RESUMEN
Parvalbumin (PV)-expressing basket interneurons in the prefrontal cortex (PFC) regulate pyramidal cell firing, synchrony, and network oscillations. Yet, it is unclear how their perisomatic inputs to pyramidal neurons are integrated into neural circuitry and adjusted postnatally. Neural cell adhesion molecule NCAM is expressed in a variety of cells in the PFC and cooperates with EphrinA/EphAs to regulate inhibitory synapse density. Here, analysis of a novel parvalbumin (PV)-Cre: NCAM F/F mouse mutant revealed that NCAM functions presynaptically in PV+ basket interneurons to regulate postnatal elimination of perisomatic synapses. Mutant mice exhibited an increased density of PV+ perisomatic puncta in PFC layer 2/3, while live imaging in mutant brain slices revealed fewer puncta that were dynamically eliminated. Furthermore, EphrinA5-induced growth cone collapse in PV+ interneurons in culture depended on NCAM expression. Electrophysiological recording from layer 2/3 pyramidal cells in mutant PFC slices showed a slower rise time of inhibitory synaptic currents. PV-Cre: NCAM F/F mice exhibited impairments in working memory and social behavior that may be impacted by altered PFC circuitry. These findings suggest that the density of perisomatic synapses of PV+ basket interneurons is regulated postnatally by NCAM, likely through EphrinA-dependent elimination, which is important for appropriate PFC network function and behavior.
Asunto(s)
Interneuronas/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neurogénesis/fisiología , Corteza Prefrontal/metabolismo , Sinapsis/fisiología , Animales , Conducta Animal , Femenino , Masculino , Memoria a Corto Plazo/fisiología , Ratones , Ratones Endogámicos C57BL , Corteza Prefrontal/crecimiento & desarrolloRESUMEN
Dendritic spines in the developing mammalian neocortex are initially overproduced and then eliminated during adolescence to achieve appropriate levels of excitation in mature networks. We show here that the L1 family cell adhesion molecule Close Homolog of L1 (CHL1) and secreted repellent ligand Semaphorin 3B (Sema3B) function together to induce dendritic spine pruning in developing cortical pyramidal neurons. Loss of CHL1 in null mutant mice in both genders resulted in increased spine density and a greater proportion of immature spines on apical dendrites in the prefrontal and visual cortex. Electron microscopy showed that excitatory spine synapses with postsynaptic densities were increased in the CHL1-null cortex, and electrophysiological recording in prefrontal slices from mutant mice revealed deficiencies in excitatory synaptic transmission. Mechanistically, Sema3B protein induced elimination of spines on apical dendrites of cortical neurons cultured from wild-type but not CHL1-null embryos. Sema3B was secreted by the cortical neuron cultures, and its levels increased when cells were treated with the GABA antagonist gabazine. In vivo CHL1 was coexpressed with Sema3B in pyramidal neuron subpopulations and formed a complex with Sema3B receptor subunits Neuropilin-2 and PlexinA4. CHL1 and NrCAM, a closely related L1 adhesion molecule, localized primarily to distinct spines and promoted spine elimination to Sema3B or Sema3F, respectively. These results support a new concept in which selective spine elimination is achieved through different secreted semaphorins and L1 family adhesion molecules to sculpt functional neural circuits during postnatal maturation.SIGNIFICANCE STATEMENT Dendritic spines in the mammalian neocortex are initially overproduced and then pruned in adolescent life through unclear mechanisms to sculpt maturing cortical circuits. Here, we show that spine and excitatory synapse density of pyramidal neurons in the developing neocortex is regulated by the L1 adhesion molecule, Close Homolog of L1 (CHL1). CHL1 mediated spine pruning in response to the secreted repellent ligand Semaphorin 3B and associated with receptor subunits Neuropilin-2 and PlexinA4. CHL1 and related L1 adhesion molecule NrCAM localized to distinct spines, and promoted spine elimination to Semaphorin 3B and -3F, respectively. These results support a new concept in which selective elimination of individual spines and nascent synapses can be achieved through the action of distinct secreted semaphorins and L1 adhesion molecules.