RESUMEN
Neurotrophins (NTs) play an essential role in modulating activity-dependent neuronal plasticity. In this context, the site and extent of NT secretion are of crucial importance. Here, we demonstrate that the activation of phospolipase C (PLC) and the subsequent mobilization of Ca(2+) from intracellular stores are essential for NT secretion initiated by both Trk and glutamate receptor activation. Mutational analysis of tyrosine residues, highly conserved in the cytoplasmic domain of all Trk receptors, revealed that the activation of PLC-gamma in cultured hippocampal neurons and nnr5 cells is necessary to mobilize Ca(2+) from intracellular stores, the key mechanism for regulated NT secretion. A similar signalling mechanism has been identified for glutamate-mediated NT secretion-which in part depends on the activation of PLC via metabotropic receptors-leading to the mobilization of Ca(2+) from internal stores by inositol trisphosphate. Thus, PLC-mediated signal transduction pathways are the common mechanisms for both Trk- and mGluRI-mediated NT secretion.
Asunto(s)
Isoenzimas/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Receptor trkA/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Transducción de Señal , Fosfolipasas de Tipo C/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Calcio/metabolismo , Línea Celular , Hipocampo/citología , Hipocampo/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Líquido Intracelular/metabolismo , Neuronas/metabolismo , Fosfolipasa C gamma , Fosforilación , Ratas , Ratas Wistar , Receptor trkA/genética , Receptores AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Tirosina/metabolismoAsunto(s)
Potenciales de Acción/fisiología , Factores de Crecimiento Nervioso/metabolismo , Plasticidad Neuronal/fisiología , Neuronas/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Compartimento Celular/fisiología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Humanos , Memoria/fisiología , Factor de Crecimiento Nervioso/metabolismo , Neuronas/citología , Proteínas Tirosina Quinasas Receptoras/efectos de los fármacos , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
A modulatory role of neurotrophins (NTs) in activity-dependent neuronal plasticity by pre- and postsynaptic mechanisms is now well established. In this context, it is important to identify the storage compartments and to localize the precise site(s) and mechanism of NT secretion in order to deduce the spatial and temporal availability of NTs. We approached these questions at the ultrastructural level, exploiting the unique property of NT6 to bind tightly to heparan sulfate proteoglycans at the neuronal surface (R. Götz et al., 1994, Nature 372, 266-269), permitting the localization of secretion sites excluding diffusion artifacts. The myc tagging of NT6 permitted glutaraldehyde fixation and hence good preservation of the membrane structure, permitting immunogold labeling of NT6myc at the neuronal surface. NT6myc is preferentially secreted from neurites compared to neuronal cell bodies. In agreement with light-microscopic observations, the ultrastructural localization of NT6myc by postembedding procedures showed a predominant localization in ER-like membrane-confined compartments, partially associated with microtubules.
Asunto(s)
Hipocampo/citología , Factores de Crecimiento Nervioso/metabolismo , Neuronas/metabolismo , Secuencia de Aminoácidos , Animales , Compartimento Celular , Membrana Celular/ultraestructura , Fijadores , Glutaral , Glicosilación , Proteoglicanos de Heparán Sulfato/metabolismo , Heparina/farmacología , Hipocampo/metabolismo , Inmunohistoquímica , Microtúbulos/ultraestructura , Modelos Moleculares , Factores de Crecimiento Nervioso/análisis , Factores de Crecimiento Nervioso/genética , Plasticidad Neuronal , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Células PC12 , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-myc/química , Proteínas Proto-Oncogénicas c-myc/genética , Ratas , Ratas Wistar , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Fracciones Subcelulares/química , Fijación del Tejido/métodos , TransfecciónRESUMEN
Neurotrophins are a family of structurally related proteins that regulate the survival, differentiation and maintenance of function of different populations of peripheral and central neurons. They are also essential for modulating activity-dependent neuronal plasticity. Here we show that neurotrophins elicit action potentials in central neurons. Even at low concentrations, brain-derived neurotrophic factor (BDNF) excited neurons in the hippocampus, cortex and cerebellum. We found that BDNF and neurotrophin-4/5 depolarized neurons just as rapidly as the neurotransmitter glutamate, even at a more than thousand-fold lower concentration. Neurotrophin-3 produced much smaller responses, and nerve growth factor was ineffective. The neurotrophin-induced depolarization resulted from the activation of a sodium ion conductance which was reversibly blocked by K-252a, a protein kinase blocker which prefers tyrosine kinase Trk receptors. Our results demonstrate a very rapid excitatory action of neurotrophins, placing them among the most potent endogenous neuro-excitants in the mammalian central nervous system described so far.
