RESUMO
Although the p38 mitogen-activated protein kinases are active in many neuronal populations in the peripheral and central nervous systems, little is known about the physiological functions of p38 in postmitotic neurons. We report that p38 activity determines in vitro and in vivo the switch from noradrenergic to cholinergic neurotransmission that occurs in sympathetic neurons on exposure to the neuropoietic cytokines CNTF and LIF. This transdifferentiation serves as a model for the plastic mechanisms that enable mature neurons to change some of their central functions without passing through the cell cycle. We demonstrate that in postmitotic neurons, p38 and STAT pathways are concurrently activated by neuropoietic cytokine treatment for at least 12 h overlapping with changes in neurotransmitter marker gene expression. Inhibition of p38 blocks the upregulation of the nuclear matrix protein Satb2 and of cholinergic markers by CNTF without affecting STAT3 phosphorylation. Conversely, overexpression of p38α or ß in the absence of cytokines stimulates cholinergic marker expression. The neurotransmitter switch in vitro is impaired in neurons isolated from p38ß(-/-) mice. Consistent with these in vitro results, a substantial loss of cells expressing cholinergic properties is observed in vivo in the stellate ganglion of mature mice deficient in the p38ß isoform.
Assuntos
Acetilcolina/fisiologia , Transdiferenciação Celular/genética , Neurônios Colinérgicos/enzimologia , Proteína Quinase 11 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/genética , Gânglio Estrelado/enzimologia , Animais , Animais Recém-Nascidos , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Transdiferenciação Celular/efeitos dos fármacos , Células Cultivadas , Neurônios Colinérgicos/citologia , Neurônios Colinérgicos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 11 Ativada por Mitógeno/deficiência , Proteína Quinase 14 Ativada por Mitógeno/deficiência , Neurotransmissores/genética , Neurotransmissores/fisiologia , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição STAT/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Gânglio Estrelado/citologia , Gânglio Estrelado/crescimento & desenvolvimentoRESUMO
Sympathetic neurons can switch their neurotransmitter phenotype from noradrenergic to cholinergic on exposure to neuropoietic cytokines in vitro and in vivo. Here, we provide evidence that this transspecification is regulated by the chromatin architecture protein Satb2. Treatment with the neuropoietic cytokines ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor rapidly and strongly increases Satb2 transcript and protein levels in cultures of rat superior cervical ganglia neurons. Knockdown of endogenous Satb2 by short interfering RNA prevents the upregulation of choline acetyltransferase (Chat) and vesicular acetylcholine transporter (Vacht) by CNTF as well as the loss of norepinephrine transporter (Net). Conversely, overexpression of Satb2 in the noradrenergic sympathetic phenotype results in a marked increase of Chat and Vacht expression and reduced Net mRNA levels in the absence of neuropoietic cytokines. Chromatin immunoprecipitation analysis in primary sympathetic neurons reveals that Satb2 binds to matrix attachment regions (MARs) within the Chat locus. In vivo, in the rat stellate ganglion, Satb2 is expressed exclusively in sudomotor cholinergic neurons innervating the sweat glands and only after establishment of contact between neurons and target. These findings demonstrate a function of the MAR-binding protein Satb2 in growth factor-dependent neurotransmitter plasticity in postmitotic neurons.
Assuntos
Proteínas de Ligação à Região de Interação com a Matriz/fisiologia , Neurotransmissores/fisiologia , Gânglio Cervical Superior/fisiologia , Fatores de Transcrição/fisiologia , Fibras Adrenérgicas/fisiologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Técnicas de Silenciamento de Genes , Proteínas de Ligação à Região de Interação com a Matriz/genética , Mitose/genética , Mitose/fisiologia , Plasticidade Neuronal/genética , Plasticidade Neuronal/fisiologia , Neurotransmissores/genética , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/fisiologia , Ratos , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: The Notch signalling pathway is conserved in pre-bilaterian animals. In the Cnidarian Hydra it is involved in interstitial stem cell differentiation and in boundary formation during budding. Experimental evidence suggests that in Hydra Notch is activated by presenilin through proteolytic cleavage at the S3 site as in all animals. However, the endogenous ligand for HvNotch has not been described yet. RESULTS: We have cloned a cDNA from Hydra, which encodes a bona-fide Notch ligand with a conserved domain structure similar to that of Jagged-like Notch ligands from other animals. Hyjagged mRNA is undetectable in adult Hydra by in situ hybridisation but is strongly upregulated and easily visible at the border between bud and parent shortly before bud detachment. In contrast, HyJagged protein is found in all cell types of an adult hydra, where it localises to membranes and endosomes. Co-localisation experiments showed that it is present in the same cells as HvNotch, however not always in the same membrane structures. CONCLUSIONS: The putative Notch ligand HyJagged is conserved in Cnidarians. Together with HvNotch it may be involved in the formation of the parent-bud boundary in Hydra. Moreover, protein distribution of both, HvNotch receptor and HyJagged indicate a more widespread function for these two transmembrane proteins in the adult hydra, which may be regulated by additional factors, possibly involving endocytic pathways.