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1.
J Cell Biol ; 123(5): 1207-22, 1993 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-7503996

RESUMEN

The time course of molecular events that accompany degeneration and death after nerve growth factor (NGF) deprivation and neuroprotection by NGF and other agents was examined in cultures of NGF-dependent neonatal rat sympathetic neurons and compared to death by apoptosis. Within 12 h after onset of NGF deprivation, glucose uptake, protein synthesis, and RNA synthesis fell precipitously followed by a moderate decrease of mitochondrial function. The molecular mechanisms underlying the NGF deprivation-induced decrease of protein synthesis and neuronal death were compared and found to be different, demonstrating that this decrease of protein synthesis is insufficient to cause death subsequently. After these early changes and during the onset of neuronal atrophy, inhibition of protein synthesis ceased to halt neuronal degeneration while readdition of NGF or a cAMP analogue remained neuroprotective for 6 h. This suggests a model in which a putative killer protein reaches lethal levels several hours before the neurons cease to respond to readdition of NGF with survival and become committed to die. Preceding loss of viability by 5 h and concurrent with commitment to die, the neuronal DNA fragmented into oligonucleosomes. The temporal and pharmacological characteristics of DNA fragmentation is consistent with DNA fragmentation being part of the mechanism that commits the neuron to die. The antimitotic and neurotoxin cytosine arabinoside induced DNA fragmentation in the presence of NGF, supporting previous evidence that it mimicked NGF deprivation-induced death closely. Thus trophic factor deprivation-induced death occurs by apoptosis and is an example of programmed cell death.


Asunto(s)
Apoptosis/fisiología , Ganglios Simpáticos/fisiología , Factores de Crecimiento Nervioso/fisiología , Neuronas/fisiología , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Células Cultivadas , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Cicloheximida/farmacología , Desoxiglucosa/metabolismo , Metabolismo Energético/efectos de los fármacos , Ganglios Simpáticos/citología , Ganglios Simpáticos/efectos de los fármacos , Factores de Crecimiento Nervioso/deficiencia , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , ARN/metabolismo , Ratas , Tionucleótidos/farmacología , Factores de Tiempo
2.
J Cell Biol ; 108(2): 729-36, 1989 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-2918029

RESUMEN

The nucleotide sequence of human thrombospondin (TS) mRNA has been determined from human fibroblast and endothelial cDNAs. The sequence of 5802 bp begins 110 bp upstream from the initiator codon and includes the entire 3' untranslated region (UTR) of the mRNA. The coding region (3510 bp) specifies a protein of 1170 amino acids with all of the known features of the TS subunit (Frazier, W. A. 1987. J. Cell Biol. 105:625-632). The long 3' UTR of 2166 nucleotides is extremely A/T-rich, particularly in the latter half. It contains 37 TATT or ATTT(A) sequences that have been suggested as mediators of the stability of mRNAs for cytokines, lymphokines, and oncogenes (Shaw, G., and R. Kamen. 1986. Cell. 46:659-667). Another unusual feature of the 3' UTR of TS mRNA is a stretch of 42 nucleotides of which 40 are thymidines (uridine in the mRNA) including an uninterrupted sequence of 26 thymidines. This region is flanked by two sets of direct repeats suggesting that it may be an insertion element of retrotranscriptional origin. Comparison of the 3' untranslated region of TS mRNA with the GenBank data base indicates the greatest degree of similarity with an alpha-interferon gene which contains a number of the TATT/ATTT consensus sites. The degree of similarity between the TS and interferon sequences is the same in regions of the interferon gene corresponding to its coding and noncoding regions suggesting that most of the TS 3' UTR may be derived from an interferon gene or pseudogene. The features of the TS mRNA 3' UTR provide a potential explanation for the rapid regulation of TS message observed in cultured cells in response to PDGF and suggest that TS is a member of a group of proteins which are intimately involved in the control of cell growth and differentiation.


