RESUMEN

An analysis of programmed cell death of several populations of developing postmitotic neurons after genetic deletion of two key members of the caspase family of pro-apoptotic proteases, caspase-3 and caspase-9, indicates that normal neuronal loss occurs. Although the amount of cell death is not altered, the death process may be delayed, and the cells appear to use a nonapoptotic pathway of degeneration. The neuronal populations examined include spinal interneurons and motor, sensory, and autonomic neurons. When examined at both the light and electron microscopic levels, the caspase-deficient neurons exhibit a nonapoptotic morphology in which nuclear changes such as chromatin condensation are absent or reduced; in addition, this morphology is characterized by extensive cytoplasmic vacuolization that is rarely observed in degenerating control neurons. There is also reduced terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling in dying caspase-deficient neurons. Despite the altered morphology and apparent temporal delay in cell death, the number of neurons that are ultimately lost is indistinguishable from that seen in control animals. In contrast to the striking perturbations in the morphology of the forebrain of caspase-deficient embryos, the spinal cord and brainstem appear normal. These results are consistent with the growing idea that the involvement of specific caspases and the occurrence of caspase-independent programmed cell death may be dependent on brain region, cell type, age, and species or may be the result of specific perturbations or pathology.

Asunto(s)

Apoptosis , Caspasas/deficiencia , Neuronas/metabolismo , Animales , Tronco Encefálico/citología , Caspasa 3 , Caspasa 9 , Caspasas/genética , Caspasas/metabolismo , Recuento de Células , Supervivencia Celular/genética , Ganglios/citología , Homocigoto , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Ratones , Ratones Endogámicos , Ratones Mutantes , Defectos del Tubo Neural/genética , Defectos del Tubo Neural/patología , Neuronas/citología , Prosencéfalo/anomalías , Prosencéfalo/patología , Médula Espinal/patología

RESUMEN

Developing motoneurons require trophic support from their target, the skeletal muscle. Despite a large number of neurotrophic molecules with survival-promoting activity for isolated embryonic motoneurons, those factors that are required for motoneuron survival during development are still not known. Cytokines of the ciliary neurotrophic factor (CNTF)-leukemia inhibitory factor (LIF) family have been shown to play a role in motoneuron (MN) survival. Importantly, in mice lacking the LIFRbeta or the CNTFRalpha there is a significant loss of MNs during embryonic development. Because genetic deletion of either (or both) CNTF or LIF fails, by contrast, to perturb MN survival before birth, it was concluded that another ligand exists that is functionally inactivated in the receptor deleted mice, resulting in MN loss during development. One possible candidate for this ligand is the CNTF-LIF family member cardiotrophin-1 (CT-1). CT-1 is highly expressed in embryonic skeletal muscle, secreted by myotubes, and promotes the survival of cultured embryonic mouse and rat MNs. Here we show that ct-1 deficiency causes increased motoneuron cell death in spinal cord and brainstem nuclei of mice during a period between embryonic day 14 and the first postnatal week. Interestingly, no further loss was detectable during the subsequent postnatal period, and nerve lesion in young adult ct-1-deficient mice did not result in significant additional loss of motoneurons, as had been previously observed in mice lacking both CNTF and LIF. CT-1 is the first bona fide muscle-derived neurotrophic factor to be identified that is required for the survival of subgroups of developing motoneurons.

Asunto(s)

Citocinas/metabolismo , Interleucina-6 , Neuronas Motoras/metabolismo , Músculo Esquelético/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Axotomía , Tronco Encefálico/embriología , Tronco Encefálico/metabolismo , Tronco Encefálico/patología , Muerte Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Embrión de Pollo , Factor Neurotrófico Ciliar/genética , Factor Neurotrófico Ciliar/metabolismo , Receptor gp130 de Citocinas , Citocinas/deficiencia , Citocinas/genética , Citocinas/farmacología , Relación Dosis-Respuesta a Droga , Nervio Facial , Inhibidores de Crecimiento/genética , Inhibidores de Crecimiento/metabolismo , Factor Inhibidor de Leucemia , Linfocinas/genética , Linfocinas/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/patología , Músculo Esquelético/embriología , Músculo Esquelético/inervación , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/patología , ARN Mensajero/biosíntesis , Receptor de Factor Neurotrófico Ciliar/genética , Receptor de Factor Neurotrófico Ciliar/metabolismo , Médula Espinal/embriología , Médula Espinal/metabolismo , Médula Espinal/patología

RESUMEN

Using two different lesion models, the spinal root avulsion and the distal nerve axotomy, the present study investigated effects of known neurotrophic factors on motoneuron survival in newborn rats. Results of the present study show that 100% of motoneurons in the lesioned spinal segment die at 1 week following root avulsion, and more than 80% of them die at 2 weeks following distal nerve axotomy. Local application of GDNF can rescue 92% of motoneurons up to 1 week from degeneration due to root avulsion and almost 100% of them up to 2 weeks from degeneration due to distal nerve axotomy. Local application of BDNF fails to prevent any motoneuron death in newborn rats following root avulsion, but it can rescue about 50% of motoneurons up to 2 weeks from degeneration due to distal nerve axotomy. CNTF and IGF-1 fail to prevent any motoneuron death following either distal nerve axotomy or root avulsion. Thus, comparing all the neurotrophic factors tested in this study, GDNF is most effective in preventing death of motoneurons following axonal injury in newborn rats.

