RESUMEN
Cyclin-dependent kinase 5 is predominantly expressed in postmitotic neurons and plays a role in neurite elongation during development. It has also been postulated to play a role in apoptosis in a variety of cells, including neurons, but little is known about the generality and functional significance of cdk5 expression in neuronal apoptosis in living brain. We have therefore examined its expression and that of its known activators, p35, p39 and p67, in models of induced apoptosis in neurons of the substantia nigra. We find that cdk5 is expressed in apoptotic profiles following intrastriatal injection of 6-hydroxydopamine and axotomy. It is expressed exclusively in profiles which are in late morphologic stages of apoptosis. In these late stages, derivation of the profiles from neurons, and localization of expression to the nucleus, can be demonstrated by co-labeling with a neuron-specific nuclear marker, NeuN. In another model of induced apoptotic death in nigra, produced by developmental striatal lesion, kinase activity increases in parallel with cell death. While mRNAs for all three cdk5 activators are expressed in nigra during development, only p35 protein is expressed in apoptotic profiles. We conclude that cdk5/p35 expression is a general feature of apoptotic neuron death in substantia nigra neurons in vivo.
Asunto(s)
Apoptosis/fisiología , Quinasas Ciclina-Dependientes/genética , Proteínas del Tejido Nervioso/genética , Neuronas/enzimología , Sustancia Negra/citología , Proteínas de Transporte Vesicular , Animales , Anticuerpos , Axotomía , Quinasa 5 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Epítopos/análisis , Epítopos/inmunología , Regulación Enzimológica de la Expresión Génica , Haz Prosencefálico Medial/fisiología , Proteínas Munc18 , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Neurofilamentos/análisis , Proteínas de Neurofilamentos/inmunología , Neuronas/citología , Oxidopamina , Fosforilación , Ácido Quinolínico , ARN Mensajero/análisis , Ratas , Sustancia Negra/metabolismo , Simpaticolíticos , Proteínas tau/análisis , Proteínas tau/inmunología , Proteínas tau/metabolismoRESUMEN
Apoptosis is a form of cell death in which genetically regulated programs intrinsic to the cell bring about its own demise. In recent years, there has been a tremendous growth of interest in apoptosis as a mechanism of disease in a wide range of human disorders including the neurodegenerative diseases, such as Parkinson's disease (PD) (1). This growth of interest has spawned an extraordinary number of recent discoveries about the molecular basis of apoptosis. It is important to emphasize, however, that much of this new knowledge has been attained in the study of relatively simple systems, such as invertebrate models or mammalian nonneural cell culture systems. Less is known about these mechanisms in neural cells, and much of what is known is based on studies of peripheral neural cells (such as sympathetic ganglia and PC 12 cells) in tissue culture. Much less is known about central neurons; in particular, we know little about the regulation of apoptotic death in central neurons in living animals. It is especially important to try to identify the mechanisms of cell death in central neurons of known phenotype, particularly those implicated in human neurodegenerative disease, such as the dopamine neurons in PD. The purpose of the models we have developed of induced apoptosis in dopamine neurons of the substantia nigra (SN) is to try to translate what is being learned about the molecular mechanisms of apoptosis in simpler systems to these neurons.
RESUMEN
In the preceding chapter we described three paradigms by which we have studied programmed cell death in the substantia nigra (SN) of living animals: natural cell death and death induced either by developmental injury to the target striatum or by dopamine terminal destruction with the neurotoxin 6-hydroxydopamine (6-OHDA). In each of these paradigms, in order to relate experimental investigations specifically to dopamine neurons, the dopaminergic phenotype must be identified in conjunction with the demonstration of apoptotic morphology. This identification is essential, because apoptosis has been recognized in diverse neuronal populations and in glia (1).
RESUMEN
We have previously shown that apoptotic cell death can be induced in substantia nigra (SN) by developmental striatal target lesion. In this model, only a portion of nigral neurons dies, so it provides a paradigm to examine not only the molecular basis of cell death, but also the cellular responses of adjacent neurons which survive. Using a differential display approach, we have found that cytosolic branched chain aminotransferase (BCATc) mRNA is upregulated in SN in this model. This upregulation is associated with an increased number of BCATc-positive neuronal profiles, demonstrated by immunostaining. BCATc-positive neurons show normal morphology and rarely contain apoptotic chromatin. We conclude that BCATc is upregulated in neurons, which are likely to survive, and plays a role in either maintenance of viability or restoration of normal function.