Asunto(s)
Factores de Crecimiento Nervioso/fisiología , Receptor trkB/fisiología , Transmisión Sináptica/fisiología , Potenciales de Acción , Animales , Factor Neurotrófico Derivado del Encéfalo/fisiología , Calcio/metabolismo , Carbazoles/farmacología , Ácido Glutámico/fisiología , Técnicas In Vitro , Alcaloides Indólicos , Potenciales de la Membrana , Neurotransmisores/fisiología , Técnicas de Placa-Clamp , Células Piramidales/fisiología , Ratas , Ratas Wistar , Canales de Sodio/fisiologíaRESUMEN
In previous experiments the activity-dependent secretion of nerve growth factor (NGF) from native hippocampal slices and from NGF-cDNA transfected hippocampal neurons showed unusual characteristics [Blochl and Thoenen (1995) Eur J Neurosci 7:1220-1228; (1996) Mol Cell Neurosci 7:173-190]. In both hippocampal slices and cultured hippocampal neurons the activity-dependent secretion proved to be independent of extracellular calcium, but dependent on the release of calcium from intracellular stores. Under different experimental conditions, Goodman et al. [(1996) Mol Cell Neurosci 7:222-238] reported that the high potassium-mediated secretion of brain-derived neurotrophic factor (BDNF) from hippocampal cultures was dependent on extracellular calcium. Mowla et al. [(1997) Proc 27th Annu Meet Soc Neurosci New Orleans 875.10] reported on even further-reaching differences between NGF and BDNF secretion, namely, that in hippocampal neurons and in pituitary cell lines NGF was secreted exclusively according to the constitutive pathway, whereas BDNF was exclusively sorted according to the activity-dependent regulated pathway. In view of the crucial importance of such potential differences between the processing, sorting, and secretory mechanisms of different neurotrophins for their modulatory roles in activity-dependent neuronal plasticity, a thorough analysis under comparable experimental conditions was mandatory. We demonstrate that in native hippocampal slices and adenoviral-transduced hippocampal neurons there are no differences between NGF and BDNF with respect to the subcellular distribution and mechanism of secretion; that the activity-dependent secretion of both NGF and BDNF is dependent on intact intracellular calcium stores; and that the differences between our own observations and those of Goodman et al. (ibid.) regarding the dependence on extracellular calcium do not reflect differences between NGF and BDNF sorting and secretion, but reflect the differing experimental conditions used.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/metabolismo , Hipocampo/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Neuronas/fisiología , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Calcio/metabolismo , Células Cultivadas , Femenino , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Hipocampo/citología , Masculino , Microscopía Confocal , Factores de Crecimiento Nervioso/genética , Neuronas/citología , Neuronas/efectos de los fármacos , Potasio/metabolismo , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Over-expression of Bcl-2 is known to reduce the extent of retinal ganglion cell death during development as well as after axotomy. Here we investigated whether retinal ganglion cell (RGC) numbers are reduced in mice with a targeted inactivation of the bcl-2 gene. Compared with wild-type mice, adult bcl-2 null mutants have lost 29% of the retinal ganglion cell axons in the optic nerve. This reduction was almost fully established at P15, but not present at P10, which marks the end of the period of naturally occurring cell death. These observations, together with the previously reported late loss of primary motoneurons and peripheral neurons, point to a general physiological requirement for Bcl-2 soon after the period of naturally occurring cell death.