Asunto(s)

Supervivencia Celular/fisiología , Neuronas Motoras/fisiología , Traumatismos de la Médula Espinal/fisiopatología , Médula Espinal/patología , Raíces Nerviosas Espinales/lesiones , Animales , Animales Recién Nacidos , Axones/fisiología , Axotomía , Muerte Celular , Laminectomía , Neuronas Motoras/citología , Neuronas Motoras/patología , Ratas , Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/patología

RESUMEN

Spinal motoneurons (MNs) in the chick embryo undergo programmed cell death coincident with the establishment of nerve-muscle connections and the onset of synaptic transmission at the neuromuscular junction. Chronic treatment of embryos during this period with nicotinic acetylcholine receptor (nAChR)-blocking agents [e.g., curare or alpha-bungarotoxin (alpha-BTX)] prevents the death of MNs. Although this rescue effect has been attributed previously to a peripheral site of action of the nAChR-blocking agents at the neuromuscular junction (NMJ), because nAChRs are expressed in both muscle and spinal cord, it has been suggested that the rescue effect may, in fact, be mediated by a direct central action of nAChR antagonists. By using a variety of different nAChR-blocking agents that target specific muscle or neuronal nAChR subunits, we find that only those agents that act on muscle-type receptors block neuromuscular activity and rescue MNs. However, paralytic, muscular dysgenic mutant chick embryos also exhibit significant increases in MN survival that can be further enhanced by treatment with curare or alpha-BTX, suggesting that muscle paralysis may not be the sole factor involved in MN survival. Taken together, the data presented here support the argument that, in vivo, nAChR antagonists promote the survival of spinal MNs primarily by acting peripherally at the NMJ to inhibit synaptic transmission and reduce or block muscle activity. Although a central action of these agents involving direct perturbations of MN activity may also play a contributory role, further studies are needed to determine more precisely the relative roles of central versus peripheral sites of action in MN rescue.

Asunto(s)

Apoptosis/efectos de los fármacos , Neuronas Motoras/efectos de los fármacos , Músculo Esquelético/embriología , Unión Neuromuscular/embriología , Antagonistas Nicotínicos/farmacología , Médula Espinal/embriología , Animales , Apoptosis/fisiología , Bungarotoxinas/farmacología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Embrión de Pollo , Curare/farmacología , Neuronas Motoras/citología , Neuronas Motoras/metabolismo , Movimiento/efectos de los fármacos , Movimiento/fisiología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/metabolismo , Nervios Periféricos/efectos de los fármacos , Nervios Periféricos/embriología , Nervios Periféricos/metabolismo , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/metabolismo , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Sinapsis/ultraestructura

RESUMEN

Because of discrepancies in previous reports regarding the role of glial cell line-derived neurotrophic factor (GDNF) in motoneuron (MN) development and survival, we have reexamined MNs in GDNF-deficient mice and in mice exposed to increased GDNF after in utero treatment or in transgenic animals overexpressing GDNF under the control of the muscle-specific promoter myogenin (myo-GDNF). With the exception of oculomotor and abducens MNs, the survival of all other populations of spinal and cranial MNs were reduced in GDNF-deficient embryos and increased in myo-GDNF and in utero treated animals. By contrast, the survival of spinal sensory neurons in the dorsal root ganglion and spinal interneurons were not affected by any of the perturbations of GDNF availability. In wild-type control embryos, all brachial and lumbar MNs appear to express the GDNF receptors c-ret and GFRalpha1 and the MN markers ChAT, islet-1, and islet-2, whereas only a small subset express GFRalpha2. GDNF-dependent MNs that are lost in GDNF-deficient animals express ret/GFRalpha1/islet-1, whereas many surviving GDNF-independent MNs express ret/GFRalpha1/GFRalpha2 and islet-1/islet-2. This indicates that many GDNF-independent MNs are characterized by the presence of GFRalpha2/islet-2. It seems likely that the GDNF-independent population represent MNs that require other GDNF family members (neurturin, persephin, artemin) for their survival. GDNF-dependent and -independent MNs may reflect subtypes with distinct synaptic targets and afferent inputs.

