RESUMEN
Although membrane trafficking pathways are involved in basic cellular functions, the evolutionally expanded number of their related family proteins suggests additional roles for membrane trafficking in higher organisms. Here, we show that several Rab-dependent trafficking pathways differentially participate in neuronal migration, an essential step for the formation of the mammalian-specific six-layered brain structure. In vivo electroporation-mediated suppression of Rab5 or dynamin to block endocytosis caused a severe neuronal migration defect in mouse cerebral cortex. Among many downstream endocytic pathways, suppression of Rab11-dependent recycling pathways exhibited a similar migration disorder, whereas inhibition of Rab7-dependent lysosomal degradation pathways affected only the final phase of neuronal migration and dendrite morphology. Inhibition of Rab5 or Rab11 perturbed the trafficking of N-cadherin, whose suppression also disturbed neuronal migration. Taken together, our findings reveal physiological roles of endocytic pathways, each of which has specific functions in distinct steps of neuronal migration and maturation during mammalian brain formation.
Asunto(s)
Cadherinas/metabolismo , Movimiento Celular/fisiología , Endocitosis/fisiología , Neuronas/fisiología , Proteínas de Unión al GTP rab/metabolismo , Animales , Cadherinas/genética , Adhesión Celular/fisiología , Comunicación Celular/fisiología , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/crecimiento & desarrollo , Corteza Cerebral/fisiología , Dendritas/fisiología , Dinaminas/genética , Dinaminas/metabolismo , Lisosomas/enzimología , Lisosomas/fisiología , MAP Quinasa Quinasa 4/metabolismo , Ratones , Ratones Endogámicos ICR , Neuronas/citología , Transducción de Señal , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión al GTP rab5/metabolismo , Proteínas de Unión a GTP rab7RESUMEN
Neuronal migration is essential for proper cortical layer formation and brain function, because migration defects result in neurological disorders such as mental retardation and epilepsy. Neuronal migration is divided into several contiguous steps: early phase (multipolar mode), locomotion mode, and terminal translocation mode. The locomotion mode covers most of the migration route and thereby is the main contributor to cortical layer formation. However, analysis of the molecular mechanisms regulating this mode is difficult due to the secondary effects of defects at the early phase of migration. In this study, we established an ex vivo chemical inhibitor screening, allowing us to directly analyze the locomotion mode of migration. Roscovitine and PP2, inhibitors for Cdk5 and Src family kinases, respectively, suppressed the locomotion mode of migration. In line with this, a small percentage of Cdk5- or Src family kinase (Fyn)-knockdown cells exhibited locomoting morphology but retarded migration, although the majority of cells were stalled at the early phase of migration. We also showed that rottlerin, widely used as a specific inhibitor for protein kinase Cdelta (PKCdelta), suppressed the locomotion mode. Unexpectedly, however, the dominant-negative form as well as RNA interference for PKCdelta hardly affected the locomotion, whereas they may disturb terminal translocation. In addition, we found JNK to be a potential downstream target of rottlerin. Taken together, our novel chemical inhibitor screening provides evidence that Cdk5 and Src family kinases regulate the locomotion mode of neuronal migration. It also uncovered roles for Fyn and PKCdelta in the early and final phases of migration, respectively.
Asunto(s)
Movimiento Celular/fisiología , Corteza Cerebral/embriología , Corteza Cerebral/enzimología , Quinasa 5 Dependiente de la Ciclina/metabolismo , Neuronas/enzimología , Proteína Quinasa C-delta/metabolismo , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Animales , Movimiento Celular/efectos de los fármacos , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 5 Dependiente de la Ciclina/genética , Femenino , MAP Quinasa Quinasa 4/antagonistas & inhibidores , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/metabolismo , Ratones , Ratones Endogámicos ICR , Embarazo , Proteína Quinasa C-delta/antagonistas & inhibidores , Proteína Quinasa C-delta/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-fyn/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-fyn/genética , Purinas/farmacología , Pirimidinas/farmacología , RoscovitinaRESUMEN
p27(kip1), a cyclin-dependent kinase (CDK) inhibitor (CKI), generally suppresses CDK activity in proliferating cells. Although another role of p27 in cell migration has been recently suggested in vitro, the physiological importance of p27 in cell migration remains elusive, as p27-deficient mice have not shown any obvious migration-defect-related phenotypes. Here, we show that Cdk5, an unconventional neuronal CDK, phosphorylates and stabilizes p27 as an upstream regulator, maintaining the amount of p27 in post-mitotic neurons. In vivo RNA interference (RNAi) experiments showed that reduced amounts of p27 caused inhibition of cortical neuronal migration and decreased the amount of F-actin in the processes of migrating neurons. The Cdk5-p27 pathway activates an actin-binding protein, cofilin, which is also shown to be involved in cortical neuronal migration in vivo. Our findings shed light on a previously unknown new relationship between CDK and CKI in G0-arrested cells that regulates cytoskeletal reorganization and neuronal migration during corticogenesis.
