RESUMEN
Neurturin (NTN) a structural and functional relative of glial cell line-derived neurotrophic factor, was originally identified based on its ability to support the survival of sympathetic neurons in culture. Similar to glial cell line-derived neurotrophic factor (GDNF), Neurturin has been shown to bind to a high affinity glycosylphosphatidylinositol (GPI)-linked receptor (GFRalpha2) and induce phosphorylation of the tyrosine kinase receptor Ret, resulting in the activation of the mitogen activated protein kinase (MAPK) signalling pathway. A panel of six novel murine monoclonal antibodies (MAbs) specific to human Neurturin has been developed and characterized. Four of the MAbs tested inhibit, to varying degrees, binding of NTN to the GPI-linked GFRalpha2 receptor. Three MAbs cross-react with the murine homolog. These antibodies have been shown to be useful reagents for Western blotting, immunohistochemistry, and also for the development of a sensitive, quantitative enzyme-linked immunosorbent assay (ELISA) for human NTN. Novel, specific MAbs with varying epitope specificities and blocking activity will be valuable tools for both the in vitro and in vivo characterization of NTN and its relationship to the GFRalpha2 and Ret receptors.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos/inmunología , Factores de Crecimiento Nervioso/inmunología , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Afinidad de Anticuerpos/inmunología , Unión Competitiva/inmunología , Western Blotting , Supervivencia Celular/fisiología , Cricetinae , Reacciones Cruzadas/inmunología , Inhibidores Enzimáticos/inmunología , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial , Humanos , Inmunización , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Proteínas del Tejido Nervioso/inmunología , Neuritas/fisiología , Neuroblastoma/inmunología , Neuroblastoma/patología , Neuroblastoma/ultraestructura , Neurturina , Ratas , Sustancia Negra/citología , Sustancia Negra/inmunología , Ganglio Cervical Superior/inmunologíaRESUMEN
Neurturin (NRTN) and glial cell line-derived neurotrophic factor (GDNF) are members of a family of trophic factors with similar actions in vitro on certain neuronal classes. Retrograde transport of GDNF and NRTN was compared in peripheral sensory, sympathetic, and motor neurons to determine whether in vivo these factors are transported selectively by different neuronal populations. After sciatic nerve injections, NRTN was transported by sensory neurons of the dorsal root ganglion (DRG). Competition studies demonstrated only limited cross-competition between NRTN and GDNF, indicating selective receptor-mediated transport of these factors. By using immunohistochemistry, we identified two populations of NRTN-transporting DRG neurons: a major population of small, RET-positive, IB4-positive, non-TrkA-expressing neurons that also show the ability to transport GDNF and a minor population of calretinin-expressing neurons that fail to transport GDNF. Spinal motor neurons in the adult showed relatively less ability to transport NRTN than to transport GDNF, although NRTN prevented the cell death of neonatal motor neurons in a manner very similar to GDNF (Yan et al., 1995) and persephin (PSPN) (Milbrandt et al., 1998). Last, NRTN, like GDNF, was not transported to sympathetic neurons of the adult superior cervical ganglion (SCG) after injection into the anterior eye chamber. These data reveal a high degree of functional selectivity of GDNF family receptor-alpha (GFRalpha) coreceptor subtypes for NRTN and GDNF in vivo.