Asunto(s)
Proteínas Proto-Oncogénicas c-bcl-2/deficiencia , Células Ganglionares de la Retina/citología , Envejecimiento/fisiología , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Animales Recién Nacidos/fisiología , Axones/ultraestructura , Recuento de Células , Muerte Celular/fisiología , Ratones , Ratones Noqueados/anatomía & histología , Ratones Noqueados/genética , Microscopía Electrónica , Mutación/fisiología , Nervio Óptico/ultraestructura , Proteínas Proto-Oncogénicas c-bcl-2/genética , Valores de Referencia , Células Ganglionares de la Retina/fisiología , Células Ganglionares de la Retina/ultraestructuraRESUMEN
Cytoplasmic calcium-binding proteins are thought to shield neurons against damage induced by excessive Ca2+ elevations. Yet, in theory, a mobile cellular Ca2+ buffer could just as well promote neuronal injury by facilitating the rapid dispersion of Ca2+ throughout the cytoplasm. In sharp contrast to controls, in mice lacking the gene for calbindin-D28k, synaptic responses of hippocampal CA1 pyramidal neurons which are normally extremely vulnerable to ischemia, recovered significantly faster and more completely after a transient oxygen-glucose deprivation in vitro, and sustained less cellular damage following a 12 min carotid artery occlusion in vivo. Other cellular and synaptic properties such as the altered adaptation of action potential firing, and altered paired-pulse and frequency potentiation at affected synapses in calbindin-D28k-deficient mice were consistent with a missing intraneuronal Ca2+ buffer. Our findings provide direct experimental evidence against a neuroprotective role for calbindin-D28k.
Asunto(s)
Isquemia Encefálica/prevención & control , Calcio/metabolismo , Hipocampo/patología , Proteínas del Tejido Nervioso/farmacología , Proteína G de Unión al Calcio S100/farmacología , Animales , Isquemia Encefálica/metabolismo , Tampones (Química) , Calbindina 1 , Calbindinas , Citoplasma/metabolismo , Glucosa/deficiencia , Hipocampo/irrigación sanguínea , Hipocampo/efectos de los fármacos , Técnicas In Vitro , Canales Iónicos/efectos de los fármacos , Canales Iónicos/metabolismo , Ratones , Ratones Endogámicos , Ratones Noqueados , N-Metilaspartato/metabolismo , Oxígeno/metabolismoRESUMEN
Brain-derived neurotrophic factor (BDNF) is a survival factor for certain sensory neurons during development. Using electrophysiology in BDNF-deficient mice, we show here that slowly adapting mechanoreceptors (SAM), but not other types of cutaneous afferents, require BDNF in postnatal life for normal mechanotransduction. Neurons lacking BDNF did not die, but instead showed a profound and specific reduction in their mechanical sensitivity, which was quantitatively the same in BDNF -/- and BDNF +/- animals. Postnatal treatment of BDNF +/- mice with recombinant BDNF completely rescued the mechanosensitivity deficit. Therefore BDNF is important for regulating SAM mechanosensitivity, independent of any survival-promoting function.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/fisiología , Mecanorreceptores/fisiología , Sensación/fisiología , Adaptación Fisiológica/fisiología , Animales , Axones/fisiología , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/farmacología , Senescencia Celular/fisiología , Mecanorreceptores/efectos de los fármacos , Ratones , Ratones Noqueados/genética , Vaina de Mielina/fisiología , Neuronas Aferentes/fisiología , Proteínas Recombinantes , Sensación/efectos de los fármacos , Piel/inervación , Factores de TiempoRESUMEN
Neurotrophins, secreted in an activity-dependent manner, are thought to be involved in the activity-dependent refinement of synaptic connections. Here we demonstrate that in hippocampal neurons and the rat pheochromocytoma cell line PC12 application of exogenous neurotrophins induces secretion of neurotrophins, an effect that is mediated by the activation of tyrosine kinase neurotrophin receptors (Trks). Like activity-dependent secretion of neurotrophins, neurotrophin-induced neurotrophin secretion requires mobilization of calcium from intracellular stores. Because neurotrophins are likely to be released from both dendrites and axons, neurotrophin-induced neurotrophin release represents a potential positive feedback mechanism, contributing to the reinforcement and stabilization of synaptic connections.