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Espinas Dendríticas/fisiología , Corteza Prefrontal/fisiología , Semaforinas/fisiología , Corteza Visual/fisiología , Envejecimiento/fisiología , Animales , Moléculas de Adhesión Celular/deficiencia , Células Cultivadas , Femenino , Agonistas del GABA/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/fisiología , Neuropilina-2/fisiología , Técnicas de Placa-Clamp , Corteza Prefrontal/citología , Corteza Prefrontal/crecimiento & desarrollo , Mapeo de Interacción de Proteínas , Células Piramidales/efectos de los fármacos , Células Piramidales/fisiología , Células Piramidales/ultraestructura , Piridazinas/farmacología , Receptores de Superficie Celular/fisiología , Transmisión Sináptica , Corteza Visual/citología , Corteza Visual/crecimiento & desarrolloRESUMEN
Neuron-glial related cell adhesion molecule NrCAM is a newly identified negative regulator of spine density that genetically interacts with Semaphorin3F (Sema3F), and is implicated in autism spectrum disorders (ASD). To investigate a role for NrCAM in spine pruning during the critical adolescent period when networks are established, we generated novel conditional, inducible NrCAM mutant mice (Nex1Cre-ERT2: NrCAMflox/flox). We demonstrate that NrCAM functions cell autonomously during adolescence in pyramidal neurons to restrict spine density in the visual (V1) and medial frontal cortex (MFC). Guided by molecular modeling, we found that NrCAM promoted clustering of the Sema3F holoreceptor complex by interfacing with Neuropilin-2 (Npn2) and PDZ scaffold protein SAP102. NrCAM-induced receptor clustering stimulated the Rap-GAP activity of PlexinA3 (PlexA3) within the holoreceptor complex, which in turn, inhibited Rap1-GTPase and inactivated adhesive ß1 integrins, essential for Sema3F-induced spine pruning. These results define a developmental function for NrCAM in Sema3F receptor signaling that limits dendritic spine density on cortical pyramidal neurons during adolescence.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Espinas Dendríticas/fisiología , Lóbulo Frontal/crecimiento & desarrollo , Proteínas de la Membrana/fisiología , Proteínas del Tejido Nervioso/fisiología , Células Piramidales/fisiología , Corteza Visual/crecimiento & desarrollo , Animales , Guanilato-Quinasas/fisiología , Ratones Transgénicos , Modelos Moleculares , Transducción de SeñalRESUMEN
Establishment of a proper balance of excitatory and inhibitory connectivity is achieved during development of cortical networks and adjusted through synaptic plasticity. The neural cell adhesion molecule (NCAM) and the receptor tyrosine kinase EphA3 regulate the perisomatic synapse density of inhibitory GABAergic interneurons in the mouse frontal cortex through ephrin-A5-induced growth cone collapse. In this study, it was demonstrated that binding of NCAM and EphA3 occurred between the NCAM Ig2 domain and EphA3 cysteine-rich domain (CRD). The binding interface was further refined through molecular modeling and mutagenesis and shown to be comprised of complementary charged residues in the NCAM Ig2 domain (Arg-156 and Lys-162) and the EphA3 CRD (Glu-248 and Glu-264). Ephrin-A5 induced co-clustering of surface-bound NCAM and EphA3 in GABAergic cortical interneurons in culture. Receptor clustering was impaired by a charge reversal mutation that disrupted NCAM/EphA3 association, emphasizing the importance of the NCAM/EphA3 binding interface for cluster formation. NCAM enhanced ephrin-A5-induced EphA3 autophosphorylation and activation of RhoA GTPase, indicating a role for NCAM in activating EphA3 signaling through clustering. NCAM-mediated clustering of EphA3 was essential for ephrin-A5-induced growth cone collapse in cortical GABAergic interneurons, and RhoA and a principal effector, Rho-associated protein kinase, mediated the collapse response. This study delineates a mechanism in which NCAM promotes ephrin-A5-dependent clustering of EphA3 through interaction of the NCAM Ig2 domain and the EphA3 CRD, stimulating EphA3 autophosphorylation and RhoA signaling necessary for growth cone repulsion in GABAergic interneurons in vitro, which may extend to remodeling of axonal terminals of interneurons in vivo.