Asunto(s)
Proteínas de Drosophila , Ganglios Espinales/fisiología , Neuronas Motoras/fisiología , Factores de Crecimiento Nervioso/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas Aferentes/fisiología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Tirosina Quinasas Receptoras/fisiología , Nervio Ciático/fisiología , Transducción de Señal/fisiología , Médula Espinal/fisiología , Animales , Animales Recién Nacidos , Transporte Axonal , Transporte Biológico , Tamaño de la Célula , Ganglios Espinales/citología , Factor Neurotrófico Derivado de la Línea Celular Glial , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial , Inmunohistoquímica , Radioisótopos de Yodo , Masculino , Neuronas Motoras/citología , Neuronas Aferentes/citología , Neurturina , Proteínas Proto-Oncogénicas c-ret , Ratas , Ratas Sprague-Dawley , Médula Espinal/citologíaRESUMEN
Neurturin (NTN) and glial cell line-derived neurotrophic factor (GDNF) are the first two members of the GDNF family (GF) of neurotrophic factors. These two proteins are potent survival factors for several populations of central and peripheral neurons in mature and developing rodents. The receptor for these factors is a multicomponent complex that includes the RET (rearranged during transfection) tyrosine kinase receptor and one of two glycosyl phosphatidylinositol (GPI)-linked ligand-binding components called GDNF family receptor alphas (GFRalpha-1 and GFRalpha-2). We have used in situ hybridization to study the mRNA expression of NTN, GDNF, RET, GFRalpha-1, and GFRalpha-2 in the central nervous system (CNS) of adult mice. GF receptors are expressed in several areas in which neuronal populations known to respond to NTN and GDNF are located, including the ventral horn of the spinal cord and the compacta region of the substantia nigra. In addition, we have demonstrated receptor expression in other areas of the brain including the thalamus and hypothalamus. Neurons in these areas express GF receptors, and therefore, may respond to NTN or GDNF. NTN and GDNF are expressed in targets of neurons that express GF receptors. The pattern of GF factor and receptor expression in the adult brain suggests a role for these factors in maintaining neuronal circuits in the mature CNS.
Asunto(s)
Proteínas de Drosophila , Ratones Endogámicos ICR/fisiología , Factores de Crecimiento Nervioso/genética , Proteínas del Tejido Nervioso/genética , Fármacos Neuroprotectores/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Factores de Edad , Animales , Química Encefálica/fisiología , Tronco Encefálico/química , Tronco Encefálico/citología , Cerebelo/química , Cerebelo/citología , Femenino , Expresión Génica/fisiología , Factor Neurotrófico Derivado de la Línea Celular Glial , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial , Hipotálamo/química , Hipotálamo/citología , Hibridación in Situ , Mesencéfalo/química , Mesencéfalo/citología , Ratones , Neurturina , Bulbo Olfatorio/química , Bulbo Olfatorio/citología , Prosencéfalo/química , Prosencéfalo/citología , Proteínas Proto-Oncogénicas c-ret , ARN Mensajero/análisis , Médula Espinal/química , Médula Espinal/citología , Tálamo/química , Tálamo/citologíaRESUMEN
Signaling through the c-Ret tyrosine kinase and the endothelin B receptor pathways is known to be critical for development of the enteric nervous system. To clarify the role of these receptors in enteric nervous system development, the effect of ligands for these receptors was examined on rat enteric neuron precursors in fully defined medium in primary culture. In this culture system, dividing Ret-positive cells differentiate, cluster into ganglia containing neurons and enteric glia, and create extensive networks reminiscent of the enteric plexus established in vivo. Glial cell-line-derived neurotrophic factor (GDNF) and neurturin both potently support survival and proliferation of enteric neuron precursors in this system. Addition of either neurturin or GDNF to these cultures increased the number of both neurons and enteric glia. Persephin, a third GDNF family member, shares many properties with neurturin and GDNF in the central nervous system and in kidney development. By contrast, persephin does not promote enteric neuron precursor proliferation or survival in these cultures. Endothelin-3 also does not increase the number of enteric neurons or glia in these cultures.