Asunto(s)
Hipocampo/metabolismo , Factores de Crecimiento Nervioso/fisiología , Plasticidad Neuronal/fisiología , Animales , Calcio/metabolismo , Células Cultivadas , Ácido Glutámico/fisiología , Hipocampo/citología , Técnicas In Vitro , Factores de Crecimiento Nervioso/metabolismo , Células PC12 , Ratas , Ratas WistarRESUMEN
Ciliary neurotrophic factor (CNTF) is an abundant cytosolic molecule in myelinating Schwann cells of adult rodents. In newborn animals in which CNTF is not yet expressed, exogenous CNTF that is locally administered very effectively protects motoneurons from degeneration by axotomy. To evaluate whether endogenous CNTF, released after nerve injury from the cytosol of Schwann cells, supports motoneuron survival, we transected the facial nerve in 4-week-old pmn mice. In this mouse mutant a rapidly progressing degenerative disease of motoneurons starts by the third postnatal week at the hindlimbs and progresses to the anterior parts of the body, leading to death by the seventh to eighth week. Apoptotic death of motoneurons can be observed during this period, as revealed by TUNEL staining. In 6-week-old unlesioned pmn mice approximately 40% of facial motoneurons have degenerated. Facial nerve lesion dramatically increased the number of surviving motoneurons in pmn mice. This protective effect was absent in pmn mice lacking endogenous CNTF. Quantitative analysis of leukemia inhibitory factor (LIF) mRNA expression revealed that the dramatic upregulation seen in wild-type mice after peripheral nerve lesion did not occur in pmn mice. Therefore, endogenous LIF cannot compensate for the lack of CNTF in pmn crossbred with CNTF knock-out mice. Thus, endogenous CNTF released from lesioned Schwann cells supports the survival of axotomized motoneurons under conditions in which motoneurons are in the process of rapid degeneration.
Asunto(s)
Axones/fisiología , Interleucina-6 , Neuronas Motoras/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Apoptosis , Supervivencia Celular , Factor Neurotrófico Ciliar , Nervio Facial/metabolismo , Femenino , Inhibidores de Crecimiento/genética , Inhibidores de Crecimiento/metabolismo , Factor Inhibidor de Leucemia , Linfocinas/genética , Linfocinas/metabolismo , Masculino , Ratones , Ratones Noqueados , ARN Mensajero/metabolismo , Nervio Ciático/metabolismo , Regulación hacia ArribaRESUMEN
Intracellular calcium-binding proteins are abundantly expressed in many neuronal populations. Previous evidence suggests that calcium-binding proteins can modulate various neuronal properties, presumably by their action as calcium buffers. The importance of calcium-binding proteins for nervous system function in an intact integrated system is, however, less clear. To investigate the physiological role of a major endogenous calcium-binding protein, calbindin D28k (calbindin) in vivo, we have generated calbindin null mutant mice by gene targeting. Surprisingly, calbindin deficiency does not affect general parameters of development and behavior or the structure of the nervous system at the light microscopic level. Null mutants are, however, severely impaired in tests of motor coordination, suggesting functional deficits in cerebellar pathways. Purkinje neurons, the only efferent of the cerebellar cortex, and inferior olive neurons, the source of the climbing fiber afferent, have previously been shown to express calbindin. Correlated with this unusual type of ataxia, confocal calcium imaging of Purkinje cells in cerebellar slices revealed marked changes of synaptically evoked postsynaptic calcium transients. Their fast, but not their slow, decay component had larger amplitudes in null mutant than in wild-type mice. We conclude that endogenous calbindin is of crucial importance for integrated nervous system function.
