrópicos de Glutamato/metabolismo , Transducción de Señal , Secuencias de Aminoácidos , Animales , Proteínas de Unión al Calcio/metabolismo , Membrana Celular/metabolismo , Potenciales Postsinápticos Excitadores , Femenino , Masculino , Ratones , Proteínas del Tejido Nervioso/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Precursores de Proteínas/metabolismo , Receptores AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/metabolismoRESUMEN
Synaptotagmin-11 (Syt11) is a Synaptotagmin isoform that lacks an apparent ability to bind calcium, phospholipids, or SNARE proteins. While human genetic studies have linked mutations in the Syt11 gene to schizophrenia and Parkinson's disease, the localization or physiological role of Syt11 remain unclear. We found that in neurons, Syt11 resides on abundant vesicles that differ from synaptic vesicles and resemble trafficking endosomes. These vesicles recycle via the plasma membrane in an activity-dependent manner, but their exocytosis is slow and desynchronized. Constitutive knockout mice lacking Syt11 died shortly after birth, suggesting Syt11-mediated membrane transport is required for survival. In contrast, selective ablation of Syt11 in excitatory forebrain neurons using a conditional knockout did not affect life span but impaired synaptic plasticity and memory. Syt11-deficient neurons displayed normal secretion of fast neurotransmitters and peptides but exhibited a reduction of long-term synaptic potentiation. Hence, Syt11 is an essential component of a neuronal vesicular trafficking pathway that differs from the well-characterized synaptic vesicle trafficking pathway but is also essential for life.
Asunto(s)
Plasticidad Neuronal/genética , Neuronas/fisiología , Vesículas Sinápticas/metabolismo , Sinaptotagminas/genética , Sinaptotagminas/metabolismo , Animales , Corteza Cerebral/embriología , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Técnicas de Sustitución del Gen , Hipocampo/fisiopatología , Memoria/fisiología , Ratones , Ratones Noqueados , Neurotransmisores/metabolismo , Prosencéfalo/citología , Prosencéfalo/fisiología , Potenciales Sinápticos/genética , Transmisión Sináptica , Vesículas Sinápticas/genética , Sinaptotagminas/deficienciaRESUMEN
Cytoplasmic dynein is essential for intracellular transport. Despite extensive in vitro characterizations, how the dynein motors transport vesicles by processive steps in live cells remains unclear. To dissect the molecular mechanisms of dynein, we develop optical probes that enable long-term single-particle tracking in live cells with high spatiotemporal resolution. We find that the number of active dynein motors transporting cargo switches stochastically between one and five dynein motors during long-range transport in neuronal axons. Our very bright optical probes allow the observation of individual molecular steps. Strikingly, these measurements reveal that the dwell times between steps are controlled by two temperature-dependent rate constants in which two ATP molecules are hydrolyzed sequentially during each dynein step. Thus, our observations uncover a previously unknown chemomechanical cycle of dynein-mediated cargo transport in living cells.
RESUMEN
Synaptotagmins Syt1, Syt2, Syt7, and Syt9 act as Ca(2+)-sensors for synaptic and neuroendocrine exocytosis, but the function of other synaptotagmins remains unknown. Here, we show that olfactory bulb neurons secrete IGF-1 by an activity-dependent pathway of exocytosis, and that Syt10 functions as the Ca(2+)-sensor that triggers IGF-1 exocytosis in these neurons. Deletion of Syt10 impaired activity-dependent IGF-1 secretion in olfactory bulb neurons, resulting in smaller neurons and an overall decrease in synapse numbers. Exogenous IGF-1 completely reversed the Syt10 knockout phenotype. Syt10 colocalized with IGF-1 in somatodendritic vesicles of olfactory bulb neurons, and Ca(2+)-binding to Syt10 caused these vesicles to undergo exocytosis, thereby secreting IGF-1. Thus, Syt10 controls a previously unrecognized pathway of Ca(2+)-dependent exocytosis that is spatially and temporally distinct from Ca(2+)-dependent synaptic vesicle exocytosis controlled by Syt1. Our findings thereby reveal that two different synaptotagmins can regulate functionally distinct Ca(2+)-dependent membrane fusion reactions in the same neuron.
Asunto(s)
Exocitosis , Factor I del Crecimiento Similar a la Insulina/metabolismo , Bulbo Olfatorio/metabolismo , Sinaptotagminas/metabolismo , Animales , Células Cultivadas , Técnicas In Vitro , Ratones , Ratones Noqueados , Neuronas/metabolismo , Bulbo Olfatorio/citologíaRESUMEN
At a synapse, fast synchronous neurotransmitter release requires localization of Ca(2+) channels to presynaptic active zones. How Ca(2+) channels are recruited to active zones, however, remains unknown. Using unbiased yeast two-hybrid screens, we here identify a direct interaction of the central PDZ domain of the active-zone protein RIM with the C termini of presynaptic N- and P/Q-type Ca(2+) channels but not L-type Ca(2+) channels. To test the physiological significance of this interaction, we generated conditional knockout mice lacking all multidomain RIM isoforms. Deletion of RIM proteins ablated most neurotransmitter release by simultaneously impairing the priming of synaptic vesicles and by decreasing the presynaptic localization of Ca(2+) channels. Strikingly, rescue of the decreased Ca(2+)-channel localization required the RIM PDZ domain, whereas rescue of vesicle priming required the RIM N terminus. We propose that RIMs tether N- and P/Q-type Ca(2+) channels to presynaptic active zones via a direct PDZ-domain-mediated interaction, thereby enabling fast, synchronous triggering of neurotransmitter release at a synapse.