Asunto(s)

Apoptosis/fisiología , Encéfalo/embriología , Proteínas de Drosophila , Neuronas Motoras/fisiología , Factores de Crecimiento Nervioso , Proteínas del Tejido Nervioso/fisiología , Médula Espinal/embriología , Animales , Encéfalo/citología , Supervivencia Celular/efectos de los fármacos , Cruzamientos Genéticos , Desarrollo Embrionario y Fetal , Edad Gestacional , Factor Neurotrófico Derivado de la Línea Celular Glial , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Neuronas Motoras/citología , Neuronas Motoras/efectos de los fármacos , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas del Tejido Nervioso/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-ret , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/fisiología , Médula Espinal/citología

RESUMEN

Hepatocyte growth factor/scatter factor (HGF/SF) is expressed in the developing limb muscles of the chick embryo during the period of spinal motoneuron (MN) programmed cell death, and its receptor c-met is expressed in lumbar MNs during this same period. Although cultured motoneurons from brachial, thoracic, and lumbar segments are all rescued from cell death by chick embryo muscle extract (CMX) as well as by other specific trophic agents, HGF/SF only promotes the survival of lumbar MNs. Similarly, treatment of embryos in ovo with exogenous HGF/SF rescues lumbar but not other somatic MNs from cell death. Blocking antibodies to HGF/SF (anti-HGF) reduce the effects of CMX on MN survival in vitro and decrease the number of lumbar MNs in vivo. The expression of c-met on MNs in vivo is regulated by a limb-derived trophic signal distinct from HGF/SF. HGF/SF is a potent, select, and physiologically relevant survival factor for a subpopulation of developing spinal MNs in the lumbar segments of the chick embryo.

Asunto(s)

Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/farmacología , Neuronas Motoras/citología , Médula Espinal/citología , Animales , Anticuerpos/farmacología , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Embrión de Pollo , Nervios Craneales/citología , Nervios Craneales/embriología , Regulación del Desarrollo de la Expresión Génica , Factor de Crecimiento de Hepatocito/antagonistas & inhibidores , Hibridación in Situ , Esbozos de los Miembros/embriología , Esbozos de los Miembros/inervación , Esbozos de los Miembros/fisiología , Neuronas Motoras/química , Neuronas Motoras/efectos de los fármacos , Proteínas Proto-Oncogénicas c-met/análisis , Proteínas Proto-Oncogénicas c-met/biosíntesis , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Médula Espinal/embriología

RESUMEN

The regulation of survival of spinal motoneurons (MNs) has been shown to depend during development and after injury on a variety of neurotrophic molecules produced by skeletal muscle target tissue. Increasing evidence also suggests that other sources of trophic support prevent MNs from undergoing naturally occurring or injury-induced death. We have examined the role of endogenous and exogenous androgens on the survival of developing avian lumbar spinal MNs during their period of programmed cell death (PCD) between embryonic day (E)6 and E11 or after axotomy on E12. We found that although treatment with testosterone, dihydrotestosterone (DHT), or the androgen receptor antagonist flutamide (FL) failed to affect the number of these MNs during PCD, administration of DHT from E12 to E15 following axotomy on E12 significantly attenuated injury-induced MN death. This effect was inhibited by cotreatment with FL, whereas treatment with FL alone did not affect MN survival. Finally, we examined the spinal cord at various times during development and following axotomy on E12 for the expression of androgen receptor using the polyclonal PG-21 antibody. Our results suggest that exogenously applied androgens are capable of rescuing MNs from injury-induced cell death and that they act directly on these cells via an androgen receptor-mediated mechanism. By contrast, endogenous androgens do not appear to be involved in the regulation of normal PCD of developing avian MNs.

Asunto(s)

Andrógenos/farmacología , Neuronas Motoras/citología , Neuronas Motoras/fisiología , Médula Espinal/embriología , Antagonistas de Andrógenos/farmacología , Animales , Apoptosis/efectos de los fármacos , Axotomía , Muerte Celular , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Embrión de Pollo , Dihidrotestosterona/farmacología , Flutamida/farmacología , Región Lumbosacra , Neuronas Motoras/efectos de los fármacos , Receptores Androgénicos/análisis , Médula Espinal/citología , Testosterona/farmacología

RESUMEN

The long-term effect of a single dose of Brain-derived neurotrophic factor (BDNF) treatment on adult motoneuron survival and on expression of nitric oxide synthase (NOS) following nerve injury (avulsion) was investigated and compared with that of continuous BDNF treatment. By 6 weeks post-injury, more than 80% of motoneurons survived in animals treated with either a single dose or continuous treatment of BDNF, while only 30% of motoneurons survived in control animals (avulsion only). There were no significant differences in motoneuron survival between animals receiving a single dose and those with continuous treatment of BDNF. Additionally, the expression of NOS in avulsed motoneurons was almost completely inhibited in all BDNF treatment groups regardless of the mode of administration (single vs. continuous). These data indicate that treatment with a single dose of BDNF at the time of injury can inhibit NOS expression and provide the first evidence that in this situation BDNF has a long-term rescue effect on adult motoneuron survival after root avulsion.

