RESUMEN
Ca(2+) signaling plays important roles during both axonal and dendritic growth. Yet whether and how Ca(2+) rises may trigger and contribute to the development of long-range cortical connections remains mostly unknown. Here, we demonstrate that two separate limbs of the Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK)-CaMKI cascades, CaMKK-CaMKIalpha and CaMKK-CaMKIgamma, critically coordinate axonal and dendritic morphogenesis of cortical neurons, respectively. The axon-specific morphological phenotype required a diffuse cytoplasmic localization and a strikingly alpha-isoform-specific kinase activity of CaMKI. Unexpectedly, treatment with muscimol, a GABA(A) receptor agonist, selectively stimulated elongation of axons but not of dendrites, and the CaMKK-CaMKIalpha cascade critically mediated this axonogenic effect. Consistent with these findings, during early brain development, in vivo knockdown of CaMKIalpha significantly impaired the terminal axonal extension and thereby perturbed the refinement of the interhemispheric callosal projections into the contralateral cortices. Our findings thus indicate a novel role for the GABA-driven CaMKK-CaMKIalpha cascade as a mechanism critical for accurate cortical axon pathfinding, an essential process that may contribute to fine-tuning the formation of interhemispheric connectivity during the perinatal development of the CNS.
Asunto(s)
Axones/fisiología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Corteza Cerebral/fisiología , Dendritas/fisiología , Neuronas/fisiología , Ácido gamma-Aminobutírico/metabolismo , Animales , Axones/enzimología , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/enzimología , Citoplasma/enzimología , Citoplasma/metabolismo , Dendritas/enzimología , Isoenzimas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/citología , Neuronas/enzimología , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/metabolismo , Transducción de SeñalRESUMEN
The neuronal immediate early gene Arc/Arg-3.1 is widely used as one of the most reliable molecular markers for intense synaptic activity in vivo. However, the cis-acting elements responsible for such stringent activity dependence have not been firmly identified. Here we combined luciferase reporter assays in cultured cortical neurons and comparative genome mapping to identify the critical synaptic activity-responsive elements (SARE) of the Arc/Arg-3.1 gene. A major SARE was found as a unique approximately 100-bp element located at >5 kb upstream of the Arc/Arg-3.1 transcription initiation site in the mouse genome. This single element, when positioned immediately upstream of a minimal promoter, was necessary and sufficient to replicate crucial properties of endogenous Arc/Arg-3.1's transcriptional regulation, including rapid onset of transcription triggered by synaptic activity and low basal expression during synaptic inactivity. We identified the major determinants of SARE as a unique cluster of neuronal activity-dependent cis-regulatory elements consisting of closely localized binding sites for CREB, MEF2, and SRF. Consistently, a SARE reporter could readily trace and mark an ensemble of cells that have experienced intense activity in the recent past in vivo. Taken together, our work uncovers a novel transcriptional mechanism by which a critical 100-bp element, SARE, mediates a predominant component of the synapse-to-nucleus signaling in ensembles of Arc/Arg-3.1-positive activated neurons.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Transducción de Señal , Sinapsis/genética , Animales , Sitios de Unión , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Regulación de la Expresión Génica , Genómica/métodos , Factores de Transcripción MEF2 , Ratones , Factores Reguladores Miogénicos , Regiones Promotoras Genéticas/genética , Factor de Respuesta Sérica , Sinapsis/metabolismoRESUMEN
Trafficking of immune cells is controlled by directed migration of relevant cells toward chemotactic signals. Actin cytoskeleton undergoes continuous remodeling and serves as machinery for cell migration. The mDia family of formins and the Wiskott-Aldrich syndrome protein (WASP)-Arp2/3 system are two major actin nucleating-polymerizing systems in mammalian cells, with the former producing long straight actin filaments and the latter producing branched actin meshwork. Although much is known about the latter, the physiological functions of mDia proteins are unclear. We generated mice deficient in one mDia isoform, mDia1. Although mDia1(-/-) mice were born and developed without apparent abnormality, mDia1(-/-) T lymphocytes exhibited impaired trafficking to secondary lymphoid organs in vivo and showed reduced chemotaxis, little actin filament formation, and impaired polarity in response to chemotactic stimuli in vitro. Similarly, mDia1(-/-) thymocytes showed reduced chemotaxis and impaired egression from the thymus. These results suggest that mDia1 plays a distinct role in chemotaxis in T lymphocyte trafficking.
