RESUMEN
Classically, neurexins are thought to mediate synaptic connections through trans interactions with a number of different postsynaptic partners. Neurexins are cleaved by metalloproteases in an activity-dependent manner, releasing the soluble extracellular domain. Here, we report that in both immature (before synaptogenesis) and mature (after synaptogenesis) hippocampal neurons, the soluble neurexin-1ß ectodomain triggers acute Ca2+-influx at the dendritic/postsynaptic side. In both cases, neuroligin-1 expression was required. In immature neurons, calcium influx required N-type calcium channels and stimulated dendritic outgrowth and neuronal survival. In mature glutamatergic neurons the neurexin-1ß ectodomain stimulated calcium influx through NMDA-receptors, which increased presynaptic release probability. In contrast, prolonged exposure to the ectodomain led to inhibition of synaptic transmission. This secondary inhibition was activity- and neuroligin-1 dependent and caused by a reduction in the readily-releasable pool of vesicles. A synthetic peptide modeled after the neurexin-1ß:neuroligin-1 interaction site reproduced the cellular effects of the neurexin-1ß ectodomain. Collectively, our findings demonstrate that the soluble neurexin ectodomain stimulates growth of neurons and exerts acute and chronic effects on trans-synaptic signaling involved in setting synaptic strength.
Asunto(s)
Proteínas de Unión al Calcio/farmacología , Calcio/metabolismo , Células Dendríticas/efectos de los fármacos , Células Dendríticas/fisiología , Moléculas de Adhesión de Célula Nerviosa/farmacología , Transmisión Sináptica/efectos de los fármacos , Animales , Proteínas de Unión al Calcio/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Células Cultivadas , Hipocampo/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuronas/metabolismo , Ratas Wistar , Receptores de N-Metil-D-Aspartato/metabolismo , Solubilidad , Estimulación QuímicaRESUMEN
Excitatory and inhibitory neurons are connected into microcircuits that generate circuit output. Central in the hippocampal CA3 microcircuit is the mossy fiber (MF) synapse, which provides powerful direct excitatory input and indirect feedforward inhibition to CA3 pyramidal neurons. Here, we dissect its cell-surface protein (CSP) composition to discover novel regulators of MF synaptic connectivity. Proteomic profiling of isolated MF synaptosomes uncovers a rich CSP composition, including many CSPs without synaptic function and several that are uncharacterized. Cell-surface interactome screening identifies IgSF8 as a neuronal receptor enriched in the MF pathway. Presynaptic Igsf8 deletion impairs MF synaptic architecture and robustly decreases the density of bouton filopodia that provide feedforward inhibition. Consequently, IgSF8 loss impairs excitation/inhibition balance and increases excitability of CA3 pyramidal neurons. Our results provide insight into the CSP landscape and interactome of a specific excitatory synapse and reveal IgSF8 as a critical regulator of CA3 microcircuit connectivity and function.
Asunto(s)
Región CA3 Hipocampal/fisiología , Proteínas Portadoras/metabolismo , Potenciales Postsinápticos Excitadores/fisiología , Proteínas de la Membrana/metabolismo , Fibras Musgosas del Hipocampo/metabolismo , Células Piramidales/fisiología , Animales , Proteínas Portadoras/genética , Células Cultivadas , Células HEK293 , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Técnicas de Placa-Clamp , Cultivo Primario de Células , Proteómica , Ratas , Sinaptosomas/metabolismoRESUMEN
The pre- and postsynaptic membranes comprising the synaptic junction differ in protein composition. The membrane trafficking mechanisms by which neurons control surface polarization of synaptic receptors remain poorly understood. The sorting receptor Sortilin-related CNS expressed 1 (SorCS1) is a critical regulator of trafficking of neuronal receptors, including the presynaptic adhesion molecule neurexin (Nrxn), an essential synaptic organizer. Here, we show that SorCS1 maintains a balance between axonal and dendritic Nrxn surface levels in the same neuron. Newly synthesized Nrxn1α traffics to the dendritic surface, where it is endocytosed. Endosomal SorCS1 interacts with the Rab11 GTPase effector Rab11 family-interacting protein 5 (Rab11FIP5)/Rab11 interacting protein (Rip11) to facilitate the transition of internalized Nrxn1α from early to recycling endosomes and bias Nrxn1α surface polarization towards the axon. In the absence of SorCS1, Nrxn1α accumulates in early endosomes and mispolarizes to the dendritic surface, impairing presynaptic differentiation and function. Thus, SorCS1-mediated sorting in dendritic endosomes controls Nrxn axonal surface polarization required for proper synapse development and function.
