RESUMEN
Neurexins (NRXNs) are key presynaptic cell adhesion molecules that regulate synapse formation and function via trans-synaptic interaction with postsynaptic ligands. Here, we generate cerebellar granule cell (CGC)-specific Nrxn triple-knockout (TKO) mice for complete deletion of all NRXNs. Unexpectedly, most CGCs die in these mice, and this requirement for NRXNs for cell survival is reproduced in cultured CGCs. The axons of cultured Nrxn TKO CGCs that are not in contact with a postsynaptic structure show defects in the formation of presynaptic protein clusters and in action-potential-induced Ca2+ influxes. These cells also show impaired secretion of depolarization-induced, fluorescence-tagged brain-derived neurotrophic factor (BDNF) from their axons, and the cell-survival defect is rescued by the application of BDNF. These results suggest that CGC survival is maintained by autocrine neurotrophic factors and that NRXNs organize the presynaptic protein clusters and the autocrine neurotrophic-factor secretory machinery independent of contact with postsynaptic ligands.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo , Neuronas , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Supervivencia Celular , Células Cultivadas , Cerebelo/metabolismo , Ligandos , Ratones , Ratones Noqueados , Neuronas/metabolismo , Sinapsis/metabolismoRESUMEN
Lysophosphatidylethanolamines (LPEs) are bioactive lysophospholipids that have been suggested to play important roles in several biological processes. We performed a quantitative analysis of LPE species and showed their composition in mouse brain. We examined the roles of oleoyl-LPE (18:1 LPE), which is one of the abundant LPE species in brain. In cultured cortical neurons, application of 18:1 LPE-stimulated neurite outgrowth. The effect of 18:1 LPE on neurite outgrowth was inhibited by Gq/11 inhibitor YM-254890, phospholipase C (PLC) inhibitor U73122, protein kinase C (PKC) inhibitor Go6983 or mitogen-activated protein kinase (MAPK) inhibitor U0126. Additionally, 18:1 LPE increased the phosphorylation of MAPK/extracellular signal-regulated kinase 1/2. These results suggest that the action of 18:1 LPE on neurite outgrowth is mediated by the Gq/11/PLC/PKC/MAPK pathway. Moreover, we found that application of 18:1 LPE protects neurons from glutamate-induced excitotoxicity. This effect of 18:1 LPE was suppressed by PKC inhibitor Go6983. These results suggest that 18:1 LPE protects neurons from glutamate toxicity via PKC inhibitor Go6983-sensitive PKC subtype. Collectively, our results demonstrated that 18:1 LPE stimulates neurite outgrowth and protects against glutamate toxicity in cultured cortical neurons. Our findings provide insights into the physiological or pathological roles of 18:1 LPE in the brain.
Asunto(s)
Encéfalo/efectos de los fármacos , Ácido Glutámico/toxicidad , Lisofosfolípidos/farmacología , Proyección Neuronal/efectos de los fármacos , Neuronas/metabolismo , Animales , Encéfalo/metabolismo , Células Cultivadas , Cromatografía Liquida/métodos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Neuritas/metabolismo , Fosforilación , Proteína Quinasa C/metabolismo , Transducción de Señal/efectos de los fármacos , Espectrometría de Masa por Ionización de Electrospray/métodos , Fosfolipasas de Tipo C/metabolismoRESUMEN
Neurite outgrowth is important in neuronal circuit formation and functions, and for regeneration of neuronal networks following trauma and disease in the brain. Thus, identification and characterization of the molecules that regulate neurite outgrowth are essential for understanding how brain circuits form and function and for the development of treatment of neurological disorders. In this study, we found that structurally different lysophosphatidylethanolamine (LPE) species, palmitoyl-LPE (16:0 LPE) and stearoyl-LPE (18:0 LPE), stimulate neurite growth in cultured cortical neurons. Interestingly, YM-254890, an inhibitor of Gq/11 protein, inhibited 16:0 LPE-stimulated neurite outgrowth but not 18:0 LPE-stimulated neurite outgrowth. In contrast, pertussis toxin, an inhibitor of Gi/Go proteins, inhibited 18:0 LPE-stimulated neurite outgrowth but not 16:0 LPE-stimulated neurite outgrowth. The effects of protein kinase C inhibitors on neurite outgrowth were also different. In addition, both 16:0 LPE and 18:0 LPE activate mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 1/2, but the effect of the MAPK inhibitor differed between the 16:0 LPE- and 18:0 LPE-treated cultures. Collectively, the results suggest that the structurally different LPE species, 16:0 LPE and 18:0 LPE stimulate neurite outgrowth through distinct signaling cascades in cultured cortical neurons and that distinct G protein-coupled receptors are involved in these processes.
Asunto(s)
Lisofosfolípidos/farmacología , Proyección Neuronal/efectos de los fármacos , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Axones/efectos de los fármacos , Axones/ultraestructura , Encéfalo/citología , Butadienos/farmacología , Células Cultivadas , Dendritas/efectos de los fármacos , Dendritas/ultraestructura , Yema de Huevo/química , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/antagonistas & inhibidores , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/antagonistas & inhibidores , Proteínas de Unión al GTP Heterotriméricas/antagonistas & inhibidores , Lisofosfolípidos/química , Ratones Endogámicos ICR , Neuronas/efectos de los fármacos , Neuronas/enzimología , Nitrilos/farmacología , Péptidos Cíclicos/farmacología , Toxina del Pertussis/farmacología , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Genome editing is a powerful technique for studying gene functions. CRISPR/Cas9-mediated gene knock-in has recently been applied to various cells and organisms. Here, we successfully knocked in an EGFP coding sequence at the site immediately after the first ATG codon of the ß-actin gene in neurons in the brain by the combined use of the CRISPR/Cas9 system and in utero electroporation technique, resulting in the expression of the EGFP-tagged ß-actin protein in cortical layer 2/3 pyramidal neurons. We detected EGFP fluorescence signals in the soma and neurites of EGFP knock-in neurons. These signals were particularly abundant in the head of dendritic spines, corresponding to the localization of the endogenous ß-actin protein. EGFP knock-in neurons showed no detectable changes in spine density and basic electrophysiological properties. In contrast, exogenously overexpressed EGFP-ß-actin showed increased spine density and EPSC frequency, and changed resting membrane potential. Thus, our technique provides a potential tool to elucidate the localization of various endogenous proteins in neurons by epitope tagging without altering neuronal and synaptic functions. This technique can be also useful for introducing a specific mutation into genes to study the function of proteins and genomic elements in brain neurons.