Asunto(s)
Ataxia/fisiopatología , Calcio/metabolismo , Dendritas/fisiología , Proteínas del Tejido Nervioso/fisiología , Proteína G de Unión al Calcio S100/fisiología , Animales , Ataxia/etiología , Ataxia/genética , Conducta Animal , Calbindina 1 , Calbindinas , Proteínas de Unión al Calcio/metabolismo , Cerebelo/citología , Cerebelo/fisiología , Desarrollo Embrionario y Fetal , Potenciales Evocados , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Proteínas del Tejido Nervioso/genética , Desempeño Psicomotor , Células de Purkinje/fisiología , Proteína G de Unión al Calcio S100/genética , Transducción de Señal , Sinapsis/fisiología , Regulación hacia ArribaRESUMEN
Primary cultures of hippocampal neurons were infected with an adenovirus coding for beta-galactosidase. Expression could be detected as early as 4 h after infection and steadily increased to high levels at 24 h without evidence for a functional impairment of the infected neurons. Similarly, adenovirus-mediated gene transfer into acute hippocampal slices was detectable 4 h after infection and could be localized to discrete areas of the CA1 region by microinjection of the virus stock solution. Infected slices were still suitable for electrophysiological experiments.
Asunto(s)
Adenoviridae/genética , Técnicas de Transferencia de Gen , Hipocampo/virología , Animales , Células Cultivadas , Hipocampo/ultraestructura , Microscopía Electrónica , Ratas , Ratas Wistar , Factores de Tiempo , beta-Galactosidasa/metabolismoRESUMEN
Calbindin-D28k (calbindin) is an intracellular calcium binding protein of unknown in vivo function. It is abundantly expressed in many populations of neurons, and it can, presumably by buffering calcium overload, protect cells against excitotoxic damage. In the midbrain, calbindin is preferentially expressed in those dopamine neurons which are spared from degeneration in Parkinson's disease and its animal models. Whether calbindin itself determines neuronal vulnerability is questioned in other lesion models where calbindin expression is not positively correlated with neuronal resistance. To study the possible neuroprotective role of calbindin in vivo, we generated calbindin-deficient mice by gene targeting and assessed the viability of midbrain dopamine neurons in both a chemical and a genetic lesion paradigm. Tyrosine hydroxylase-immunoreactive neurons were counted in calbindin null-mutant mice treated with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and in a calbindin-deficient weaver strain (homozygous for weaver and the calbindin null mutation). The extent and pattern of neuron loss observed in MPTP-treated wild-type and homozygous weaver mice were as previously described. Surprisingly, no significant differences were observed between MPTP-treated calbindin null mutants and their wild-type littermates, or between calbindin-weaver double mutant mice and weaver mice. Thus, in all four groups the same subpopulation of tyrosine hydroxylase-positive midbrain neurons (i.e. those normally containing calbindin) were preferentially spared. Calretinin, a closely related calcium-binding protein, which is also expressed in some midbrain dopamine neurons, was not up-regulated in these surviving neurons. These findings indicate that the resistance of calbindin-containing neurons in the MPTP and weaver models is not causally related to the expression of calbindin, and that endogenous calbindin is not required for protection of these neurons.
Asunto(s)
Dopaminérgicos/toxicidad , Dopamina/fisiología , Intoxicación por MPTP , Mesencéfalo/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuronas/fisiología , Proteína G de Unión al Calcio S100/fisiología , Animales , Calbindina 1 , Calbindinas , Ataxia Cerebelosa/genética , Ataxia Cerebelosa/patología , Femenino , Inmunohistoquímica , Mesencéfalo/citología , Mesencéfalo/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Mutantes Neurológicos , Microscopía Confocal , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Proteína G de Unión al Calcio S100/genéticaRESUMEN
While brain-derived neurotrophic factor (BDNF) delays the death of axotomized retinal ganglion cells in rodents, it is unclear if it affects any aspect of the normal development of these cells. Here we examined the optic nerve of bdnf-/- mice. Axonal numbers were normal, but their diameter, as well as the proportion of myelinated axons, was reduced at postnatal day 20 (P20). In contrast, the facial nerve was not hypomyelinated. Expression levels of mRNAs coding for the myelin proteins PLP and MBP were substantially reduced in the hippocampus and cortex at P20, but not in the sciatic nerve. Intraventricular injections of BDNF into the ventricles of wild-type mice at P10 and P12 up-regulated expression of PLP in the hippocampus at P14. These results indicate a role of BDNF, discussed as indirect, in the control of myelination in the central nervous system.