Asunto(s)
Proteínas de Unión al Calcio/genética , Corteza Cerebral/metabolismo , Moléculas de Adhesión de Célula Nerviosa/genética , Neuronas/metabolismo , Receptores de Superficie Celular/genética , Membranas Sinápticas/metabolismo , Transmisión Sináptica/genética , Animales , Proteínas de Unión al Calcio/metabolismo , Polaridad Celular , Corteza Cerebral/citología , Embrión de Mamíferos , Endocitosis , Endosomas/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuronas/ultraestructura , Cultivo Primario de Células , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte de Proteínas , Ratas , Ratas Wistar , Receptores de Superficie Celular/metabolismo , Membranas Sinápticas/ultraestructura , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Amyloid-ß precursor protein (APP) is central to the pathogenesis of Alzheimer's disease, yet its physiological function remains unresolved. Accumulating evidence suggests that APP has a synaptic function mediated by an unidentified receptor for secreted APP (sAPP). Here we show that the sAPP extension domain directly bound the sushi 1 domain specific to the γ-aminobutyric acid type B receptor subunit 1a (GABABR1a). sAPP-GABABR1a binding suppressed synaptic transmission and enhanced short-term facilitation in mouse hippocampal synapses via inhibition of synaptic vesicle release. A 17-amino acid peptide corresponding to the GABABR1a binding region within APP suppressed in vivo spontaneous neuronal activity in the hippocampus of anesthetized Thy1-GCaMP6s mice. Our findings identify GABABR1a as a synaptic receptor for sAPP and reveal a physiological role for sAPP in regulating GABABR1a function to modulate synaptic transmission.
Asunto(s)
Precursor de Proteína beta-Amiloide/fisiología , Plasticidad Neuronal , Receptores de GABA-A/fisiología , Transmisión Sináptica , Secuencia de Aminoácidos , Animales , Células Cultivadas , Células HEK293 , Hipocampo/fisiología , Humanos , Masculino , Proteínas de la Membrana/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/citología , Péptidos , Unión Proteica , Dominios Proteicos , Proteómica , Sinapsis/fisiología , Vesículas Sinápticas/fisiologíaRESUMEN
Pyramidal neuron dendrites integrate synaptic input from multiple partners. Different inputs converging on the same dendrite have distinct structural and functional features, but the molecular mechanisms organizing input-specific properties are poorly understood. We identify the orphan receptor GPR158 as a binding partner for the heparan sulfate proteoglycan (HSPG) glypican 4 (GPC4). GPC4 is enriched on hippocampal granule cell axons (mossy fibers), whereas postsynaptic GPR158 is restricted to the proximal segment of CA3 apical dendrites receiving mossy fiber input. GPR158-induced presynaptic differentiation in contacting axons requires cell-surface GPC4 and the co-receptor LAR. Loss of GPR158 increases mossy fiber synapse density but disrupts bouton morphology, impairs ultrastructural organization of active zone and postsynaptic density, and reduces synaptic strength of this connection, while adjacent inputs on the same dendrite are unaffected. Our work identifies an input-specific HSPG-GPR158 interaction that selectively organizes synaptic architecture and function of developing mossy fiber-CA3 synapses in the hippocampus.