Asunto(s)

Factor Neurotrófico Derivado del Encéfalo/farmacología , Neuronas Motoras/citología , Raíces Nerviosas Espinales/citología , Raíces Nerviosas Espinales/lesiones , Factores de Edad , Animales , Recuento de Células , Supervivencia Celular/efectos de los fármacos , Colorantes , Masculino , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/enzimología , Rojo Neutro , Óxido Nítrico Sintasa/análisis , Óxido Nítrico Sintasa/metabolismo , Ratas , Ratas Sprague-Dawley

RESUMEN

Sonic hedgehog (Shh) is a secreted glycoprotein expressed by the notochord and floor plate that is involved in the induction and specification of ventral phenotypes in the vertebrate neural tube. Recently, Shh has also been shown to promote the survival of cultured rat embryo ventral brain and spinal cord cells. We have examined whether Shh can promote the survival of chick embryo neurons in vivo or in vitro. In the chick, Shh is expressed in notochord, floor plate, and ventral neural tube/spinal cord at several stages at which programmed cell death (PCD) occurs. However, the administration of exogenous Shh to embryos in vivo or to motoneuron cultures at these stages failed to promote the survival of several different neuronal populations, including spinal motoneurons, spinal interneurons, sympathetic preganglionic neurons, sensory neurons, and neuronal precursor cells. Rather, at the earliest stage of PCD examined here (embryonic day 3) Shh selectively induced the death of ventral neuronal precursors and floor-plate cells, resulting in a net loss of cells in the neural tube. Altered concentrations of Shh induce aberrant phenotypes that are removed by PCD. Accordingly, normal PCD in the early neural tube may play a role in dorsal-ventral patterning.

Asunto(s)

Apoptosis/genética , Inducción Embrionaria/fisiología , Proteínas/genética , Médula Espinal/citología , Médula Espinal/embriología , Transactivadores , Animales , Apoptosis/efectos de los fármacos , Recuento de Células , Células Cultivadas , Embrión de Pollo , Ganglios Espinales/citología , Ganglios Simpáticos/citología , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog , Interneuronas/química , Interneuronas/citología , Neuronas Motoras/química , Neuronas Motoras/citología , Proteínas/farmacología , ARN Mensajero/análisis

RESUMEN

Embryonic lumbar spinal motoneurons (MNs) are characterized by a period of programmed cell death (PCD) that spans several days and occurs in a rostrocaudal gradient. The generation of these MNs also takes place in a temporal-spatial gradient, such that MNs within rostral lumbar segments exit the cell cycle earlier and MNs within progressively caudal regions are born later. In vitro studies have shown that the latest born spinal MNs, presumably through the possession of endogenous "survival properties," are also the last to acquire their trophic dependence. If the birth date and therefore spinal cord location of lumbar spinal MNs influence the spatial-temporal pattern of PCD, then earlier born MNs should die sooner and be located more rostrally than those generated later. Alternatively, if the time at which MNs die during development is unrelated to their prior exit from the cell cycle, those born at various phases should die throughout the period of PCD. We report here that lumbar MNs generated during the earliest part (embryonic day 2-3) of the proliferative period in the developing chick spinal cord tend to die during the earliest stages of the PCD period and that MNs born in successive 12-h intervals die at correspondingly later periods during PCD. Furthermore, the spatial progression of PCD of these subpopulations of MNs occurs in a rostrocaudal gradient. Finally, while MNs do appear to die in a mediolateral gradient during the period of MN PCD, this pattern is only partly accounted for by MNs born in consecutive intervals. These data support the notion that the timing and rostrocaudal location of MNs undergoing PCD reflect their time of exit from the cell cycle.

Asunto(s)

Apoptosis/genética , Ciclo Celular/genética , Neuronas Motoras/metabolismo , Nervios Espinales/embriología , Animales , Bromodesoxiuridina , División Celular , Embrión de Pollo , Embrión no Mamífero/inervación , Inmunohistoquímica , Factores de Tiempo

RESUMEN

The caspases have been shown to be key components of programmed cell death (PCD) in various cell types, including neurons. Caspase-3 (CPP32) is the predominant caspase that appears to be involved in cell death in several systems. In embryonic motoneuron cultures, caspase-3 activity increases beginning at 20 h following deprivation of trophic support, as determined by the cleavage of its specific substrates. Inhibition of caspase-3 by peptide inhibitors prevents the PCD of motoneurons following trophic factor deprivation in vitro, as well as in vivo. We also investigated the cleavage of poly(ADP-ribose) polymerase (PARP) in motoneurons after trophic factor withdrawal. No PARP cleavage was detected in either viable or dying cells. These data suggest that some components of the cell death machinery such as the involvement of caspases may be conserved in different cell types undergoing PCD, whereas the activation and specific substrates of the caspases may differ from one cell type to another.