Asunto(s)
Neuronas GABAérgicas/metabolismo , Conos de Crecimiento/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Receptor EphA3/metabolismo , Transducción de Señal/fisiología , Proteínas de Unión al GTP rho/metabolismo , Quinasas Asociadas a rho/metabolismo , Animales , Efrina-A5/genética , Efrina-A5/metabolismo , Ratones , Ratones Mutantes , Moléculas de Adhesión de Célula Nerviosa/genética , Fosforilación/fisiología , Proteínas de Unión al GTP rho/genética , Quinasas Asociadas a rho/genética , Proteína de Unión al GTP rhoARESUMEN
During the development of the peripheral nervous system there is extensive apoptosis, and these neuronal corpses need to be cleared to prevent an inflammatory response. Recently, Jedi-1 and MEGF10, both expressed in glial precursor cells, were identified in mouse as having an essential role in this phagocytosis (Wu et al., 2009); however, the mechanisms by which they promote engulfment remained unknown. Both Jedi-1 and MEGF10 are homologous to the Drosophila melanogaster receptor Draper, which mediates engulfment through activation of the tyrosine kinase Shark. Here, we identify Syk, the mammalian homolog of Shark, as a signal transducer for both Jedi-1 and MEGF10. Syk interacted with each receptor independently through the immunoreceptor tyrosine-based activation motifs (ITAMs) in their intracellular domains. The interaction was enhanced by phosphorylation of the tyrosines in the ITAMs by Src family kinases (SFKs). Jedi association with Syk and activation of the kinase was also induced by exposure to dead cells. Expression of either Jedi-1 or MEGF10 in HeLa cells facilitated engulfment of carboxylated microspheres to a similar extent, and there was no additive effect when they were coexpressed. Mutation of the ITAM tyrosines of Jedi-1 and MEGF10 prevented engulfment. The SFK inhibitor PP2 or a selective Syk inhibitor (BAY 61-3606) also blocked engulfment. Similarly, in cocultures of glial precursors and dying sensory neurons from embryonic mice, addition of PP2 or knock down of endogenous Syk decreased the phagocytosis of apoptotic neurons. These results indicate that both Jedi-1 and MEGF10 can mediate phagocytosis independently through the recruitment of Syk.
Asunto(s)
Apoptosis/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal/genética , Secuencias de Aminoácidos , Animales , Proteínas de Arabidopsis/metabolismo , Recuento de Células , Células Cultivadas , Técnicas de Cocultivo , Embrión de Mamíferos , Inhibidores Enzimáticos/farmacología , Femenino , Ganglios Espinales/citología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Humanos , Inmunoprecipitación , Motivo de Activación del Inmunorreceptor Basado en Tirosina/efectos de los fármacos , Motivo de Activación del Inmunorreceptor Basado en Tirosina/genética , Motivo de Activación del Inmunorreceptor Basado en Tirosina/fisiología , Péptidos y Proteínas de Señalización Intracelular/genética , Transferasas Intramoleculares/metabolismo , Masculino , Proteínas de la Membrana/genética , Ratones , Microglía , Mutagénesis Sitio-Dirigida , Mutación/genética , Neuronas , Niacinamida/análogos & derivados , Niacinamida/farmacología , Fagocitosis/efectos de los fármacos , Fagocitosis/genética , Fosforilación/efectos de los fármacos , Fosforilación/genética , Proteínas Tirosina Quinasas/genética , Pirimidinas/farmacología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal/efectos de los fármacos , Estaurosporina/farmacología , Quinasa Syk , TransfecciónRESUMEN
Perineuronal nets (PNNs) are implicated in closure of critical periods of synaptic plasticity in the brain, but the molecular mechanisms by which PNNs regulate synapse development are obscure. A receptor complex of NCAM and EphA3 mediates postnatal remodeling of inhibitory perisomatic synapses of GABAergic interneurons onto pyramidal cells in the mouse frontal cortex necessary for excitatory/inhibitory balance. Here it is shown that enzymatic removal of PNN glycosaminoglycan chains decreased the density of GABAergic perisomatic synapses in mouse organotypic cortical slice cultures. Neurocan, a key component of PNNs, was expressed in postnatal frontal cortex in apposition to perisomatic synapses of parvalbumin-positive interneurons. Polysialylated NCAM (PSA-NCAM), which is required for ephrin-dependent synapse remodeling, bound less efficiently to neurocan than mature, non-PSA-NCAM. Neurocan bound the non-polysialylated form of NCAM at the EphA3 binding site within the immunoglobulin-2 domain. Neurocan inhibited NCAM/EphA3 association, membrane clustering of NCAM/EphA3 in cortical interneuron axons, EphA3 kinase activation, and ephrin-A5-induced growth cone collapse. These studies delineate a novel mechanism wherein neurocan inhibits NCAM/EphA3 signaling and axonal repulsion, which may terminate postnatal remodeling of interneuron axons to stabilize perisomatic synapses in vivo.