Asunto(s)
Región CA3 Hipocampal/metabolismo , Proteoglicanos de Heparán Sulfato/metabolismo , Fibras Musgosas del Hipocampo/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sinapsis/metabolismo , Animales , Región CA3 Hipocampal/embriología , Células HEK293 , Humanos , Ratones , Fibras Musgosas del Hipocampo/embriología , Neurogénesis/fisiología , Células Piramidales/metabolismo , Ratas , Ratas Long-Evans , Transmisión Sináptica/fisiologíaRESUMEN
Pyramidal neurons express rich repertoires of leucine-rich repeat (LRR)-containing adhesion molecules with similar synaptogenic activity in culture. The in vivo relevance of this molecular diversity is unclear. We show that hippocampal CA1 pyramidal neurons express multiple synaptogenic LRR proteins that differentially distribute to the major excitatory inputs on their apical dendrites. At Schaffer collateral (SC) inputs, FLRT2, LRRTM1, and Slitrk1 are postsynaptically localized and differentially regulate synaptic structure and function. FLRT2 controls spine density, whereas LRRTM1 and Slitrk1 exert opposing effects on synaptic vesicle distribution at the active zone. All LRR proteins differentially affect synaptic transmission, and their combinatorial loss results in a cumulative phenotype. At temporoammonic (TA) inputs, LRRTM1 is absent; FLRT2 similarly controls functional synapse number, whereas Slitrk1 function diverges to regulate postsynaptic AMPA receptor density. Thus, LRR proteins differentially control synaptic architecture and function and act in input-specific combinations and a context-dependent manner to specify synaptic properties.
Asunto(s)
Glicoproteínas de Membrana/fisiología , Proteínas de la Membrana/fisiología , Moléculas de Adhesión de Célula Nerviosa/fisiología , Sinapsis/fisiología , Animales , Células Cultivadas , Técnicas de Cocultivo , Potenciales Postsinápticos Excitadores/fisiología , Femenino , Células HEK293 , Humanos , Masculino , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/ultraestructura , Proteínas de la Membrana/análisis , Proteínas de la Membrana/ultraestructura , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso , Moléculas de Adhesión de Célula Nerviosa/análisis , Moléculas de Adhesión de Célula Nerviosa/ultraestructura , Ratas , Ratas Wistar , Sinapsis/química , Sinapsis/ultraestructura , Transmisión Sináptica/fisiologíaRESUMEN
Whether interactions between synaptotagmin-1 (syt-1) and the soluble NSF attachment protein receptors (SNAREs) are required during neurotransmission is debated. We examined five SNAP-25 mutations designed to interfere with syt-1 interactions. One mutation, D51/E52/E55A, targeted negative charges within region II of the primary interface (Zhou et al., 2015); two mutations targeted region I (D166A and D166/E170A) and one mutation targeted both (D51/E52/E55/D166A). The final mutation (D186/D193A) targeted C-terminal residues not expected to interact with syt-1. An in vitro assay showed that the region I, region II, and region I+II (D51/E52/E55/D166A) mutants markedly reduced the attachment between syt-1 and t-SNARE-carrying vesicles in the absence of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. In the presence of PI(4,5)P2, vesicle attachment was unaffected by mutation. When expressed in Snap-25-null mouse autaptic neurons, region I mutations reduced the size of the readily releasable pool of vesicles, whereas the region II mutation reduced vesicular release probability. Combining both in the D51/E52/E55/D166A mutation abrogated evoked release. These data point to a division of labor between region I (vesicle priming) and region II (evoked release). Spontaneous release was disinhibited by region I mutations and found to correlate with defective complexin (Cpx) clamping in an in vitro fusion assay, pointing to an interdependent role of synaptotagmin and Cpx in release clamping. Mutation in region II (D51/E52/E55A) also unclamped release, but this effect could be overcome by synaptotagmin overexpression, arguing against an obligatory role in clamping. We conclude that three synaptic release functions of syt-1, vesicle priming, spontaneous release clamping, and evoked release triggering, depend on direct SNARE complex interaction. SIGNIFICANCE STATEMENT: The function of synaptotagmin-1 (syt-1):soluble NSF attachment protein receptor (SNARE) interactions during neurotransmission remains unclear. We mutated SNAP-25 within the recently identified region I and region II of the primary synaptotagmin:SNARE interface. Using in vitro assays and rescue experiments in autaptic neurons, we show that interactions within region II of the primary interface are necessary for synchronized calcium-triggered release, whereas region I is involved in vesicle priming. Spontaneous release was disinhibited by region I mutation and found to correlate with defective complexin (Cpx) clamping in vitro, pointing to an interdependent role of synaptotagmin and Cpx in release clamping. Therefore, vesicle priming, clamping spontaneous release, and eliciting evoked release are three different functions of syt-1 that involve different interaction modes with the SNARE complex.