Asunto(s)
Proteínas de Drosophila , Sistema Nervioso Entérico/embriología , Factores de Crecimiento Nervioso/farmacología , Proteínas del Tejido Nervioso/farmacología , Células Madre/efectos de los fármacos , Animales , Muerte Celular/efectos de los fármacos , División Celular , Supervivencia Celular , Células Cultivadas , Medio de Cultivo Libre de Suero , Endotelina-3/metabolismo , Sistema Nervioso Entérico/citología , Sistema Nervioso Entérico/efectos de los fármacos , Factor Neurotrófico Derivado de la Línea Celular Glial , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial , Cresta Neural/citología , Cresta Neural/efectos de los fármacos , Neuroglía/citología , Neuroglía/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Neurturina , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-ret , Ratas , Ratas Sprague-Dawley , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de SeñalRESUMEN
Herein we report the generation of mice lacking the ubiquitously expressed Janus kinase, Jak1. Jak1-/- mice are runted at birth, fail to nurse, and die perinatally. Although Jak1-/- cells are responsive to many cytokines, they fail to manifest biologic responses to cytokines that bind to three distinct families of cytokine receptors. These include all class II cytokine receptors, cytokine receptors that utilize the gamma(c) subunit for signaling, and the family of cytokine receptors that depend on the gp130 subunit for signaling. Our results thus demonstrate that Jak1 plays an essential and nonredundant role in promoting biologic responses induced by a select subset of cytokine receptors, including those in which Jak utilization was thought to be nonspecific.
Asunto(s)
Antígenos CD/fisiología , Citocinas/farmacología , Glicoproteínas de Membrana/fisiología , Proteínas Tirosina Quinasas/fisiología , Receptores de Citocinas/fisiología , Animales , Peso al Nacer , Células Cultivadas , Receptor gp130 de Citocinas , Proteínas de Unión al ADN/metabolismo , Fibroblastos , Antígenos de Histocompatibilidad Clase I/análisis , Janus Quinasa 1 , Leucopoyesis , Ligandos , Macrófagos/citología , Ratones , Ratones Noqueados , Miocardio/citología , Neuronas Aferentes/citología , Tamaño de los Órganos , Proteínas Tirosina Quinasas/genética , Factor de Transcripción STAT3 , Transducción de Señal/fisiología , Bazo/citología , Bazo/inmunología , Timo/citología , Timo/patología , Transactivadores/metabolismoRESUMEN
A novel neurotrophic factor named Persephin that is approximately 40% identical to glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) has been identified using degenerate PCR. Persephin, like GDNF and NTN, promotes the survival of ventral midbrain dopaminergic neurons in culture and prevents their degeneration after 6-hydroxydopamine treatment in vivo. Persephin also supports the survival of motor neurons in culture and in vivo after sciatic nerve axotomy and, like GDNF, promotes ureteric bud branching. However, in contrast to GDNF and NTN, persephin does not support any of the peripheral neurons that were examined. Fibroblasts transfected with Ret and one of the coreceptors GFRalpha-1 or GFRalpha-2 do not respond to persephin, suggesting that persephin utilizes additional, or different, receptor components than GDNF and NTN.
Asunto(s)
Neuronas Motoras/química , Factores de Crecimiento Nervioso/genética , Proteínas del Tejido Nervioso/genética , Fármacos Neuroprotectores/metabolismo , Animales , Muerte Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Ganglios Espinales/citología , Regulación del Desarrollo de la Expresión Génica , Factor Neurotrófico Derivado de la Línea Celular Glial , Humanos , Mesencéfalo/citología , Ratones , Datos de Secuencia Molecular , Neuronas Motoras/fisiología , Neurturina , Ganglio Nudoso/citología , Reacción en Cadena de la Polimerasa/métodos , Ratas , Ratas Sprague-Dawley , Receptores de Factores de Crecimiento/fisiología , Receptores de Ácido Retinoico/fisiología , Homología de Secuencia de Aminoácido , Transducción de Señal/fisiología , Ganglio Cervical Superior/citología , Transfección , Ganglio del Trigémino/citología , Uréter/citología , Uréter/embriologíaRESUMEN
The glial cell line-derived neurotrophic factor (GDNF) ligands (GDNF, Neurturin [NTN], and Persephin [PSP]) signal through a multicomponent receptor system composed of a high-affinity binding component (GFRalpha1-GFRalpha4) and a common signaling component (RET). Here, we report the identification of Artemin, a novel member of the GDNF family, and demonstrate that it is the ligand for the former orphan receptor GFRalpha3-RET. Artemin is a survival factor for sensory and sympathetic neurons in culture, and its expression pattern suggests that it also influences these neurons in vivo. Artemin can also activate the GFRalpha1-RET complex and supports the survival of dopaminergic midbrain neurons in culture, indicating that like GDNF (GFRalpha1-RET) and NTN (GFRalpha2-RET), Artemin has a preferred receptor (GFRalpha3-RET) but that alternative receptor interactions also occur.