Asunto(s)
Glicoproteínas/genética , ARN Mensajero/genética , Secuencias Reguladoras de Ácidos Nucleicos , Secuencia de Aminoácidos , Secuencia de Bases , Sondas de ADN , Endotelio Vascular/análisis , Fibroblastos/análisis , Humanos , Interferón Tipo I/genética , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Biosíntesis de Proteínas , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Ácido Nucleico , Trombospondinas
3.
J Cell Biol ; 135(5): 1341-54, 1996 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-8947555

RESUMEN

Sympathetic neurons undergo programmed cell death (PCD) when deprived of NGF. We used an inhibitor to examine the function of interleukin-1 beta-converting enzyme (ICE) family proteases during sympathetic neuronal death and to assess the metabolic and genetic status of neurons saved by such inhibition. Bocaspartyl(OMe)-fluoromethylketone (BAF), a cell-permeable inhibitor of the ICE family of cysteine proteases, inhibited ICE and CPP32 (IC50 approximately 4 microM) in vitro and blocked Fas-mediated apoptosis in thymocytes (EC50 approximately 10 microM). At similar concentrations, BAF also blocked the NGF deprivation-induced death of rat sympathetic neurons in culture. Compared to NGF-maintained neurons, BAF-saved neurons had markedly smaller somas and maintained only basal levels of protein synthesis; readdition of NGF restored growth and metabolism. Although BAF blocked apoptosis in sympathetic neurons, it did not prevent the fall in protein synthesis or the increase in the expression of c-jun, c-fos, and other mRNAs that occur during neuronal PCD, implying that the ICE-family proteases function downstream of these events during PCD.NGF and BAF rescued sympathetic neurons with an identical time course, suggesting that NGF, in addition to inhibiting metabolic and genetic events associated with neuronal PCD, can act posttranslationally to abort apoptosis at a time point indistinguishable from the activation of cysteine proteases. Both poly-(ADP ribose) polymerase and pro-ICE and Ced-3 homolog-1 (ICH-1) appear to be cleaved in a BAF-inhibitable manner, although the majority of pro-CPP32 appears unchanged, suggesting that ICH-1 is activated during neuronal PCD. Potential implications of these findings for anti-apoptotic therapies are discussed.


Asunto(s)
Clorometilcetonas de Aminoácidos/farmacología , Apoptosis , Caspasas , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Factores de Crecimiento Nervioso/fisiología , Neuronas/fisiología , Animales , Apoptosis/efectos de los fármacos , Caspasa 1 , Caspasa 2 , Caspasa 3 , Núcleo Celular/metabolismo , Fragmentación del ADN/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Genes fos , Genes jun , Ratones , Ratones Endogámicos C57BL , Factores de Crecimiento Nervioso/farmacología , Neuronas/citología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas , Ganglio Cervical Superior/citología , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Factores de Tiempo , Receptor fas/fisiología
4.
Neuron ; 14(2): 303-15, 1995 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-7857640

RESUMEN

Sympathetic neurons in culture die by apoptosis when deprived of nerve growth factor (NGF). We used this model of programmed cell death to study the mechanisms that mediate neuronal apoptosis. Cultured sympathetic neurons were injected with copper/zinc superoxide dismutase protein (SOD) or with an expression vector containing an SOD cDNA. In both cases apoptosis was delayed when the neurons were deprived of NGF. The delay was similar to that seen when a bcl-2 expression vector was injected. SOD, injected 8 hr after NGF deprivation, provided no protection, indicating that superoxide production may occur early in response to trophic factor deprivation. We have demonstrated, with a redox-sensitive dye, an increase in reactive oxygen species (ROS) that peaked at 3 hr after sympathetic neurons were deprived of NGF. If NGF was added back to the culture medium after the period of peak ROS generation, apoptosis was completely prevented, suggesting that ROS production serves as an early signal, rather than a toxic agent, to mediate apoptosis.