Asunto(s)
Apoptosis , Sustancia Negra/enzimología , Transaminasas/metabolismo , Animales , Northern Blotting , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/patología , Citosol/enzimología , Datos de Secuencia Molecular , Ácido Quinolínico/farmacología , ARN Mensajero/metabolismo , Ratas , Sustancia Negra/metabolismo , Transaminasas/genética , Regulación hacia ArribaRESUMEN
Human alpha-synuclein was identified on the basis of proteolytic fragments derived from senile plaques of Alzheimer's disease, and it is the locus of mutations in some familial forms of Parkinson's disease. Its normal function and whether it may play a direct role in neural degeneration remain unknown. To explore cellular responses to neural degeneration in the dopamine neurons of the substantia nigra, we have developed a rodent model of apoptotic death induced by developmental injury to their target, the striatum. We find by mRNA differential display that synuclein is up-regulated in this model, and thus it provides an opportunity to examine directly whether synuclein plays a role in the death of these neurons or, alternatively, in compensatory responses. Up-regulation of mRNA is associated with an increase in the number of neuronal profiles immunostained for synuclein protein. At a cellular level, synuclein is almost exclusively expressed in normal neurons, rather than apoptotic profiles. Synuclein is up-regulated throughout normal postnatal development of substantia nigra neurons, but it is not further up-regulated during periods of natural cell death. We conclude that up-regulation of synuclein in the target injury model is unlikely to mediate apoptotic death and propose that it may be due to a compensatory response in neurons destined to survive.
Asunto(s)
Dopamina/metabolismo , Regulación del Desarrollo de la Expresión Génica , Degeneración Nerviosa/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Enfermedad de Parkinson/metabolismo , ARN Mensajero/biosíntesis , Sustancia Negra/metabolismo , Secuencia de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Secuencia de Bases , Northern Blotting , Western Blotting , Células Cultivadas , Perfilación de la Expresión Génica , Genes , Hibridación in Situ , Datos de Secuencia Molecular , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/genética , Proteínas del Tejido Nervioso/genética , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neurotoxinas/toxicidad , Ácido Quinolínico/toxicidad , Empalme del ARN , ARN sin Sentido/farmacología , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sustancia Negra/efectos de los fármacos , Sustancia Negra/crecimiento & desarrollo , Sustancia Negra/patología , Técnica de Sustracción , Sinucleínas , alfa-SinucleínaRESUMEN
We have previously shown that synuclein is upregulated in the substantia nigra following developmental lesion of the target striatum. In this model, synuclein is preferentially expressed in normal-appearing neurons, rather than those which undergo apoptosis. It has been proposed, however, that synuclein may mediate apoptosis in other contexts, such as that induced by neurotoxins. To examine this possibility, we have studied a model in which apoptosis is induced in dopamine neurons by intrastriatal injection of 6-hydroxydopamine. In this model synuclein mRNA and protein expression are not upregulated at any time point, but instead diminish as dopamine neurons die. We observe a complete dissociation between apoptotic morphology and synuclein protein expression. We conclude that synuclein is unlikely to play a direct role in apoptotic death in dopamine neurons and is more likely, in the target injury model, to play a role in protection or restoration of neurons which survive.
Asunto(s)
Apoptosis , Cuerpo Estriado/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Oxidopamina/farmacología , Sustancia Negra/metabolismo , Animales , Apoptosis/efectos de los fármacos , Northern Blotting , Inmunohistoquímica , Neuronas/metabolismo , ARN Mensajero/metabolismo , Ratas , Sustancia Negra/patología , SinucleínasRESUMEN
Programmed cell death has been proposed to play a role in the death of neurons in acute and chronic degenerative neurologic disease. There is now evidence that the caspases, a family of cysteine proteases, mediate programmed cell death in various cells. In neurons, caspase-3 (CPP32/Yama/apopain), in particular, has been proposed to play a role. We examined the expression of caspase-3 in three models of programmed cell death affecting neurons of the substantia nigra in the rat: natural developmental neuron death and induced developmental death following either striatal target injury with quinolinic acid or dopamine terminal lesion with intrastriatal injection of 6-hydroxydopamine. Using an antibody to the large (p17) subunit of activated caspase-3, we have found that activated enzyme is expressed in apoptotic profiles in all models. Increased p17 immunostaining correlated with increased enzyme activity. The subcellular distribution of activated caspase-3 differed among the models: In natural cell death and the target injury model, it was strictly nuclear, whereas in the toxin model, it was also cytoplasmic. We conclude that p17 immunostaining is a useful marker for programmed cell death in neurons of the substantia nigra.