Asunto(s)
Proteínas de Drosophila , Mesencéfalo/fisiología , Factores de Crecimiento Nervioso/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuronas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Tirosina Quinasas Receptoras/fisiología , Transducción de Señal/fisiología , Ganglio Cervical Superior/fisiología , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Sitios de Unión , Supervivencia Celular , Células Cultivadas , Clonación Molecular , Embrión de Mamíferos , Factor Neurotrófico Derivado de la Línea Celular Glial , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial , Humanos , Ligandos , Mesencéfalo/citología , Ratones , Modelos Químicos , Datos de Secuencia Molecular , Factores de Crecimiento Nervioso/química , Proteínas del Tejido Nervioso/química , Neuronas/citología , Proteínas Proto-Oncogénicas c-ret , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Ganglio Cervical Superior/citologíaRESUMEN
Neurturin (NTN) is a neurotrophic factor that shares homology with glial cell line-derived neurotrophic factor (GDNF). Recently, a receptor complex has been identified for GDNF that includes the Ret tyrosine kinase receptor and a glycosylphosphatidylinositol-linked protein termed "GDNFRalpha." However, differences in the phenotype of Ret and GDNF knockout animals suggest that Ret has at least one additional ligand. In this report, we demonstrate that NTN induces Ret phosphorylation in primary cultures of rat superior cervical ganglion (SCG) neurons. NTN also caused Ret phosphorylation in fibroblasts that were transfected stably with Ret and GDNFRalpha but not in cells expressing Ret alone. A glycosylphosphatidylinositol-linked protein also was important for NTN and GDNF signaling in SCG neurons; phosphatidylinositol-specific phospholipase C treatment of SCG cultures reduced the ability of NTN to phosphorylate Ret and the ability of NTN or GDNF to activate the mitogen-activated protein kinase pathway. NTN and GDNF also caused sustained activation of Ret and the mitogen-activated protein kinase pathway in SCG neurons. Finally, both NTN and GDNF activated the phosphatidylinositol 3-kinase pathway in SCG neurons, which may be important for the ability of NTN and GDNF to promote neuronal survival. These data indicate that NTN is a physiologically relevant ligand for the Ret receptor and suggest that NTN may have a critical role in the development of many neuronal populations.
Asunto(s)
Proteínas de Drosophila , Factores de Crecimiento Nervioso/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuronas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptores de Superficie Celular/fisiología , Sistema Nervioso Simpático/fisiología , Animales , Secuencia de Bases , Células Cultivadas , Factor Neurotrófico Derivado de la Línea Celular Glial , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial , Datos de Secuencia Molecular , Neurturina , Proteínas Proto-Oncogénicas c-ret , Ratas , Transducción de Señal , Sistema Nervioso Simpático/citologíaRESUMEN
Sympathetic neurons undergo programmed cell death (PCD) when deprived of NGF. We used an inhibitor to examine the function of interleukin-1 beta-converting enzyme (ICE) family proteases during sympathetic neuronal death and to assess the metabolic and genetic status of neurons saved by such inhibition. Bocaspartyl(OMe)-fluoromethylketone (BAF), a cell-permeable inhibitor of the ICE family of cysteine proteases, inhibited ICE and CPP32 (IC50 approximately 4 microM) in vitro and blocked Fas-mediated apoptosis in thymocytes (EC50 approximately 10 microM). At similar concentrations, BAF also blocked the NGF deprivation-induced death of rat sympathetic neurons in culture. Compared to NGF-maintained neurons, BAF-saved neurons had markedly smaller somas and maintained only basal levels of protein synthesis; readdition of NGF restored growth and metabolism. Although BAF blocked apoptosis in sympathetic neurons, it did not prevent the fall in protein synthesis or the increase in the expression of c-jun, c-fos, and other mRNAs that occur during neuronal PCD, implying that the ICE-family proteases function downstream of these events during PCD.NGF and BAF rescued sympathetic neurons with an identical time course, suggesting that NGF, in addition to inhibiting metabolic and genetic events associated with neuronal PCD, can act posttranslationally to abort apoptosis at a time point indistinguishable from the activation of cysteine proteases. Both poly-(ADP ribose) polymerase and pro-ICE and Ced-3 homolog-1 (ICH-1) appear to be cleaved in a BAF-inhibitable manner, although the majority of pro-CPP32 appears unchanged, suggesting that ICH-1 is activated during neuronal PCD. Potential implications of these findings for anti-apoptotic therapies are discussed.
