RESUMO
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.
Assuntos
Inibidores da Angiogênese/metabolismo , Encéfalo/metabolismo , Neurogênese , Neurônios/metabolismo , Proteínas Nogo/metabolismo , Receptores Nogo/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Adipocinas/metabolismo , Sequência de Aminoácidos , Animais , Axônios/metabolismo , Adesão Celular , Moléculas de Adesão Celular Neuronais/metabolismo , Complemento C1q/metabolismo , Dendritos/metabolismo , Glicosilação , Células HEK293 , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Ligantes , Camundongos Endogâmicos C57BL , Rede Nervosa/metabolismo , Polissacarídeos/metabolismo , Ligação Proteica , Domínios Proteicos , Deleção de Sequência , Sinapses/metabolismo , Transmissão Sináptica/fisiologiaRESUMO
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.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Terminações Pré-Sinápticas/metabolismo , Sinapsinas/metabolismo , Transmissão Sináptica , Vesículas Sinápticas/metabolismo , Animais , Células Cultivadas , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neurotransmissores/metabolismo , Receptores de Neurotransmissores/metabolismo , Transdução de SinaisRESUMO
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.
Assuntos
Toxinas Bacterianas/química , Proteínas de Membrana/química , Proteínas do Tecido Nervoso/química , Sinapses/metabolismo , Processamento Alternativo , Motivos de Aminoácidos , Animais , Axônios , Adesão Celular , Linhagem Celular , AMP Cíclico/metabolismo , Feminino , Células HEK293 , Hormônios/química , Humanos , Insetos , Proteínas de Membrana/metabolismo , Camundongos , Conformação Molecular , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Neuropeptídeos/química , Ligação Proteica , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/química , Transdução de SinaisRESUMO
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.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Vias Neurais , Sinapses , Processamento Alternativo , Animais , Transtorno Autístico/metabolismo , Transtorno Autístico/patologia , Moléculas de Adesão Celular Neuronais/química , Moléculas de Adesão Celular Neuronais/genética , Humanos , Glicoproteínas de Membrana/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Esquizofrenia/metabolismo , Esquizofrenia/patologia , Transdução de Sinais , Síndrome de Tourette/metabolismo , Síndrome de Tourette/patologiaRESUMO
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.
Assuntos
Precursor de Proteína beta-Amiloide/genética , Apolipoproteínas E/metabolismo , Sistema de Sinalização das MAP Quinases , Doença de Alzheimer/metabolismo , Animais , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Fibroblastos/metabolismo , Humanos , Camundongos , Neurônios/metabolismo , Isoformas de Proteínas/metabolismoRESUMO
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.
Assuntos
Endocanabinoides/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Vias Neurais/metabolismo , Sinapses/metabolismo , Animais , Ácidos Araquidônicos/biossíntese , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio , Endocanabinoides/biossíntese , Glicerídeos/biossíntese , Hipocampo/citologia , Hipocampo/metabolismo , Camundongos , Camundongos Knockout , Moléculas de Adesão de Célula Nervosa/genética , Neurônios/metabolismo , Neurotransmissores/metabolismo , Transdução de SinaisRESUMO
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.
Assuntos
Tonsila do Cerebelo , Preferências Alimentares , Consolidação da Memória , Memória de Longo Prazo , Comportamento Social , Animais , Masculino , Camundongos , Tonsila do Cerebelo/fisiologia , Tonsila do Cerebelo/citologia , Consolidação da Memória/fisiologia , Memória de Longo Prazo/fisiologia , Camundongos Endogâmicos C57BL , Odorantes/análise , Bulbo Olfatório/fisiologia , Bulbo Olfatório/citologia , Análise de Célula Única , Sinapses/metabolismo , Transcriptoma , Preferências Alimentares/fisiologia , Preferências Alimentares/psicologiaRESUMO
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.
Assuntos
Processamento Alternativo , Receptores Acoplados a Proteínas G , Receptores de Peptídeos , Sinapses , Animais , Camundongos , Processamento Alternativo/genética , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP , Subunidades alfa Gs de Proteínas de Ligação ao GTP , Ligantes , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/deficiência , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Sinapses/metabolismo , Transdução de SinaisRESUMO
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.