Asunto(s)
Axones/fisiología , Factor Neurotrófico Derivado del Encéfalo/deficiencia , Factor Neurotrófico Derivado del Encéfalo/genética , Vaina de Mielina/genética , Vaina de Mielina/fisiología , Células Ganglionares de la Retina/fisiología , Animales , Apoproteínas/biosíntesis , Apoproteínas/efectos de los fármacos , Apoproteínas/genética , Factor Neurotrófico Derivado del Encéfalo/fisiología , Recuento de Células/efectos de los fármacos , Tamaño de la Célula/genética , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Nervio Facial/fisiología , Nervio Facial/ultraestructura , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Ratones , Ratones Noqueados , Proteína Proteolipídica de la Mielina/biosíntesis , Proteína Proteolipídica de la Mielina/efectos de los fármacos , Proteína Proteolipídica de la Mielina/genética , Nervio Óptico/fisiología , Nervio Óptico/ultraestructura , ARN Mensajero/biosíntesis , Células Ganglionares de la Retina/citología , Hormonas Tiroideas/genética , Hormonas Tiroideas/metabolismoRESUMEN
In view of the protective effect of brain derived neurotrophic factor (BDNF) against metabolic/excitotoxic insults in vitro, we investigated whether BDNF could limit infarct size after permanent occlusion of the middle cerebral artery in rat (MCAO). BDNF was delivered into the territory of the middle cerebral artery via an osmotic mini-pump (1 microg/h). Infusion of BDNF was started shortly after MCAO, and 24 h later brains were removed for infarct volume determination. Intraoperative and postoperative measurements of physiological variables revealed no differences among vehicle-treated and BDNF-infused animals. However, we found a 33% reduction in total infarct volume in BDNF-treated animals (p<0.05), and a 37% reduction in cortical infarct volume (p<0.05).
Asunto(s)
Isquemia Encefálica/tratamiento farmacológico , Factor Neurotrófico Derivado del Encéfalo/uso terapéutico , Infarto Cerebral/prevención & control , Animales , Encéfalo/patología , Isquemia Encefálica/patología , Masculino , Ratas , Ratas Endogámicas F344RESUMEN
Long-term potentiation (LTP) has been shown to be impaired in mice deficient in the brain-derived neurotrophic factor (BDNF) gene, as well as in a number of other knockout animals. Despite its power the gene-targeting approach is always fraught with the danger of looking at the cumulative direct and indirect effects of the absence of a particular gene rather than its immediate function. The re-expression of a specific gene at a selective time point and at a specific site in gene-defective mutants presents a potent procedure to overcome this limitation and to evaluate the causal relationship between the absence of a particular gene and the impairment of a function in gene-defective animals. Here we demonstrate that the re-expression of the BDNF gene in the CA1 region almost completely restores the severely impaired LTP in hippocampal slices of BDNF-deficient mice. The results therefore provide strong evidence for the direct involvement of BDNF in the process of LTP.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/genética , Técnicas de Transferencia de Gen , Hipocampo/metabolismo , Potenciación a Largo Plazo , Adenoviridae , Animales , Northern Blotting , Factor Neurotrófico Derivado del Encéfalo/fisiología , Vectores Genéticos , Ratones , Ratones MutantesRESUMEN
Gene targeting is a powerful method for introducing mutations into the genome of embryonic stem cells. The most widely used approach is the positive-negative selection method in which a gene encoding a negative selection marker is cloned into the replacement vector to obtain an enrichment of properly targeted clones. Here, we present an alternative means to introduce any given negative selection marker at the ends of a replacement vector using a single ligation step, thereby avoiding laborious cloning procedures. Our results demonstrate that this fast and simple method consistently provides a high level of enrichment of appropriately targeted clones.