Asunto(s)
Apoptosis/fisiología , Neuronas/citología , Especies Reactivas de Oxígeno , Ganglio Cervical Superior/citología , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa/farmacología , Animales , Apoptosis/efectos de los fármacos , Secuencia de Bases , Bovinos , Células Cultivadas , Cartilla de ADN , ADN Complementario , Expresión Génica , Vectores Genéticos , Humanos , Cinética , Datos de Secuencia Molecular , Factores de Crecimiento Nervioso/farmacología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Proteínas Tirosina Quinasas/biosíntesis , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2 , Especies Reactivas de Oxígeno/farmacología , Ganglio Cervical Superior/fisiología , Superóxido Dismutasa/biosíntesis , Factores de Tiempo , Transfección
5.
Neuron ; 17(3): 401-11, 1996 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-8816704

RESUMEN

Members of the BCL2-related family of proteins either promote or repress programmed cell death. BAX, a death-promoting member, heterodimerizes with multiple death-repressing molecules, suggesting that it could prove critical to cell death. We tested whether Bax is required for neuronal death by trophic factor deprivation and during development. Neonatal sympathetic neurons and facial motor neurons from Bax-deficient mice survived nerve growth factor deprivation and disconnection from their targets by axotomy, respectively. These salvaged neurons displayed remarkable soma atrophy and reduced elaboration of neurities; yet they responded to readdition of trophic factor with soma hypertrophy and enhanced neurite outgrowth. Bax-deficient superior cervical ganglia and facial nuclei possessed increased numbers of neurons. Our observations demonstrate that trophic factor deprivation-induced death of sympathetic and motor neurons depends on Bax.


Asunto(s)
Neuronas Motoras/citología , Factores de Crecimiento Nervioso/farmacología , Ovario/patología , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Proto-Oncogénicas/genética , Testículo/patología , Animales , Animales Recién Nacidos , Axones/fisiología , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Linaje de la Célula/fisiología , Supervivencia Celular/efectos de los fármacos , Nervio Facial/citología , Nervio Facial/cirugía , Femenino , Regulación del Desarrollo de la Expresión Génica/fisiología , Hiperplasia/genética , Hibridación in Situ , Linfocitos/fisiología , Masculino , Ratones , Ratones Noqueados , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/fisiología , ARN Mensajero/metabolismo , Sistema Nervioso Simpático/citología , Proteína X Asociada a bcl-2
6.
Cell Death Differ ; 10(9): 1045-58, 2003 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-12934079

RESUMEN

Cytosine arabinoside (ara-C) is a nucleoside analog used in the treatment of hematologic malignancies. One of the major side effects of ara-C chemotherapy is neurotoxicity. In this study, we have further characterized the cell death induced by ara-C in sympathetic neurons. Similar to neurons undergoing trophic factor deprivation-induced apoptosis, ara-C-exposed neurons became hypometabolic before death and upregulated c-myb, c-fos, and Bim. Bax deletion delayed, but did not prevent, ara-C toxicity. Neurons died by apoptosis, indicated by the release of mitochondrial cytochrome-c and caspase-3 activation. p53-deficient neurons demonstrated decreased sensitivity to ara-C, but neither p53 nor multiple p53-regulated genes were induced. Mature neurons showed increased ara-C resistance. These results demonstrate that molecular mechanisms underlying ara-C-induced death are similar to those responsible for trophic factor deprivation-induced apoptosis. However, substantial differences in neuronal death after these two distinct stress stimuli exist since ara-C toxicity, unlike the developmental death, can proceed in the absence of Bax.


Asunto(s)
Antimetabolitos Antineoplásicos/toxicidad , Apoptosis , Citarabina/toxicidad , Neuronas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Proto-Oncogénicas/fisiología , Ganglio Cervical Superior/citología , Animales , Caspasa 3 , Caspasas/metabolismo , Diferenciación Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citocromos c/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Eliminación de Gen , Genes p53 , Cinética , Masculino , Ratones , Mitocondrias/metabolismo , Neuronas/citología , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Regulación hacia Arriba , Proteína X Asociada a bcl-2
7.
Brain Pathol ; 6(4): 397-409, 1996 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-8944313

RESUMEN

Extensive neuronal death occurs in the developing nervous system. Death of neurons during this process is apoptotic and appears to utilize a pathway that is conserved in various mammalian cells and organisms. Recent evidence suggests that neuronal death during trauma, stroke, or neurodegenerative diseases may also occur by a similar mechanism. This review discusses the molecular mechanism of developmental neuronal death by examining the biochemical and molecular events associated with neuronal death after trophic factor withdrawal. The ability to inhibit neuronal death by manipulating the Bcl-2 or the ICE-family proteins demonstrates the importance of these proteins in the neuronal apoptotic pathway. The utility of inhibiting neuronal death by blocking the apoptotic pathway as therapy in neuropathological situations is discussed.


