RESUMEN
Binding of the neurotrophin brain-derived neurotrophic factor (BDNF) to the TrkB receptor is a major survival mechanism during embryonic development. In the aged brain, however, BDNF levels are low, suggesting that if TrkB is to play a role in survival at this stage additional mechanisms must have developed. We here show that TrkB activity is most robust in the hippocampus of 21-d-old BDNF-knockout mice as well as in old, wild-type, and BDNF heterozygous animals. Moreover, robust TrkB activity is evident in old but not young hippocampal neurons differentiating in vitro in the absence of any exogenous neurotrophin and also in neurons from BDNF -/- embryos. Age-associated increase in TrkB activity correlated with a mild yet progressive loss of cholesterol. This, in turn, correlated with increased expression of the cholesterol catabolic enzyme cholesterol 24-hydroxylase. Direct cause-effect, cholesterol loss-high TrkB activity was demonstrated by pharmacological means and by manipulating the levels of cholesterol 24-hydroxylase. Because reduced levels of cholesterol and increased expression of choleseterol-24-hydroxylase were also observed in the hippocampus of aged mice, changes in cellular cholesterol content may be used to modulate receptor activity strength in vivo, autonomously or as a way to complement the natural decay of neurotrophin production.
Asunto(s)
Senescencia Celular , Colesterol/deficiencia , Hipocampo/citología , Neuronas/citología , Neuronas/enzimología , Receptor trkB/metabolismo , Transducción de Señal , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Factor Neurotrófico Derivado del Encéfalo/farmacología , Diferenciación Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Células Cultivadas , Senescencia Celular/efectos de los fármacos , Colesterol 24-Hidroxilasa , Detergentes/farmacología , Activación Enzimática/efectos de los fármacos , Hipocampo/enzimología , Ligandos , Ratones , Neuronas/efectos de los fármacos , Ratas , Transducción de Señal/efectos de los fármacos , Esteroide Hidroxilasas/metabolismoRESUMEN
Appropriate neurogenesis and patterning of the forebrain requires the transcription factor Pax6, yet it is largely unknown how Pax6 exerts its effects at the molecular level. To characterize Pax6-mediated regulation of gene expression during murine forebrain neurogenesis, we performed microarray analysis with tissue from the dorsal Pax6-dependent telencephalon and the ventral Pax6-negative telencephalon at the onset of neurogenesis (E12) and at mid-neurogenesis (E15) in wild-type and Pax6-deficient mutant littermates. In the Pax6-deficient cortex the expression levels of various transcription factors involved in neurogenesis (like Satb2, Nfia, AP-2gamma, NeuroD6, Ngn2, Tbr2, Bhlhb5) and the retinoic acid signalling molecule Rlbp1 were reduced. Regulation by Pax6 could be confirmed upon electroporation of a Pax6- and a dominant-negative Pax6-containing vector into embryonic cortex. Taken together, our data reveal novel insights into the molecular pathways regulated by Pax6 during cortical neurogenesis. Most intriguingly, this analysis revealed time- and region-specific differences in Pax6-mediated transcription, explaining the specific function of Pax6 at early and later stages of neurogenesis.
Asunto(s)
Diferenciación Celular/fisiología , Proteínas del Ojo/genética , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de Homeodominio/genética , Neuronas/metabolismo , Factores de Transcripción Paired Box/genética , Proteínas Represoras/genética , Células Madre/metabolismo , Telencéfalo/embriología , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Regulación hacia Abajo/genética , Ratones , Ratones Noqueados , Mutación/genética , Neuronas/citología , Análisis de Secuencia por Matrices de Oligonucleótidos , Factor de Transcripción PAX6 , Transducción de Señal/genética , Células Madre/citología , Telencéfalo/citología , Telencéfalo/metabolismo , Factores de Transcripción/genética , Tretinoina/metabolismoRESUMEN
In the present study, we investigated the involvement of rhombomere 1 patterning proteins in the regulation of the major noradrenergic centre of the brain, the locus coeruleus. Primary cultures of rat embryonic day 13.5 locus coeruleus were treated with fibroblast growth factor-8, noggin and members of the bone morphogenetic and Wnt protein families. We show that bone morphogenetic proteins 2, 5 and 7 increase and noggin decreases the number of tyrosine hydroxylase-positive locus coeruleus neurons. Interestingly, from all Wnts expressed in the first rhombomere by embryonic day 12.5 in the mice, we only found expression of wnt5a mRNA in the vicinity of the locus coeruleus. In agreement with this finding, from all Wnts studied in vitro, only Wnt5a increased the number of tyrosine hydroxylase-positive neurons in locus coeruleus cultures. Finally, we also found that fibroblast growth factor-8 increased the number of tyrosine hydroxylase-positive cells in locus coeruleus cultures. Neither of the identified factors affected the survival of tyrosine hydroxylase-positive locus coeruleus noradrenergic neurons or the proliferation of their progenitors or neurogenesis. Instead, our results suggest that these patterning signals of rhombomere 1 may work to promote the differentiation of noradrenergic progenitors at later stages of development.