Asunto(s)

Caspasas/fisiología , Neuronas Motoras/fisiología , Proteínas del Tejido Nervioso/deficiencia , Animales , Caspasa 3 , Caspasas/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Células Cultivadas , Embrión de Pollo , Inhibidores de Cisteína Proteinasa/farmacología , Activación Enzimática/fisiología , Neuronas Motoras/efectos de los fármacos , Factores de Crecimiento Nervioso , Oligopéptidos/farmacología , Poli(ADP-Ribosa) Polimerasas/fisiología , Especificidad por Sustrato

RESUMEN

During development of the avian neuromuscular system, lumbar spinal motoneurons (MNs) innervate their muscle targets in the hindlimb coincident with the onset and progression of MN programmed cell death (PCD). Paralysis (activity blockade) of embryos during this period rescues large numbers of MNs from PCD. Because activity blockade also results in enhanced axonal branching and increased numbers of neuromuscular synapses, it has been postulated that following activity blockade, increased numbers of MNs can gain access to muscle-derived trophic agents that prevent PCD. An assumption of the access hypothesis of MN PCD is the presence of an activity-dependent, muscle-derived sprouting or branching agent. Several previous studies of sprouting in the rodent neuromuscular system indicate that insulin-like growth factors (IGFs) are candidates for such a sprouting factor. Accordingly, in the present study we have begun to test whether the IGFs may play a similar role in the developing avian neuromuscular system. Evidence in support of this idea includes the following: (a) IGFs promote MN survival in vivo but not in vitro; (b) neutralizing antibodies against IGFs reduce MN survival in vivo; (c) both in vitro and in vivo, IGFs increase neurite growth, branching, and synapse formation; (d) activity blockade increases the expression of IGF-1 and IGF-2 mRNA in skeletal muscles in vivo; (e) in vivo treatment of paralyzed embryos with IGF binding proteins (IGF-BPs) that interfere with the actions of endogenous IGFs reduce MN survival, axon branching, and synapse formation; (f) treatment of control embryos in vivo with IGF-BPs also reduces synapse formation; and (g) treatment with IGF-1 prior to the major period of cell death (i.e., on embryonic day 6) increases subsequent synapse formation and MN survival and potentiates the survival-promoting actions of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) administered during the subsequent 4- to 5-day period of PCD. Collectively, these data provide new evidence consistent with the role of the IGFs as activity-dependent, muscle-derived agents that play a role in regulating MN survival in the avian embryo.

Asunto(s)

Apoptosis/fisiología , Neuronas Motoras/fisiología , Músculo Esquelético/embriología , Músculo Esquelético/inervación , Somatomedinas/fisiología , Animales , Recuento de Células , Células Cultivadas , Embrión de Pollo , Miembro Posterior/inervación , Inmunohistoquímica , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/genética , Factor II del Crecimiento Similar a la Insulina/genética , Oligonucleótidos/farmacología , Terminales Presinápticos/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Somatomedinas/genética , Sinapsis/fisiología

RESUMEN

Experimental lesions have been used widely to induce motoneuron (MN) degeneration as a model to test the ability of different trophic molecules to prevent lesion-induced alterations. However, the morphological mechanisms of spinal MN death following different types of lesions is not clear at the present time. In this study, we have characterized the morphological characteristics of MN cell death by examining DNA fragmentation and the ultrastructural and light microscopic morphological features of MNs following different types of spinal nerve injury (i.e., axotomy and avulsion) in the developing and adult mouse. In neonatal mice, axotomy induced cell death as well as the atrophy of MNs that survived the injury. DNA fragmentation could be detected by using the terminal deoxynucleotidyl transferase (TUNEL) method during the cell death process following neonatal axotomy, whereas TUNEL labeling was not observed following either neonatal or adult avulsion. However, with the exception of TUNEL labeling, the morphological characteristics of MN death following neonatal axotomy and avulsion were similar, and both resembled most closely the form of programmed cell death termed cytoplasmic or type 3B, which exhibits similarities as well as differences with currently accepted definitions of apoptosis. By contrast, adult avulsion resulted in a type of degeneration that resembled necrosis more closely. However, even there, the morphology was mixed, showing characteristics of both apoptosis and necrosis. These results indicate that the mode of MN degeneration is complex and is related to developmental age and type of lesion.

Asunto(s)

Neuronas Motoras/patología , Degeneración Nerviosa/patología , Traumatismos de los Nervios Periféricos , Nervios Espinales/patología , Animales , Animales Recién Nacidos , Apoptosis/genética , Axotomía , Fragmentación del ADN , ADN Nucleotidilexotransferasa , Técnicas Genéticas , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica , Necrosis , Nervios Espinales/crecimiento & desarrollo

RESUMEN

We describe here a binary transgenic system based on Cre-mediated DNA recombination for genetic cell ablation in mice that enabled us to obtain skeletal muscle-deficient embryos by mating two phenotypically normal transgenic lines. In those embryos, skeletal muscles are eliminated as a consequence of the expression of the gene encoding the diphtheria toxin A fragment. Cell ablation occurs gradually beginning approximately on embryonic day (E) 12.5, and by E18-5 almost all skeletal muscle is absent. Analysis of the consequences of muscle cell ablation revealed that almost all spinal motoneurons are lost by E18.5, providing strong evidence that survival of spinal motoneurons during embryogenesis is dependent on signals from their target tissue, skeletal muscle, and that trophic signals produced by nonmuscle sources are sufficient to support survival of no more than 10% of embryonic spinal motoneurons in the absence of muscle-derived signals. There was also substantial loss of cranial (hypoglossal and facial) motoneurons in the muscle-deficient embryos, thus indicating that cranial motoneuron survival is also dependent on trophic signals produced by their target tissue. Although spinal motoneurons are a major target of spinal interneurons, the loss of motoneurons did not affect interneuron survival. Muscle-deficient embryos had a cleft palate and abnormalities of the lower jaw, raising the possibility that they might serve as a mouse model for the human disorder, Robin sequence. The data reported here demonstrate the utility of a binary transgenic system for obtaining mouse embryos in which a specific cell population has been ablated, so that its role in embryonic development can be studied.