Asunto(s)
Clorometilcetonas de Aminoácidos/farmacología , Apoptosis , Caspasas , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Factores de Crecimiento Nervioso/fisiología , Neuronas/fisiología , Animales , Apoptosis/efectos de los fármacos , Caspasa 1 , Caspasa 2 , Caspasa 3 , Núcleo Celular/metabolismo , Fragmentación del ADN/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Genes fos , Genes jun , Ratones , Ratones Endogámicos C57BL , Factores de Crecimiento Nervioso/farmacología , Neuronas/citología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas , Ganglio Cervical Superior/citología , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Factores de Tiempo , Receptor fas/fisiologíaRESUMEN
The normal development of the vertebrate nervous system entails the death of 30-70% of the neurons originally generated in most neuronal populations. This naturally occurring cell death is regulated by specific neurotrophic factors that promote neuronal survival and which are produced in limiting quantities by target cells, glial cells and neurons. These factors are also of potential utility as therapeutic agents for neurodegenerative diseases. Here we describe the purification and cloning of a new neurotrophic factor, identified on the basis of its ability to support the survival of sympathetic neurons in culture. This factor, neurturin, is structurally related to glial-cell-line-derived neurotrophic factor (GDNF). These factors can each activate the MAP kinase signalling pathway in cultured sympathetic neurons and support the survival of sympathetic neurons, as well as of sensory neurons of the nodose and dorsal root ganglia. Thus, neurturin and GDNF together now define a new family of neurotrophic factors.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos , Factores de Crecimiento Nervioso/aislamiento & purificación , Proteínas del Tejido Nervioso/química , Células 3T3 , Secuencia de Aminoácidos , Animales , Células CHO , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Supervivencia Celular/fisiología , Células Cultivadas , Cromatografía de Afinidad , Clonación Molecular , Cricetinae , Medios de Cultivo Condicionados , ADN Complementario , Activación Enzimática , Ganglios Espinales/citología , Factor Neurotrófico Derivado de la Línea Celular Glial , Humanos , Ratones , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Datos de Secuencia Molecular , Factores de Crecimiento Nervioso/química , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/fisiología , Neuroglía/fisiología , Neuronas/fisiología , Neurturina , Ganglio Nudoso/citología , Ratas , Ratas Sprague-Dawley , Homología de Secuencia de Aminoácido , Ganglio Cervical Superior/citologíaRESUMEN
Rat sympathetic neurons undergo programmed cell death (PCD) in vitro and in vivo when they are deprived of nerve growth factor (NGF). Chronic depolarization of these neurons in cell culture with elevated concentrations of extracellular potassium ([K+]o) prevents this death. The effect of prolonged depolarization on neuronal survival is thought to be mediated by a rise of intracellular calcium concentration ([Ca2+]i) caused by Ca2+ influx through voltage-gated channels. In this report we investigate the effects of chronic treatment of rat sympathetic neurons with thapsigargin, an inhibitor of intracellular Ca2+ sequestration. In medium containing a normal concentration of extracellular Ca2+ ([Ca2+]o), thapsigargin caused a sustained rise of intracellular Ca2+ concentration and partially blocked death of NGF-deprived cells. Elevating [Ca2+]o in the presence of thapsigargin further increased [Ca2+]i, suggesting that the sustained rise of [Ca2+]i was caused by a thapsigargin-induced Ca2+ influx. This treatment potentiated the effect of thapsigargin on survival. The dihydropyridine Ca2+ channel antagonist, nifedipine, blocked both a sustained elevation of [Ca2+]i and enhanced survival caused by depolarization with elevated [K+]o, suggesting that these effects are mediated by Ca2+ influx through L-type channels. Nifedipine did not block the sustained rise of [Ca2+]i or enhanced survival caused by thapsigargin treatment, indicating that these effects were not mediated by influx of Ca2+ through L-type channels. These results provide additional evidence that increased [Ca2+]i can suppress neuronal PCD and identify a novel method for chronically raising neuronal [Ca2+]i for investigation of this and other Ca(2+)-dependent phenomena.