Asunto(s)
Potenciales de Acción/fisiología , Transducción de Señal/fisiología , Transmisión Sináptica/fisiología , Vesículas Sinápticas/fisiología , Proteína 25 Asociada a Sinaptosomas/metabolismo , Sinaptotagmina I/metabolismo , Animales , Sitios de Unión , Señalización del Calcio/fisiología , Femenino , Ratones , Ratones Noqueados , Mutagénesis Sitio-Dirigida , Unión Proteica , Relación Estructura-Actividad , Proteína 25 Asociada a Sinaptosomas/genética , Sinaptotagmina I/genéticaRESUMEN
The formation, function, and plasticity of synapses require dynamic changes in synaptic receptor composition. Here, we identify the sorting receptor SorCS1 as a key regulator of synaptic receptor trafficking. Four independent proteomic analyses identify the synaptic adhesion molecule neurexin and the AMPA glutamate receptor (AMPAR) as major proteins sorted by SorCS1. SorCS1 localizes to early and recycling endosomes and regulates neurexin and AMPAR surface trafficking. Surface proteome analysis of SorCS1-deficient neurons shows decreased surface levels of these, and additional, receptors. Quantitative in vivo analysis of SorCS1-knockout synaptic proteomes identifies SorCS1 as a global trafficking regulator and reveals decreased levels of receptors regulating adhesion and neurotransmission, including neurexins and AMPARs. Consequently, glutamatergic transmission at SorCS1-deficient synapses is reduced due to impaired AMPAR surface expression. SORCS1 mutations have been associated with autism and Alzheimer disease, suggesting that perturbed receptor trafficking contributes to synaptic-composition and -function defects underlying synaptopathies.
Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuronas/metabolismo , Receptores AMPA/metabolismo , Receptores de Superficie Celular/fisiología , Animales , Proteínas de Unión al Calcio , Células Cultivadas , Técnicas de Silenciamiento del Gen , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/análisis , Moléculas de Adhesión de Célula Nerviosa/análisis , Neuronas/química , Transporte de Proteínas/fisiología , Ratas , Ratas Long-Evans , Receptores AMPA/análisis , Receptores de Superficie Celular/análisisRESUMEN
The role of spontaneously occurring release events in glutamatergic and GABAergic neurons and their regulation is intensely debated. To study the interdependence of glutamatergic and GABAergic spontaneous release, we compared reciprocally connected "mixed" glutamatergic/GABAergic neuronal pairs from mice cultured on astrocyte islands with "homotypic" glutamatergic or GABAergic pairs and autaptic neurons. We measured mEPSC and mIPSC frequencies simultaneously from both neurons. Neuronal pairs formed both interneuronal synaptic and autaptic connections indiscriminately. We find that whereas mEPSC and mIPSC frequencies did not deviate between autaptic and synaptic connections, the frequency of mEPSCs in mixed pairs was strongly depressed compared with either autaptic neurons or glutamatergic pairs. Simultaneous imaging of synapses, or comparison to evoked release amplitudes, showed that this decrease was not caused by fewer active synapses. The mEPSC frequency was negatively correlated with the mIPSC frequency, indicating interdependence. Moreover, the reduction in mEPSC frequency was abolished when established pairs were exposed to bicuculline for 3 d, but not by long-term incubation with tetrodotoxin, indicating that spontaneous GABA release downregulates mEPSC frequency. Further investigations showed that knockout of synaptotagmin-1 did not affect mEPSC frequencies in either glutamatergic autaptic neurons or in glutamatergic pairs. However, in mixed glutamatergic/GABAergic pairs, mEPSC frequencies were increased by a factor of four in the synaptotagmin-1-null neurons, which is in line with data obtained from mixed cultures. The effect persisted after incubation with BAPTA-AM. We conclude that spontaneous GABA release exerts control over mEPSC release, and GABAergic innervation of glutamatergic neurons unveils the unclamping phenotype of the synaptotagmin-1-null neurons.