Asunto(s)

Apoptosis/genética , ADN Recombinante/genética , Neuronas Motoras/citología , Músculo Esquelético/embriología , Proteínas Virales , Animales , Secuencia de Bases , Encéfalo/anomalías , Encéfalo/citología , Encéfalo/embriología , Toxina Diftérica/genética , Femenino , Humanos , Integrasas/genética , Integrasas/metabolismo , Interneuronas/citología , Masculino , Ratones , Ratones Transgénicos , Modelos Biológicos , Músculo Esquelético/anomalías , Músculo Esquelético/inervación , Miogenina/genética , Fragmentos de Péptidos/genética , Recombinación Genética , Transducción de Señal , Médula Espinal/anomalías , Médula Espinal/citología , Médula Espinal/embriología

RESUMEN

CEP-1347, also known as KT7515, a derivative of a natural product indolocarbazole, inhibited motor neuronal death in vitro, inhibited activation of the stress-activated kinase JNK1 (c-jun NH terminal kinase) in cultured spinal motor neurons, but had no effect on the mitogen-activated protein kinase ERK1 in these cells. Results reported here profile the functional activity of CEP-1347/KT7515 in vivo in models of motor neuronal death or dedifferentiation. Application of CEP-1347/KT7515 to the chorioallantoic membrane of embryonic chicks rescued 40% of the lumbar motor neurons that normally die during the developmental period assessed. Peripheral administration of low doses (0.5 and 1 mg/kg daily) of CEP-1347/KT7515 reduced death of motor neurons of the spinal nucleus of the bulbocavernosus in postnatal female rats, with efficacy comparable to testosterone. Strikingly, daily administration of CEP-1347/KT7515 during the 4-day postnatal window of motor neuronal death resulted in persistent long-term motor neuronal survival in adult animals that received no additional CEP-1347/KT7515. In a model of adult motor neuronal dedifferentiation following axotomy, local application of CEP-1347/KT7515 to the transected hypoglossal nerve substantially reduced the loss of choline acetyl transferase immunoreactivity observed 7 days postaxotomy compared to untreated animals. Results from these experiments demonstrate that a small organic molecule that inhibits a signaling pathway associated with stress and injury also reduces neuronal death and degeneration in vivo.

Asunto(s)

Apoptosis/efectos de los fármacos , Axotomía , Carbazoles/farmacología , Indoles/farmacología , Proteínas Quinasas Activadas por Mitógenos , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/fisiología , Animales , Animales Recién Nacidos/fisiología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Diferenciación Celular/efectos de los fármacos , Embrión de Pollo , Colina O-Acetiltransferasa/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Nervio Hipogloso/efectos de los fármacos , Nervio Hipogloso/enzimología , Nervio Hipogloso/patología , Proteínas Quinasas JNK Activadas por Mitógenos , Neuronas Motoras/patología , Ratas , Ratas Sprague-Dawley

RESUMEN

Unilateral limb-bud removal (LBR) before the outgrowth of sensory or motor neurons to the leg of chick embryos was used to examine the role of limb (target)-derived signals in the development and survival of lumbar motoneurons and sensory neurons in the dorsal root ganglia (DRG). After LBR, motor and sensory neurons underwent normal initial histological differentiation, and cell growth in both populations was unaffected. Before their death, target-deprived motoneurons also expressed a cell-specific marker, the homeodomain protein islet-1. Proliferation of sensory and motor precursor cells was also unaffected by LBR, and the migration of neural crest cells to the DRG and of motoneurons into the ventral horn occurred normally. During the normal period of programmed cell death (PCD), increased numbers of both sensory and motor neurons degenerated after LBR. However, whereas motoneuron loss increased by 40-50% (90% total), only approximately 25% more sensory neurons degenerated after LBR. A significant number of the surviving sensory neurons projected to aberrant targets in the tail after LBR, and many of these were lost after ablation of both the limb and tail. Treatment with neurotrophic factors (or muscle extract) rescued sensory and motor neurons from cell death after LBR without affecting precursor proliferation of either population. Activity blockade with curare failed to rescue motoneurons after LBR, and combined treatment with curare plus muscle extract was no more effective than muscle extract alone. Treatment with the antioxidant N-acetylcysteine rescued motoneurons from normal cell death but not after LBR. Two specific inhibitors of the interleukin beta1 converting enzyme (ICE) family of cysteine proteases also failed to prevent motoneuron death after LBR. Taken together these data provide definitive evidence that the loss of spinal neurons after LBR cannot be attributed to altered proliferation, migration, or differentiation. Rather, in the absence of limb-derived trophic signals, the affected neurons fail to survive and undergo PCD. Although normal cell death and cell death after target deprivation share many features in common, the intracellular pathways of cell death in the two may be distinct.