Assuntos
Astrócitos , Comunicação Celular , Perfilação da Expressão Gênica , Memória de Longo Prazo , Neurônios , Astrócitos/citologia , Astrócitos/metabolismo , Astrócitos/fisiologia , Complexo Nuclear Basolateral da Amígdala/citologia , Complexo Nuclear Basolateral da Amígdala/metabolismo , Complexo Nuclear Basolateral da Amígdala/fisiologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Memória de Longo Prazo/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Neurônios/fisiologia , Análise de Sequência de RNA , Imagem Individual de Molécula , Análise da Expressão Gênica de Célula Única , UbiquitinaçãoRESUMO
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.
Assuntos
Transtorno Autístico/genética , Transtorno Autístico/fisiopatologia , Moléculas de Adesão Celular Neuronais/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Animais , Transtorno Autístico/metabolismo , Gânglios da Base/metabolismo , Gânglios da Base/fisiopatologia , Moléculas de Adesão Celular Neuronais/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Mutação , Proteínas do Tecido Nervoso/metabolismo , Núcleo Accumbens/metabolismo , Teste de Desempenho do Rota-RodRESUMO
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.
Assuntos
Processamento Alternativo , Neurônios/metabolismo , Receptores de AMPA/metabolismo , Animais , Endocitose , Técnicas de Introdução de Genes , Hipocampo/metabolismo , Potenciação de Longa Duração , Camundongos , Proteínas do Tecido Nervoso/genética , SinapsesRESUMO
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.
Assuntos
Reprogramação Celular , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Redes Reguladoras de Genes , Neurônios/citologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Cromatina/metabolismo , Fibroblastos/metabolismo , Estudo de Associação Genômica Ampla , Humanos , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Fatores do Domínio POU/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Segmento Inicial do Axônio , Polaridade Celular , Endocitose , Animais , Segmento Inicial do Axônio/metabolismo , Caenorhabditis elegans , Membrana Celular/metabolismo , Dendritos/metabolismo , Difusão , Endossomos/metabolismo , Humanos , Camundongos , Transporte Proteico , Proteólise , Ratos , Receptores de Superfície Celular/metabolismoRESUMO
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.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Hipocampo , Proteínas do Tecido Nervoso , Receptores de Peptídeos , Animais , Hipocampo/metabolismo , Hipocampo/embriologia , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Receptores de Peptídeos/metabolismo , Receptores de Peptídeos/genética , Feminino , Camundongos Endogâmicos C57BL , Masculino , Tenascina , Receptores Acoplados a Proteínas GRESUMO
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.
Assuntos
Glutamato Desidrogenase/metabolismo , Receptores Ionotrópicos de Glutamato/metabolismo , Transdução de Sinais , Motivos de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Membrana Celular/metabolismo , Potenciais Pós-Sinápticos Excitadores , Feminino , Masculino , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Precursores de Proteínas/metabolismo , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismoRESUMO
Generation of defined neuronal subtypes from human pluripotent stem cells remains a challenge. The proneural factor NGN2 has been shown to overcome experimental variability observed by morphogen-guided differentiation and directly converts pluripotent stem cells into neurons, but their cellular heterogeneity has not been investigated yet. Here, we found that NGN2 reproducibly produces three different kinds of excitatory neurons characterized by partial coactivation of other neurotransmitter programs. We explored two principle approaches to achieve more precise specification: prepatterning the chromatin landscape that NGN2 is exposed to and combining NGN2 with region-specific transcription factors. Unexpectedly, the chromatin context of regionalized neural progenitors only mildly altered genomic NGN2 binding and its transcriptional response and did not affect neurotransmitter specification. In contrast, coexpression of region-specific homeobox factors such as EMX1 resulted in drastic redistribution of NGN2 including recruitment to homeobox targets and resulted in glutamatergic neurons with silenced nonglutamatergic programs. These results provide the molecular basis for a blueprint for improved strategies for generating a plethora of defined neuronal subpopulations from pluripotent stem cells for therapeutic or disease-modeling purposes.
Assuntos
Genes Homeobox , Neurônios , Humanos , Cromatina , Neurotransmissores , ProsencéfaloRESUMO
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.
Assuntos
Plasticidade Neuronal/genética , Neurônios/fisiologia , Vesículas Sinápticas/metabolismo , Sinaptotagminas/genética , Sinaptotagminas/metabolismo , Animais , Córtex Cerebral/embriologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Introdução de Genes , Hipocampo/fisiopatologia , Memória/fisiologia , Camundongos , Camundongos Knockout , Neurotransmissores/metabolismo , Prosencéfalo/citologia , Prosencéfalo/fisiologia , Potenciais Sinápticos/genética , Transmissão Sináptica , Vesículas Sinápticas/genética , Sinaptotagminas/deficiênciaRESUMO
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.
RESUMO
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.