Asunto(s)
División Celular/efectos de los fármacos , Terpenos , Fibras Adrenérgicas/patología , Fibras Adrenérgicas/ultraestructura , Animales , Calcio , Muerte Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Factores de Crecimiento Nervioso/fisiología , Neuronas , Potasio , Ratas , Ratas Sprague-Dawley , TapsigarginaRESUMEN
Embryonic rat sympathetic neurons undergo programmed cell death upon NGF deprivation. We show that during postnatal development, these neurons acquire the ability to be supported in vitro by LIF and CNTF as well as NGF. LIF and CNTF do not promote the long-term survival of embryonic day 21 sympathetic neurons in vitro. However, after 5 days of culture in the presence of NGF, the majority of embryonic day 21 sympathetic neurons can be supported by either of these factors. Furthermore, postnatal day 6 sympathetic neurons can be immediately supported by LIF and CNTF, indicating that acquisition of survival responsiveness occurs in vivo as well as in vitro. During this period, neuronal expression of LIF and CNTF receptor mRNAs remains constant, suggesting that sympathetic neurons alter their responsiveness to LIF and CNTF by allowing additional intracellular signaling pathways to promote survival.
Asunto(s)
Inhibidores de Crecimiento/farmacología , Interleucina-6 , Linfocinas/farmacología , Proteínas del Tejido Nervioso/farmacología , Neuronas/citología , Sistema Nervioso Simpático/citología , Animales , Muerte Celular , Supervivencia Celular , Células Cultivadas , Factor Neurotrófico Ciliar , Expresión Génica , Inhibidores de Crecimiento/genética , Factor Inhibidor de Leucemia , Linfocinas/genética , Proteínas del Tejido Nervioso/genética , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Sistema Nervioso Simpático/embriología , Sistema Nervioso Simpático/crecimiento & desarrolloRESUMEN
Young sympathetic neurons die when deprived of nerve growth factor (NGF). Under such circumstances, cell death is appropriate to the developing nervous system and requires RNA and protein synthesis. We have hypothesized the existence of an endogenous death program within neurons that is suppressed by trophic factors. The extent and timing of required changes in the synthetic events that comprise the death program are unknown. In an effort to characterize the biochemical events that mediate the death program further, we performed several experiments on embryonic rat sympathetic neurons in vitro. The death program was blocked with cycloheximide when total protein synthesis was inhibited > or = 80%. When protein synthesis was inhibited within 22 +/- 4 h of NGF deprivation, death was prevented in half the neurons. Hence, we define the commitment point for protein synthesis to be 22 +/- 4 h. Analogously, the commitment point for RNA synthesis was 26 +/- 4 h and that for NGF rescue, 24 +/- 4 h. We tested the ability of a wide variety of chemicals to interfere with the death program. Most compounds tested were unable to prevent neuronal death. Some treatments, however, did save NGF-deprived neurons and were subsequently characterized. These included ultraviolet light and agents that raise intracellular concentrations of cAMP. Finally, we looked for the neuronal expression in vitro and in vivo of genes that have been associated with programmed death in other cell types, including TRPM-2/SGP-2, polyubiquitin, TGF beta-1, c-fos, and c-myc. None of these genes showed significant activation associated with neuronal death.