Asunto(s)
Apoptosis , Caspasas/metabolismo , Modelos Biológicos , Neuronas/enzimología , Sustancia Negra/citología , Animales , Northern Blotting , Caspasa 3 , Caspasas/genética , Cuerpo Estriado/efectos de los fármacos , Dopamina/farmacología , Activación Enzimática , Expresión Génica , Oxidopamina/farmacología , Ácido Quinolínico/farmacología , ARN Mensajero/análisis , Ratas , Sustancia Negra/efectos de los fármacos , Sustancia Negra/enzimologíaRESUMEN
The transcription factors c-fos and c-jun have been proposed to play a role in the initiation of programmed cell death in neurons. We have shown that programmed cell death, with the morphology of apoptosis, occurs in dopamine neurons of the substantia nigra (SN) during normal postnatal development and that this death event can be induced by early striatal target injury. We have investigated the relationship between c-fos and c-jun protein expression and induced death in neurons of the SN. Although c-fos is induced, it is unlikely to play a role in cell death, because its expression is not well correlated with apoptotic death either temporally or at a cellular level. Expression of c-jun, however, is both temporally and regionally correlated with induction of death, and, at a cellular level, it colocalizes with apoptotic morphology. The increased expression of c-jun is likely to be functionally significant, because it is associated with increased c-jun N-terminal kinase (JNK) and phosphorylated c-jun expression. JNK expression also colocalizes with apoptotic morphology. We conclude that c-jun is likely to play a role in the initiation of apoptotic cell death in these neurons.
Asunto(s)
Apoptosis/fisiología , Proteínas Quinasas JNK Activadas por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos , Neuronas/enzimología , Proteínas Quinasas/biosíntesis , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Proteínas Proto-Oncogénicas c-jun/biosíntesis , Animales , Dopamina/fisiología , Femenino , MAP Quinasa Quinasa 4 , Degeneración Nerviosa/inducido químicamente , Neuronas/citología , Neurotoxinas/farmacología , Enfermedad de Parkinson/metabolismo , Embarazo , Ácido Quinolínico , Ratas , Sustancia Negra/citología , Sustancia Negra/embriologíaRESUMEN
Many developing neural systems with peripheral projections depend on their target for trophic support during a critical period of natural cell death. Much less is known about central systems. That dopaminergic neurons of the substantia nigra may depend on their target, the striatum, during development is suggested by the presence of a natural apoptotic cell death event in these neurons that can be augmented by an early developmental axon-sparing striatal injury. To further assess the target dependence of these neurons, we have used the selective neurotoxin 6-hydroxydopamine to lesion their terminals within the striatum during development, while sparing intrinsic striatal target neurons. This lesion results in an induction of apoptotic cell death in phenotypically defined dopaminergic neurons that appears on the third postlesion day and persists until the tenth. This inducibility of cell death is dependent on developmental age: it is most marked before postnatal day (PND) 14. As late as PND42, inducibility is still detectable but much less so. In addition, at day 42 the morphology of cell death changes and becomes nonapoptotic in some cells. We conclude that terminal injury during a critical period of postnatal development, like axon-sparing target injury, induces augmented apoptotic death in mesencephalic dopaminergic neurons. These results suggest that these neurons have a period of target dependence. Regulation of this dependence is likely to influence the mature adult number of dopaminergic neurons.
Asunto(s)
Apoptosis/fisiología , Cuerpo Estriado/crecimiento & desarrollo , Dopamina/metabolismo , Terminales Presinápticos/fisiología , Sustancia Negra/crecimiento & desarrollo , Sustancia Negra/fisiología , Animales , Femenino , Oxidopamina/farmacología , Embarazo , RatasRESUMEN
We have previously shown that apoptotic natural cell death occurs within the substantia nigra (SN) pars compacta of the rat postnatally. However, the occurrence of natural cell death in phenotypically defined dopaminergic neurons has not previously been identified, nor has its time course been defined in pre- or postnatal development. We therefore examined the SN at intervals from E19 to P28 using immunostaining for tyrosine hydroxylase with a Nissl counterstain to identify intranuclear apoptotic chromatin clumps. We have found that natural cell death in dopaminergic neurons is biphasic. An initial, broad peak begins at E20, reaches maximum at P2, and abates by P8. A second peak occurs at P14. We conclude that most of the natural cell death in this neuronal population occurs in the early postnatal period.