Asunto(s)
Actinas/metabolismo , Movimiento Celular , Quinasa 5 Dependiente de la Ciclina/fisiología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/fisiología , Neuronas/fisiología , Factores Despolimerizantes de la Actina/metabolismo , Animales , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Quinasa 5 Dependiente de la Ciclina/química , Quinasa 5 Dependiente de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Ratones , Ratones Endogámicos ICR , Modelos Biológicos , Fosforilación , TransfecciónRESUMEN
Rho-family GTPases play key roles in regulating cytoskeletal reorganization, contributing to many aspects of nervous system development. Their activities are known to be regulated by guanine nucleotide exchange factors (GEFs), in response to various extracellular cues. P-Rex1, a GEF for Rac, has been mainly investigated in neutrophils, in which this molecule contributes to reactive oxygen species formation. However, its role in the nervous system is essentially unknown. Here we describe the expression profile and a physiological function of P-Rex1 in nervous system development. In situ hybridization revealed that P-Rex1 is dynamically expressed in a variety of cells in the developing mouse brain, including some cortical and DRG neurons. In migrating neurons in the intermediate zone, P-Rex1 protein was found to localize in the leading process and adjacent cytoplasmic region. When transfected in pheochromocytoma PC12 cells, P-Rex1 can be activated by NGF, causing an increase in GTP-bound Rac1 and cell motility. Deletion analyses suggested roles for distinct domains of this molecule. Experiments using a P-Rex1 mutant lacking the Dbl-homology domain, a dominant-negative-like form, and small interfering RNA showed that endogenous P-Rex1 was involved in cell migration of PC12 cells and primary cultured neurons from the embryonic day 14 cerebral cortices, induced by extracellular stimuli (NGF, BDNF, and epidermal growth factor). Furthermore, in utero electroporation of the mutant protein into the embryonic cerebral cortex perturbed radial neuronal migration. These findings suggest that P-Rex1, which is expressed in a variety of cell types, is activated by extracellular cues such as neurotrophins and contributes to neuronal migration in the developing nervous system.
Asunto(s)
Movimiento Celular/fisiología , Factores de Intercambio de Guanina Nucleótido/fisiología , Factores de Crecimiento Nervioso/metabolismo , Neuronas/fisiología , Transducción de Señal/fisiología , Actinas/metabolismo , Animales , Northern Blotting/métodos , Encéfalo/anatomía & histología , Encéfalo/embriología , Encéfalo/metabolismo , Células Cultivadas , Embrión de Mamíferos , Técnica del Anticuerpo Fluorescente/métodos , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Hibridación in Situ/métodos , Ratones , Ratones Endogámicos ICR , Mutagénesis/fisiología , Factor de Crecimiento Nervioso/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Ratas , Receptor trkB/metabolismo , Factores de Tiempo , Transfección/métodos , Proteínas de Unión al GTP rac/metabolismo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Mode I phosphorylated MAP1B is observed in developing and pathogenic brains. Although Cdk5 has been believed to phosphorylate MAP1B in the developing cerebral cortex, we show that a Cdk5 inhibitor does not suppress mode I phosphorylation of MAP1B in primary and slice cultures, while a JNK inhibitor does. Coincidently, an increase in phosphorylated MAP1B was not observed in COS7 cells when Cdk5 was cotransfected with p35, but this did occur with p25 which is specifically produced in pathogenic brains. Our primary culture studies showed an involvement of Cdk5 in regulating microtubule dynamics without affecting MAP1B phosphorylation status. The importance of regulating microtubule dynamics in neuronal migration was also demonstrated by in utero electroporation experiments. These findings suggest that mode I phosphorylation of MAP1B is facilitated by JNK but not Cdk5/p35 in the developing cerebral cortex and by Cdk5/p25 in pathogenic brains, contributing to various biological events.
Asunto(s)
Corteza Cerebral/enzimología , Quinasas Ciclina-Dependientes/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Células COS , Corteza Cerebral/embriología , Chlorocebus aethiops , Quinasa 5 Dependiente de la Ciclina , Ratones , FosforilaciónRESUMEN
The coordinated migration of neurons is a pivotal step for functional architectural formation of the mammalian brain. To elucidate its molecular mechanism, gene transfer by means of in utero electroporation was applied in the developing murine brain, revealing the crucial roles of Rac1, its activators, STEF/Tiam1, and its downstream molecule, c-Jun N-terminal kinase (JNK), in the cerebral cortex. Functional repression of these molecules resulted in inhibition of radial migration of neurons without affecting their proper differentiation. Interestingly, distinct morphological phenotypes were observed; suppression of Rac1 activity caused loss of the leading process, whereas repression of JNK activity did not, suggesting the complexity of the signaling cascade. In cultured neurons from the intermediate zone, activated JNK was detected along microtubules in the processes. Application of a JNK inhibitor caused irregular morphology and increased stable microtubules in processes, and decreased phosphorylation of microtubule associated protein 1B, raising a possibility of the involvement of JNK in controlling tubulin dynamics in migrating neurons. Our data thus provide important clues for understanding the intracellullar signaling machinery for cortical neuronal migration.