Asunto(s)
Apoptosis/fisiología , Factores de Crecimiento Nervioso/deficiencia , Neuronas/citología , Sistema Nervioso Simpático/citología , Adenilato Quinasa/metabolismo , Animales , Apoptosis/genética , Apoptosis/efectos de la radiación , Northern Blotting , Células Cultivadas , AMP Cíclico/metabolismo , Regulación de la Expresión Génica/fisiología , Metionina/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Neuronas/efectos de la radiación , Ratas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Sistema Nervioso Simpático/efectos de la radiación , Rayos UltravioletaRESUMEN
Data are presented for 16 enzymes from 8 metabolic systems in cell cultures consisting of approximately 95% astrocytes and 5% oligodendrocytes. Nine of these enzymes were also measured in cultures of oligodendrocytes, Schwann cells, and neurons prepared from both cerebral cortex and superior cervical ganglia. Activities, in mature astrocyte cultures, expressed as percentage of their activity in brain, ranged from 9% for glycerol-3-phosphate dehydrogenase to over 300% for glucose-6-phosphate dehydrogenase. Creatine phosphokinase activity in astrocytes was about the same as in brain, half as high in oligodendrocytes, but 7% or less of the brain level in Schwann cells and superior cervical ganglion neurons and only 16% of brain in cortical neurons. Three enzymes which generate NADPH, the dehydrogenases for glucose-6-phosphate and 6-phosphogluconate, and the NADP-requiring isocitrate dehydrogenase, were present in astrocytes at levels at least twice that of brain. Oligodendrocytes had enzyme levels only 30% to 70% of those of astrocytes. Schwann cells had much higher lactate dehydrogenase and 6-phosphogluconate dehydrogenase activities than oligodendrocytes, but showed a remarkable similarity in enzyme pattern to those of cortical and superior cervical ganglion neurons.
Asunto(s)
Corteza Cerebral/enzimología , Ganglios Simpáticos/enzimología , Neuronas/enzimología , Animales , Astrocitos/enzimología , Células Cultivadas , Corteza Cerebral/citología , Ganglios Simpáticos/citología , Oligodendroglía/enzimología , Ratas , Ratas Endogámicas , Células de Schwann/enzimologíaRESUMEN
Nerve growth factor (NGF) is known to be essential for survival and maintenance of sympathetic ganglia and of embryonic sensory ganglia of neural crest origin. The present study examined the physiological and pharmacological roles of NGF in the postnatal development of sensory neurons in the dorsal root ganglion (DRG). In contrast to what is generally stated in the literature, administration of NGF antiserum to newborn rats for a period of 7 days resulted in a significant (approximately 20%) reduction of neuronal number in the lumbar DRG. Size spectrum analysis of surviving neurons revealed a shift toward larger sizes, presumably due to a preferential loss of small cells. The number of neurons in the L5 DRG was studied at various times after unilateral sciatic nerve crush in 1-day-old rats. Axotomy resulted in a substantial loss (40 to 50%) of neurons in the immature DRG. Administration of NGF antiserum to animals with axotomized DRG did not increase cell death when compared with the axotomized controls. However, the number of neurons in the antiserum-treated ganglia decreased by the same percentage (20%) when compared with the control serum-treated ganglia before and after axotomy. Treatment with NGF initially prevented the loss of neurons in the axotomized DRG. However, some neurons died during the first week despite continued NGF administration; and, subsequent to NGF withdrawal, neuronal number decreased to the same level as in control animals. Thus, removal of exogenous NGF resulted in the death of the sensory neurons which had been maintained.(ABSTRACT TRUNCATED AT 250 WORDS)