Asunto(s)
Apoptosis , Muerte Celular , Sistema Nervioso/crecimiento & desarrollo , Neuronas/fisiología , Animales , Trastornos Cerebrovasculares/patología , Humanos , Modelos Neurológicos , Factores de Crecimiento Nervioso/fisiología , Sistema Nervioso/citología , Sistema Nervioso/patología , Enfermedades del Sistema Nervioso/patología , Neuronas/citología , Neuronas/patología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Heridas y Lesiones/patología
8.
Brain Pathol ; 11(3): 273-81, 2001 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-11414470

RESUMEN

Expression of apoptosis-associated proteins p53, bcl-2, bax, and caspase-3/CPP32, activation of caspase-3, and modification of proteins via poly(ADP-ribosyl)ation was studied in pontosubicular neuron necrosis (PSN), a form of perinatal brain damage revealing the morphological hallmarks of neuronal apoptosis. Immunoreactivity for p53 was completely absent. The majority of cells stained with the bax and procaspase-3 antibodies did not show morphological signs of apoptosis. In contrast, an antibody against activated caspase-3 almost exclusively stained cells with apoptotic morphology. Poly(ADP-ribosyl)ated proteins were only rarely detected in cells with apoptotic morphology. The expression patterns of bax, procaspase-3, bcl-2, and p53 in PSN were similar to that found in age-matched control brains. However, activated caspase-3 and poly-ADP-ribosylated proteins were exclusively found in apoptotic cells. These data indicate that detection of active caspase-3 is a reliable marker for apoptosis in formalin-fixed human tissue, and that neuronal apoptosis in pontosubicular neuron necrosis is accompanied by a pronounced activation of caspase-3.


Asunto(s)
Apoptosis/fisiología , Caspasas/biosíntesis , Precursores Enzimáticos/biosíntesis , Hipoxia Fetal/patología , Hipocampo/patología , Proteínas del Tejido Nervioso/biosíntesis , Neuronas/patología , Puente/patología , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas/biosíntesis , Proteína p53 Supresora de Tumor/biosíntesis , Biomarcadores , Caspasa 3 , Caspasas/genética , Activación Enzimática , Precursores Enzimáticos/genética , Femenino , Muerte Fetal/metabolismo , Muerte Fetal/patología , Hipoxia Fetal/metabolismo , Fijadores/farmacología , Formaldehído/farmacología , Perfilación de la Expresión Génica , Genes bcl-2 , Genes p53 , Hipocampo/metabolismo , Humanos , Recién Nacido , Masculino , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Poli Adenosina Difosfato Ribosa/análisis , Poli(ADP-Ribosa) Polimerasas/metabolismo , Puente/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteína X Asociada a bcl-2
9.
Invest Ophthalmol Vis Sci ; 39(9): 1713-20, 1998 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-9699561

RESUMEN

PURPOSE: Bax, a member of the Bcl2 family of cell death regulators, induces cell death by promoting the induction of apoptosis. Bax-deficient mice were examined in this study to determine whether Bax is required for cell death in the developing retina and for pathologic apoptotic photoreceptor degeneration resulting from the rd mutation. METHODS: Retinas from Bax-deficient mice and their wild-type siblings were harvested at postnatal day (P) 7 and processed for TdT-dUTP terminal nick-end labeling (TUNEL) staining, and the number of nuclei containing fragmented DNA were counted. Adult retinas and optic nerves were processed for plastic-embedded 1-microm sections, and the cross-sectional area was determined. The mutant Bax allele was outbred onto the C3H mouse strain, which carries the rd allele. Retinas from these animals were examined histologically at P21 after most of the photoreceptor cell death had occurred. RESULTS: At P7, around the time of peak cell death in the inner nuclear layer (INL), significantly fewer neurons in INL and ganglion cell layer (GCL) were TUNEL positive in Bax-deficient mice than in their wild-type siblings. In adult Bax-deficient mice, the cross-sectional area of the optic nerve was approximately 50% larger than in wild-type siblings, and the total number of retinal ganglion cell axons was increased to 226%. The INL of Bax-deficient mice was thicker than normal. The Bax genotype did not affect the thickness or histologic appearance of the outer nuclear layer in retinas of mice with wild-type or mutant rd alleles. CONCLUSIONS: In the absence of the expression of the Bax gene, there is a profound increase in the survival of retinal ganglion cells that lasts into adulthood. Similarly, death of INL cells is diminished but not completely abolished. The absence of Bax does not, however, protect photoreceptors from naturally occurring cell death or degeneration induced by the rd mutation. This shows that Bax is involved to a variable degree in the control of developmental cell death in the retina and that not all apoptotic retinal cell deaths are controlled by Bax.