Asunto(s)
ADN Ligasas/metabolismo , Marcación de Gen/métodos , Marcadores Genéticos , Vectores Genéticos , Selección Genética , Animales , Clonación Molecular/métodos , Factor 5 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/genética , Ratones , Proteínas Proto-Oncogénicas c-bcl-2/genética , Células MadreRESUMEN
The proto-oncogene bcl-2 and its family members, bcl-x and bax are recognized as major regulators of cell death and survival. Although Bcl-2 and Bcl-x are expressed in brain, little is known how they are regulated in neurons. Here we have studied the expression of bcl-2, bcl-xL and bax mRNA in rat cerebellar granule neurons cultured under conditions which influence neuron survival. Insulin-like growth factor-1 and brain-derived neurotrophic factor supported the survival of these neurons, but affected neither the expression of bcl-2, bcl-xL nor bax mRNA. In contrast, bcl-2 and bcl-xL mRNAs were up-regulated in cerebellar granule neurons plated at high density exhibiting an increased neuronal survival. Western blots showed that cell density also increased Bcl-2 protein level. However, conditioned medium from dense cultures did not affect the level of bcl-2 mRNA nor survival of the neurons. This suggests that cell density promotes survival and regulates Bcl-2 expression in cerebellar granule neurons through a signaling pathway different from known neurotrophic factors.
Asunto(s)
Cerebelo/citología , Expresión Génica , Genes bcl-2 , Neuronas/citología , Neuronas/fisiología , Proteínas Proto-Oncogénicas/genética , Animales , Western Blotting , Factor Neurotrófico Derivado del Encéfalo/farmacología , Recuento de Células , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Factor I del Crecimiento Similar a la Insulina/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Mensajero/metabolismo , Ratas , Proteína X Asociada a bcl-2 , Proteína bcl-XRESUMEN
Although ciliary neurotrophic factor (CNTF) is a potent survival factor for many types of neurons and glial cells in vitro, there is currently no evidence that it participates in normal development. Here we show that CNTF greatly enhances the rate of oligodendrocyte generation. Proliferation of oligodendrocyte precursor cells purified from rodent optic nerves and cultured in platelet-derived growth factor-containing medium is significantly increased by CNTF. Similarly, the number of proliferating oligodendrocyte precursor cells in developing optic nerves of transgenic mice lacking CNTF is decreased by up to threefold and the number of oligodendrocytes is transiently decreased; proliferation is restored to normal by the delivery of exogenous CNTF into the developing optic nerve. Both oligodendrocyte number and myelination ultimately attain wild-type values in CNTF-deficient adult mice, indicating that CNTF is not necessary for either oligodendrocyte differentiation or myelination, although it normally accelerates oligodendrocyte development by enhancing the proliferation of oligodendrocyte precursor cells.
RESUMEN
In contrast to sympathetic and sensory neurons in the peripheral nervous system, the neurotrophic requirements for neurons in the central nervous system (CNS) have not been clearly identified. The inactivation of specific neurotrophic factors and their receptors by gene targeting has shown that there are no major changes in neuron numbers in the CNS. This suggests an overlap between the action of different neurotrophic factors in the brain during development. Here we have studied the survival of hippocampal neurons prepared from embryonic rats using different culture conditions. Whereas the hippocampal neurons survive well in culture when plated at high density, they die at lower cell densities in the absence of appropriate neurotrophic factors. Under the latter conditions, both insulin-like growth factor-1 (IGF-1) and neurotrophins - brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) - rescued a large proportion of cultured neurons. In addition, hippocampal neurons from BDNF knockout mice exhibited enhanced cell death compared with cells from wild-type animals. BDNF and IGF-1 both increased the survival of the hippocampal neurons lacking BDNF, showing complementary action for these factors in supporting survival. Blocking antibodies against NT-3 and IGF-1 decreased hippocampal neuron survival at low cell densities, showing autocrine or paracrine action of the factors. At higher cell densities, however, the antibodies had no effect, demonstrating that there is a sufficient amount of endogenous factors in supporting survival. Blocking antibodies against NT-3 and IGF-1 decreased hippocampal neurons depend for survival on local neurotrophic factors such as IGF-1, BDNF and NT-3, which act in an autocrine/paracrine manner. The multifactorial support of hippocampal neurons ensures a maximal degree of neuron survival even in the absence of an individual factor