Asunto(s)
Proteínas Morfogenéticas Óseas/fisiología , Desarrollo Embrionario/fisiología , Factor 8 de Crecimiento de Fibroblastos/fisiología , Locus Coeruleus/embriología , Neuronas/fisiología , Proteínas Wnt/fisiología , Animales , Apoptosis , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Separación Celular , Medios de Cultivo Condicionados , Femenino , Etiquetado Corte-Fin in Situ , Locus Coeruleus/citología , Neuronas/citología , Embarazo , Ratas , Ratas Sprague-DawleyRESUMEN
The role of glial cell-line derived neurotrophic factor (GDNF) and neurotrophins in the development of locus coeruleus noradrenergic neurons was evaluated. We found that two neurotrophic factors previously reported to prevent the degeneration of lesioned adult central noradrenergic neurons, GDNF and neurotrophin 3 (NT3), do not play significant roles in the prenatal development of locus coeruleus noradrenergic neurons, as demonstrated by: (1) the lack of alterations in double Gdnf/Nt3 null mutant mice; and (2) the lack of survival-promoting effects of GDNF and/or NT3 in rat E13.5 primary cultures. In contrast, null mutant mice for TrkB, the tyrosine kinase receptor for brain-derived neurotrophic factor and neurotrophin 4, displayed a clear loss of locus coeruleus noradrenergic neurons. In accordance with this, treatment of rat E13.5 primary cultures with TrkB ligands prevented the early loss of noradrenergic neurons and maintained their survival for up to 6 days in vitro. Moreover, an additional 5-10-fold increase in the number of tyrosine hydroxylase positive noradrenergic neurons was detected after 12 hours in culture. This second effect of TrkB ligands involved neither proliferation nor survival, because the number of BrdU- or TUNEL-positive noradrenergic neurons did not change and the effect was elicited by delayed administration of either factor. Because TrkB ligands increased the number of tyrosine hydroxylase-positive cells expressing Phox2a, a paired homeodomain protein required for the development of locus coeruleus noradrenergic neurons, but did not affect the number of Phox2a-positive tyrosine hydroxylase-negative cells, our results suggest that the second effect of TrkB ligands may involve promoting or inducing a noradrenergic phenotype. In summary, our findings suggest that, unlike NT3 and GDNF, TrkB ligands are required and sufficient to promote the development of central noradrenergic neurons.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Locus Coeruleus/embriología , Glicoproteínas de Membrana/metabolismo , Neuronas/metabolismo , Proteínas Tirosina Quinasas , Animales , Factor Neurotrófico Derivado de la Línea Celular Glial , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial , Ligandos , Ratones , Mutación , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-ret , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor trkB/metabolismo , Receptor trkC/metabolismoRESUMEN
Noradrenergic neurons of the locus coeruleus (LC) express the receptor tyrosine kinase c-ret, which binds ligands of the glial cell line-derived neurotrophic factor (GDNF) family. In the present study, we evaluated the function of neurturin (NTN), a GDNF family ligand whose function on LC neurons is unknown. Interestingly, we found that tyrosine hydroxylase (TH)-positive neurons in the LC express both GFRalpha1 and 2 receptors in a developmentally regulated fashion, suggesting a function for their preferred ligands: GDNF and NTN, respectively. Moreover, our results show that NTN mRNA expression is developmentally down-regulated in the LC and peaks in the postnatal hippocampus and cerebral cortex, during the target innervation period. In order to examine the function of NTN, we next performed LC primary cultures, and found that neither GDNF nor NTN promoted the survival of TH-positive neurons. However, both factors efficiently induced neurite outgrowth in noradrenergic neurons (147% and 149% over controls, respectively). Similarly, grafting of fibroblast cell lines engineered to express high levels of NTN did not prevent the loss of LC noradrenergic neurons in a 6-hydroxydopamine (6-OHDA) lesion model, but induced the sprouting of TH-positive cells. Thus our findings show that NTN does not promote the survival of LC noradrenergic neurons, but induces neurite outgrowth in developing noradrenergic neurons in vitro and in a model of neurodegeneration in vivo. These data, combined with data in the literature, suggest that GDNF family ligands are able to independently regulate neuronal survival and/or neuritogenesis.
Asunto(s)
Envejecimiento/fisiología , Encéfalo/embriología , Encéfalo/fisiología , Proteínas de Drosophila , Factores de Crecimiento Nervioso/fisiología , Neuritas/fisiología , Neuronas/fisiología , Norepinefrina/fisiología , Animales , Encéfalo/citología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Embrión de Mamíferos/fisiología , Factor Neurotrófico Derivado de la Línea Celular Glial , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial , Locus Coeruleus/citología , Locus Coeruleus/embriología , Locus Coeruleus/enzimología , Locus Coeruleus/metabolismo , Masculino , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/farmacología , Proteínas del Tejido Nervioso/farmacología , Neuritas/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neurturina , Oxidopamina/farmacología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-ret , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas F344 , Ratas Sprague-Dawley , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Superficie Celular/metabolismoRESUMEN
The locus coeruleus (LC) is a major target of several neurodegenerative disorders, including Parkinson's and Alzheimer's diseases. However, very little is known of the trophic requirements of LC neurons. In the present work, we have studied the biological activity of neurotrophic factors from different families in E15 primary cultures of LC neurons. In agreement with previous results, neurotrophin-3 (NT-3) and also glial cell line- derived neurotrophic factor (GDNF) increased the number of embryonic LC noradrenergic neurons in the presence of serum. In serum-free conditions, none of the factors tested, including NT-3, GDNF, neurturin, basic fibroblast growth factor (bFGF), or bone morphogenetic protein-2 (BMP-2), promoted the survival of tyrosine hydroxylase (TH)-immunoreactive neurons at 6 days in vitro. However, when BMP-2 was coadministered with any of these factors the number of LC TH-positive neurons increased twofold. Similar results were obtained by cotreatment of LC neurons with forskolin and NT-3, bFGF, or BMP-2. The strongest effect (a fourfold increase in the number of TH-positive cells) was induced by cotreatment with forskolin, BMP-2, and GDNF. Thus, our results show that LC neurons require multiple factors for their survival and development, and suggest that activation of LC neurons by bone morphogenetic proteins and cAMP plays a decisive role in conferring noradrenergic neuron responsiveness to several trophic factors.