Asunto(s)

Neuronas Motoras/citología , Neuronas Aferentes/citología , Médula Espinal/citología , Médula Espinal/embriología , Acetilcisteína/farmacología , Clorometilcetonas de Aminoácidos/farmacología , Animales , Axones/fisiología , Factor Neurotrófico Derivado del Encéfalo/farmacología , Recuento de Células , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Embrión de Pollo , Inhibidores de Cisteína Proteinasa/farmacología , Depuradores de Radicales Libres/farmacología , Ganglios Espinales/citología , Esbozos de los Miembros/citología , Esbozos de los Miembros/cirugía , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/ultraestructura , Factores de Crecimiento Nervioso/farmacología , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/ultraestructura , Neurotrofina 3 , Oligopéptidos/farmacología

RESUMEN

The neurons of the dorsal root ganglia (DRG) that supply muscle spindles require target-derived factors for survival. One necessary factor for these neurons is neurotrophin-3 (NT3). To determine whether NT3 can promote the survival of these neurons in the absence of other target-derived factors, we analyzed the effects of exogenous NT3 after early limb bud deletion in the chick. In control embryos, limb bud deletion eliminated approximately 90% of the trkC-positive (trkC+) neurons in lumbar DRG on the deleted side. In addition, the deletion led to a dramatic loss of collateral sensory projections to motoneurons. Exogenous NT3 restored a normal population of trkC+ neurons in lumbar DRG on the deleted side and increased the number of trkC+ neurons in DRG with normal targets (contralateral lumbar and thoracic). The effect was highly selective; NT3 increased the number of trkC+ neurons without significantly changing the number of either trkA+ or trkB+ neurons. The effect of NT3 was attributable to the rescue of DRG neurons from cell death, because exogenous NT3 reduced the number of pyknotic nuclei without significantly altering proliferation. Analysis of spinal projections showed further that many of the trkC+ neurons rescued by NT3 projected to the ventral spinal cord. These neurons thus had central projections characteristic of muscle spindle afferents. Together, our results indicate that NT3 signaling is both necessary and sufficient for the development of the proprioceptive phenotype, even in the absence of other signals from limb muscle.

Asunto(s)

Husos Musculares/citología , Factores de Crecimiento Nervioso/farmacología , Neuronas Aferentes/citología , Animales , Especificidad de Anticuerpos , Diferenciación Celular/efectos de los fármacos , Línea Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Embrión de Pollo , Ganglios Espinales/citología , Humanos , Riñón/citología , Esbozos de los Miembros/cirugía , Husos Musculares/efectos de los fármacos , Husos Musculares/fisiología , Neuronas Aferentes/química , Neuronas Aferentes/efectos de los fármacos , Neurotrofina 3 , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas/inmunología , Proteínas Tirosina Quinasas Receptoras/análisis , Proteínas Tirosina Quinasas Receptoras/inmunología , Receptor de Factor Neurotrófico Ciliar , Receptor trkA , Receptor trkC , Receptores de Factor de Crecimiento Nervioso/análisis , Receptores de Factor de Crecimiento Nervioso/inmunología , Médula Espinal/citología , Factores de Tiempo , Transfección

RESUMEN

Neuromuscular transmission and muscle activity during early stages of embryonic development are known to influence the differentiation and survival of motoneurons and to affect interactions with their muscle targets. We have examined neuromuscular development in an avian genetic mutant, crooked neck dwarf (cn/cn), in which a major phenotype is the chronic absence of the spontaneous, neurally mediated movements (motility) that are characteristic of avian and other vertebrate embryos and fetuses. The primary genetic defect in cn/cn embryos responsible for the absence of motility appears to be the lack of excitation-contraction coupling. Although motility in mutant embryos is absent from the onset of activity on embryonic days (E) 3-4, muscle differentiation appears histologically normal up to about E8. After E8, however, previously separate muscles fuse or coalesce secondarily, and myotubes exhibit a progressive series of histological and ultrastructural degenerative changes, including disarrayed myofibrils, dilated sarcoplasmic vesicles, nuclear membrane blebbing, mitochondrial swelling, nuclear inclusions, and absence of junctional end feet. Mutant muscle cells do not develop beyond the myotube stage, and by E18-E20 most muscles have almost completely degenerated. Prior to their breakdown and degeneration, mutant muscles are innervated and synaptic contacts are established. In fact, quantitative analysis indicates that, prior to the onset of muscle degeneration, mutant muscles are hyperinnervated. There is increased branching of motoneuron axons and an increased number of synaptic contacts in the mutant muscle on E8. Naturally occurring cell death of limb-innervating motoneurons is also significantly reduced in cn/cn embryos. Mutant embryos have 30-40% more motoneurons in the brachial and lumbar spinal cord by the end of the normal period of cell death. Electrophysiological recordings (electromyographic and direct records form muscle nerves) failed to detect any differences in the activity of control vs. mutant embryos despite the absence of muscular contractile activity in the mutant embryos. The alpha-ryanodine receptor that is genetically abnormal in homozygote cn/cn embryos is not normally expressed in the spinal cord. Taken together, these data argue against the possibility that the mutant phenotype described here is caused by the perturbation of a central nervous system (CNS)-expressed alpha-ryanodine receptor. The hyperinnervation of skeletal muscle and the reduction of motoneuron death that are observed in cn/cn embryos also occur in genetically paralyzed mouse embryos and in pharmacologically paralyzed avian and rat embryos. Because a primary common feature in all three of these models is the absence of muscle activity, it seems likely that the peripheral excitation of muscle by motoneurons during normal development is a major factor in regulating retrograde muscle-derived (or muscle-associated) signals that control motoneuron differentiation and survival.