Asunto(s)
Actinas/metabolismo , Proteínas Portadoras/metabolismo , Movimiento Celular , Linfocitos T/citología , Animales , Recuento de Células , Movimiento Celular/efectos de los fármacos , Polaridad Celular/efectos de los fármacos , Quimiocinas/farmacología , Forminas , Inmunidad Celular/efectos de los fármacos , Tejido Linfoide/citología , Tejido Linfoide/efectos de los fármacos , Tejido Linfoide/inmunología , Ratones , Transducción de Señal/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
Ca(2+) signaling plays a central role in activity-dependent regulation of dendritic arborization, but key molecular mechanisms downstream of calcium elevation remain poorly understood. Here we show that the C-terminal region of the Ca(2+)/calmodulin-dependent protein kinase CLICK-III (CL3)/CaMKIgamma, a membrane-anchored CaMK, was uniquely modified by two sequential lipidification steps: prenylation followed by a kinase-activity-regulated palmitoylation. These modifications were essential for CL3 membrane anchoring and targeting into detergent-resistant lipid microdomains (or rafts) in the dendrites. We found that CL3 critically contributed to BDNF-stimulated dendritic growth. Raft insertion of CL3 specifically promoted dendritogenesis of cortical neurons by acting upstream of RacGEF STEF and Rac, both present in lipid rafts. Thus, CL3 may represent a key element in the Ca(2+)-dependent and lipid-raft-delineated switch that turns on extrinsic activity-regulated dendrite formation in developing cortical neurons.
Asunto(s)
Señalización del Calcio/fisiología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Diferenciación Celular/fisiología , Corteza Cerebral/embriología , Dendritas/metabolismo , Microdominios de Membrana/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Factor Neurotrófico Derivado del Encéfalo/farmacología , Células COS , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Chlorocebus aethiops , Dendritas/ultraestructura , Glicosilfosfatidilinositoles/metabolismo , Ratones , Ácido Palmítico/metabolismo , Prenilación de Proteína/fisiología , Procesamiento Proteico-Postraduccional/fisiología , Estructura Terciaria de Proteína/fisiología , Ratas , Ratas Sprague-Dawley , Proteínas de Unión al GTP rac/metabolismoRESUMEN
Despite the critical importance of Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaMK) II signaling in neuroplasticity, only a limited amount of work has so far been available regarding the presence and significance of another predominant CaMK subfamily, the CaMKI/CaMKIV family, in the central nervous system. We here searched for kinases with a core catalytic structure similar to CaMKI and CaMKIV. We isolated full-length cDNAs encoding three mouse CaMKI/CaMKIV-related kinases, CLICK-I (CL1)/doublecortin and CaM kinase-Like (DCAMKL)1, CLICK-II (CL2)/DCAMKL2, and CLICK-I,II-related (CLr)/DCAMKL3, the kinase domains of which had an intermediate homology not only to CaMKI/CaMKIV but also to CaMKII. Furthermore, CL1, CL2, and CLr were highly expressed in the central nervous system, in a neuron-specific fashion. CL1alpha and CL1beta were shorter isoforms of DCAMKL1, which lacked the doublecortin-like domain (Dx). In contrast, CL2alpha and CL2beta contained a full N-terminal Dx, whereas CLr only possessed a partial and dysfunctional Dx. Interestingly, despite a large similarity in the kinase domain, CL1/CL2/CLr had an impact on CRE-dependent gene expression distinct from that of the related CaMKI/CaMKIV and CaMKII. Although these were previously shown to activate Ca(2+)/cAMP-response element-binding protein (CREB)-dependent transcription, we here show that CL1 and CL2 were unable to significantly phosphorylate CREB Ser-133 and rather inhibited CRE-dependent gene expression by a dominant mechanism that bypassed CREB and was mediated by phosphorylated TORC2.