Asunto(s)
Neuronas GABAérgicas/metabolismo , Interneuronas/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neurocano/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Animales , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Humanos , Ratones , Moléculas de Adhesión de Célula Nerviosa/química , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Tirosina Quinasas Receptoras/química , Receptor EphA3RESUMEN
The neural cell adhesion molecule (NCAM), has been shown to be an obligate regulator of synaptic stability and pruning during critical periods of cortical maturation. However, the functional consequences of NCAM deletion on the organization of inhibitory circuits in cortex are not known. In vesicular gamma-amino butyric acid (GABA) transporter (VGAT)-channelrhodopsin2 (ChR2)-enhanced yellow fluorescent protein (EYFP) transgenic mice, NCAM is expressed postnatally at perisomatic synaptic puncta of EYFP-labeled parvalbumin, somatostatin and calretinin-positive interneurons, and in the neuropil in the anterior cingulate cortex (ACC). To investigate how NCAM deletion affects the spatial organization of inhibitory inputs to pyramidal cells, we used laser scanning photostimulation in brain slices of VGAT-ChR2-EYFP transgenic mice crossed to either NCAM-null or wild type (WT) mice. Laser scanning photostimulation revealed that NCAM deletion increased the strength of close-in inhibitory connections to layer 2/3 pyramidal cells of the ACC. In addition, in NCAM-null mice, the intrinsic excitability of pyramidal cells increased, whereas the intrinsic excitability of GABAergic interneurons did not change. The increase in inhibitory tone onto pyramidal cells, and the increased pyramidal cell excitability in NCAM-null mice will alter the delicate coordination of excitation and inhibition (E/I coordination) in the ACC, and may be a factor contributing to circuit dysfunction in diseases such as schizophrenia and bipolar disorder, in which NCAM has been implicated.
Asunto(s)
Fenómenos Electrofisiológicos/fisiología , Giro del Cíngulo/fisiología , Moléculas de Adhesión de Célula Nerviosa/fisiología , Células Piramidales/fisiología , Animales , Giro del Cíngulo/citología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Transgénicos , Microscopía Confocal , Inhibición Neural/fisiología , Técnicas de Placa-Clamp , Células Piramidales/citologíaRESUMEN
During the development of the peripheral nervous system, the large number of apoptotic neurons generated are phagocytosed by glial precursor cells. This clearance is mediated, in part, through the mammalian engulfment receptor Jedi-1. However, the mechanisms by which Jedi-1 mediates phagocytosis are poorly understood. Here we demonstrate that Jedi-1 associates with GULP, the mammalian homologue of CED-6, an adaptor protein required for phagocytosis mediated by the nematode engulfment receptor CED-1. Silencing GULP or mutating the NPXY motif in Jedi-1, which is required for GULP binding, prevents Jedi-1-mediated phagocytosis. How GULP promotes engulfment is not known. Of interest, we find that Jedi-1-induced phagocytosis requires GULP binding to clathrin heavy chain (CHC). During engulfment, CHC is tyrosine phosphorylated, which is required for Jedi-mediated engulfment. Both phosphoclathrin and actin accumulate around engulfed microspheres. Furthermore, knockdown of CHC in HeLa cells prevents Jedi-1-mediated engulfment of microspheres, and knockdown in glial precursors prevents the engulfment of apoptotic neurons. Taken together, these results reveal that Jedi-1 signals through recruitment of GULP, which promotes phagocytosis through a noncanonical phosphoclathrin-dependent mechanism.