Asunto(s)

Embrión de Pollo/fisiología , Neuronas Motoras/citología , Mutación , Unión Neuromuscular/fisiología , Animales , Especificidad de Anticuerpos , Canales de Calcio/análisis , Canales de Calcio/inmunología , Proteínas de Unión a Calmodulina/análisis , Recuento de Células , Muerte Celular/fisiología , Supervivencia Celular/fisiología , Electrofisiología , Heterocigoto , Microscopía Electrónica , Neuronas Motoras/química , Neuronas Motoras/fisiología , Fibras Musculares Esqueléticas/ultraestructura , Proteínas Musculares/análisis , Proteínas Musculares/inmunología , Músculo Esquelético/citología , Músculo Esquelético/embriología , Músculo Esquelético/inervación , Enfermedades del Sistema Nervioso/fisiopatología , Unión Neuromuscular/ultraestructura , Parálisis/genética , Canal Liberador de Calcio Receptor de Rianodina , Médula Espinal/embriología , Médula Espinal/fisiopatología

RESUMEN

During neuromuscular development, neuronal contact with peripheral targets is associated with an increase in synaptic vesicle protein (SVP) gene expression, suggesting that target contact and upregulation of SVP genes are causally related. To test this idea, we analyzed the developmental expression pattern of synaptotagmin (syt) mRNAs in the chick lateral motor column (LMC) using in situ hybridization. Syt I mRNA in the LMC is upregulated from Embryonic Day 4.5 (E4.5) to E5.5, coincident with the time these neurons begin to make contact with their muscle targets. In contrast, levels of mRNA for neurofilament do not change during this time. Extirpation of the limb bud prior to motor axon outgrowth eliminates the increase in syt I mRNA ipsilaterally. Later in development, there is a switch in syt isoform abundance in the LMC, with syt II mRNA being upregulated between E15 and E20 and syt I mRNA being downregulated. Our results suggest that contact with targets upregulates syt I gene expression during neuromuscular synapse formation in vivo, and that a later stage of synaptic maturation involves changes in SVP isoform abundance.

Asunto(s)

Proteínas de Unión al Calcio , Regulación de la Expresión Génica , Glicoproteínas de Membrana/genética , Neuronas Motoras/fisiología , Proteínas del Tejido Nervioso/genética , Médula Espinal/embriología , Animales , Embrión de Pollo/fisiología , Desarrollo Embrionario y Fetal , Proteínas de la Membrana/genética , Vías Nerviosas/embriología , Proteínas de Neurofilamentos/genética , ARN Mensajero/metabolismo , Médula Espinal/citología , Sinaptotagmina II , Sinaptotagminas

RESUMEN

During normal development of many vertebrate species, substantial numbers of neurons in the central and peripheral nervous system undergo naturally occurring (or programmed) cell death. For example, approximately 50% of spinal motoneurons degenerate and die at a time when these cells are establishing synaptic connections with their target muscles in the chick, mouse, rat, and human. It is generally thought that the survival of developing motoneurons depends on access to trophic molecules. Motoneurons that survive the period of programmed cell death may also die following injury in the developing or adult animal. Increasing evidence suggests that glial-cell-line-derived neurotrophic factor (GDNF) plays a physiological and/or pharmacological role in the survival of various neuronal cell types, including motoneurons. In this paper, we review the survival and growth-promoting effects of GDNF on spinal motoneurons during the period of programmed cell death and following injury.

Asunto(s)

Neuronas Motoras/efectos de los fármacos , Factores de Crecimiento Nervioso , Proteínas del Tejido Nervioso/farmacología , Fármacos Neuroprotectores/farmacología , Médula Espinal/citología , Animales , Axones/fisiología , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Embrión de Pollo , Factor Neurotrófico Derivado de la Línea Celular Glial , Ratones , Ratones Endogámicos BALB C , Neuronas Motoras/citología , Neuronas Motoras/ultraestructura , Degeneración Nerviosa/efectos de los fármacos , Rizotomía , Médula Espinal/embriología , Médula Espinal/cirugía