Asunto(s)
Neuronas GABAérgicas/metabolismo , Ácido Glutámico/metabolismo , Fenotipo , Sinaptotagmina I/deficiencia , Animales , Animales Recién Nacidos , Células Cultivadas , Potenciales Postsinápticos Excitadores/fisiología , Femenino , Hipocampo/metabolismo , Masculino , Ratones , Ratones Noqueados , Sinapsis/metabolismoRESUMEN
Diacylglycerol (DAG) is a prominent endogenous modulator of synaptic transmission. Recent studies proposed two apparently incompatible pathways, via protein kinase C (PKC) and via Munc13. Here we show how these two pathways converge. First, we confirm that DAG analogs indeed continue to potentiate transmission after PKC inhibition (the Munc13 pathway), but only in neurons that previously experienced DAG analogs, before PKC inhibition started. Second, we identify an essential PKC pathway by expressing a PKC-insensitive Munc18-1 mutant in munc18-1 null mutant neurons. This mutant supported basic transmission, but not DAG-induced potentiation and vesicle redistribution. Moreover, synaptic depression was increased, but not Ca2+-independent release evoked by hypertonic solutions. These data show that activation of both PKC-dependent and -independent pathways (via Munc13) are required for DAG-induced potentiation. Munc18-1 is an essential downstream target in the PKC pathway. This pathway is of general importance for presynaptic plasticity.
Asunto(s)
Diglicéridos/fisiología , Plasticidad Neuronal/fisiología , Proteína Quinasa C/fisiología , Receptores Presinapticos/fisiología , Transducción de Señal/fisiología , Animales , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Células Cromafines/metabolismo , Diglicéridos/metabolismo , Electrofisiología , Inhibidores Enzimáticos/farmacología , Femenino , Hipocampo/citología , Hipocampo/efectos de los fármacos , Cinética , Lentivirus/genética , Ratones , Ratones Noqueados , Microscopía Electrónica , Proteínas Munc18/genética , Proteínas Munc18/metabolismo , Mutación/fisiología , Neuronas/citología , Neuronas/metabolismo , Neuronas/ultraestructura , Técnicas de Placa-Clamp , Ésteres del Forbol/farmacología , Fosforilación , Embarazo , Proteína Quinasa C/antagonistas & inhibidores , Receptores Presinapticos/ultraestructuraRESUMEN
During the female reproductive cycle, hypothalamic oxytocin (OT) neurons undergo sharp changes in excitability. In lactating mammals, bursts of electrical activity of OT neurons result in the release of large amounts of OT in the bloodstream, which causes milk ejection. One hypothesis is that OT neurons regulate their own firing activity and that of nearby OT neurons by somatodendritic release of OT. In this study, we show that OT neuron activity strongly reduces inhibitory synaptic transmission to these neurons. This effect is blocked by antagonists of both adenosine and OT receptors and is mimicked by OT application. Inhibition of soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex formation by tetanus toxin completely blocked the stimulation-induced reduction in inhibitory input, as did the calcium chelator BAPTA. During lactation, the readily releasable pool of secretory vesicles in OT cell bodies was doubled, and calcium currents were upregulated. This resulted in an increased inhibition of GABAergic synaptic transmission by somatodendritic release during lactation compared with the adult virgin stage. These results demonstrate that somatodendritic release is augmented during lactation, which is a novel form of plasticity to change the strength of synaptic transmission.