Asunto(s)
Dopamina/análisis , Neuronas/química , Sustancia Negra/crecimiento & desarrollo , Tirosina 3-Monooxigenasa/análisis , Animales , Muerte Celular/fisiología , Desarrollo Embrionario y Fetal/fisiología , Inmunohistoquímica , Neuronas/citología , Neuronas/ultraestructura , Fenotipo , Ratas , Coloración y Etiquetado , Sustancia Negra/citología , Sustancia Negra/embriología , Factores de TiempoRESUMEN
Weaver is a spontaneous mutation in mice characterized by the postnatal loss of external granule cells in the cerebellum and dopaminergic neurons of the midbrain, especially in the substantia nigra. We have shown previously that natural cell death with the morphology of apoptosis occurs in the substantia nigra of normal rodents during postnatal development. We therefore sought to determine whether the loss of dopaminergic neurons in homozygous weaver mice occurs during the period of natural cell death in the substantia nigra and whether it has the morphology of apoptosis. We have found, using a silver stain technique, that although apoptotic cell death does occur early postnatally in homozygous weaver substantia nigra, it also does so with equal magnitude in wild-type and heterozygous weaver littermates. Unique to homozygous weavers is the occurrence of degenerating neurons in the nigra that are not apoptotic. These degenerating neurons are observed at postnatal day 7, and they are most abundant on postnatal days 24-25. The nonapoptotic nature of this cell death is confirmed by negative in situ end labeling of nuclear DNA fragmentation and by ultrastructural analysis. Ultrastructural studies reveal irregular chromatin aggregates in the nucleus, as well as marked cytoplasmic changes, including the formation of vacuoles and distinctive stacks of dilated cisternae of endoplasmic reticulum. We interpret these changes as indicative of either a variant morphology of programmed cell death or a pathological degenerative process mediated by an as yet unknown mechanism related to the recently described mutation in the GIRK2 potassium channel.
Asunto(s)
Ratones Mutantes Neurológicos/fisiología , Neuronas/fisiología , Sustancia Negra/citología , Envejecimiento/fisiología , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Animales Recién Nacidos/fisiología , Apoptosis , Muerte Celular , Ratones , Ratones Mutantes Neurológicos/crecimiento & desarrollo , Microscopía Electrónica , Neuronas/ultraestructura , Plata , Coloración y Etiquetado , Sustancia Negra/ultraestructuraRESUMEN
We have previously shown that developmental hypoxic-ischemic injury in a unilateral rodent model leads to an increase in both morphologic and biochemical indices of striatal cholinergic neurons. To investigate the functional significance these changes, we have used the in vivo microdialysis technique to examine the regulation of striatal acetylcholine release in awake, adult rats following postnatal hypoxic-ischemic injury. We have found that injury does not alter basal release or acetylcholine, but it results in a marked augmentation in the increase of acetylcholine release normally observed after infusion of atropine or pirenzepine.
Asunto(s)
Acetilcolina/metabolismo , Isquemia Encefálica/tratamiento farmacológico , Cuerpo Estriado/efectos de los fármacos , Hipoxia Encefálica/tratamiento farmacológico , Antagonistas Muscarínicos/farmacología , Análisis de Varianza , Animales , Atropina/metabolismo , Atropina/farmacología , Metabolismo Basal , Isquemia Encefálica/metabolismo , Cuerpo Estriado/crecimiento & desarrollo , Cuerpo Estriado/metabolismo , Femenino , Hipoxia Encefálica/metabolismo , Microdiálisis , Antagonistas Muscarínicos/metabolismo , Pirenzepina/metabolismo , Pirenzepina/farmacología , Ratas , Ratas Sprague-Dawley , Estimulación QuímicaRESUMEN
We have previously observed that either hypoxic-ischemic or excitotoxic striatal injury during development is associated with a reduction in the adult number of dopaminergic neurons in the substantia nigra. This decrease occurs in the presence of preserved striatal dopaminergic markers and in the absence of direct nigral injury. We have also observed that natural cell death, with the morphology of apoptosis, occurs in the substantia nigra, and that there is an induced apoptotic cell death event following early striatal excitotoxic injury. We now report that forebrain hypoxic-ischemic injury is also associated with an induced cell death event in the substantia nigra, with both morphological and histochemical features of apoptosis. Induced apoptotic cell death occurs in immunohistochemically defined dopaminergic neurons. While the mechanisms for this induced cell death are not yet known, in the case of the pars compacta it may be related to the loss of normal striatal target-derived developmental support. Since dopaminergic neurons are postmitotic at the time of the injury, we conclude that this induced cell death is responsible for the diminished adult number of dopaminergic neurons. We also conclude that hypoxic-ischemic injury to the developing brain in general causes not only direct, necrotic injury to vulnerable regions, but also induced apoptotic death at remote sites. The significance of this finding is that apoptosis is a distinct death mechanism, with unique regulatory pathways, which can potentially be modified by approaches different from those which might influence cell death in regions of direct injury. In view of the present finding that apoptosis can occur in the setting of hypoxic-ischemic injury, and our previous work demonstrating its occurrence following excitotoxic injury, it seems likely that it may occur following other forms of injury to the immature brain in which excitotoxic injury plays a role, such as seizures, head trauma and hypoglycemia.