RESUMEN
Primary cultures of granule cells (GC) from rat cerebellar cortex were used to determine whether bioelectric activity, via a Ca(2+)/calmodulin-dependent kinase (CaMK) signaling cascade, modulates expression and exon selection in the inositol trisphosphate receptor type 1 (IP(3)R1). IP(3)R1 contains or lacks three exons (S1, S2, and S3) that are regulated in a regionally and temporally specific manner. The neuronal, or long, form of IP(3)R1 is distinguished from peripheral tissues by inclusion of the S2 exon. Although previous studies indicated that IP(3)R1 are undetectable in the cerebellar granular layer in vivo, receptor protein and mRNA are induced in cultured GC grown in medium supplemented with 25 mM KCl or NMDA, two trophic agents that promote long-term survival, compared with GC grown in 5 mM KCl. IP(3)R1 induction in response to 25 mM KCl or NMDA is attenuated by coaddition of voltage-sensitive calcium channel or NMDA receptor antagonists, respectively. Actinomycin D, CaMK, and calcineurin antagonists likewise suppress induction. Unlike the major variants of IP(3)R1 in Purkinje neurons, which lack S1 and S3, GC grown with trophic agents express mRNA containing these exons. Both neuronal types contain S2. Evidence obtained using mutant mice with Purkinje cell lesions, laser-microdissected GC neurons from slices, and explant cultures indicates that GC predominantly express the S1-containing variant of IP(3)R1 in vivo.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Cerebelo/metabolismo , Neuronas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal/fisiología , Animales , Animales Recién Nacidos , Western Blotting , Canales de Calcio/genética , Señalización del Calcio/fisiología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Técnica del Anticuerpo Fluorescente , Receptores de Inositol 1,4,5-Trifosfato , N-Metilaspartato/farmacología , Neuronas/efectos de los fármacos , Fenotipo , Cloruro de Potasio/farmacología , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cultured cerebellar granule neurons were used to examine the hypothesis that NMDA or KCl-dependent Ca2+ influx activates a signaling cascade mediating transcription, translation and assembly of neurofilaments. The data indicate that 25 mM KCl and 140 microM NMDA induce neurofilament protein, mRNA, and assembly in neurites, and these require Ca2+ entry through voltage-sensitive Ca2+ channels or NMDA receptors, respectively. The CaM kinase and transcriptional inhibitors, KN-62 and actinomycin D, respectively, attenuate the stimulatory effect of Ca2+. Granular neurons are a valuable model to explore the molecular basis for transcriptional regulation of neurofilament gene expression by neuronal activity.