Asunto(s)
Células Fotorreceptoras/patología , Proteínas Proto-Oncogénicas c-bcl-2/deficiencia , Proteínas Proto-Oncogénicas/deficiencia , Degeneración Retiniana/patología , Animales , Muerte Celular/fisiología , Supervivencia Celular , Daño del ADN , Fragmentación del ADN , Femenino , Eliminación de Gen , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Transgénicos , Neuronas/patología , Nervio Óptico/patología , Células Fotorreceptoras/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Retina/crecimiento & desarrollo , Retina/metabolismo , Retina/patología , Degeneración Retiniana/genética , Degeneración Retiniana/metabolismo , Células Ganglionares de la Retina/patología , Proteína X Asociada a bcl-2
10.
Ann N Y Acad Sci ; 679: 121-31, 1993 May 28.
Artículo en Inglés | MEDLINE | ID: mdl-8512180

RESUMEN

Deprivation of sympathetic neurons of their physiological neurotrophic factor, nerve growth factor (NGF), leads to degeneration of soma and neurites, followed by loss of viability. The progression of degeneration and death are dependent upon macromolecular synthesis indicating an active participation of neuronal metabolism. Loss of viability begins only after a considerable delay after onset of NGF deprivation suggesting the presence of a sequence of degenerative events that triggers death. Such a sequence of degenerative events predicts that the activity of neuroprotective agents functioning by different mechanisms will be restricted to particular windows in time. The time-course of commitment to die as measured by the ability of NGF-deprived neurons to respond to NGF with long-term survival precedes the time-course of loss of viability by only a few hours, demonstrating that NGF displays neuroprotective properties for most of the time between onset of deprivation and death. Furthermore, NGF repairs and reverses the degenerative changes caused by prolonged periods of NGF deprivation. Because of these two aspects of NGF action, NGF demonstrates superior properties as a neuroprotective agents. NGF deprivation initiates DNA fragmentation of the neuronal genome into oligonucleosomal fragments in close temporal association with the onset of commitment to die. This is consistent with the idea that DNA fragmentation may be instrumental in causing the commitment to die. Thus, DNA fragmentation may serve as a marker of the physiologically most relevant critical step occurring during degeneration and may indicate the end of the period during which trophic factors are useful as neuroprotective agents. These results may be transferable to neurodegenerative diseases or sequelae of neuronal injury because of similarities in the phenomenology of degeneration and death.


Asunto(s)
Muerte Celular , Degeneración Nerviosa/fisiología , Factores de Crecimiento Nervioso/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuronas/fisiología , Animales , Humanos , Factores de Crecimiento Nervioso/farmacología , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Enfermedades del Sistema Nervioso/fisiopatología , Neuronas/efectos de los fármacos , Neuronas/patología
11.
J Neurol Sci ; 128(2): 157-66, 1995 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-7738592

RESUMEN

Degeneration of cholinergic neurons in the basal forebrain (CBF) is a prominent neuropathological feature of Alzheimer's disease and is thought responsible for some cognitive deficits seen in patients. An animal model of pure CBF degeneration would be valuable for analysis of the function of these neurons and testing therapeutic strategies. CBF neurons express receptors for nerve growth factor. In order to selectively destroy these neurons, we developed an immunotoxin using monoclonal antibody (192 IgG) to rat NGF receptor (p75NGFr) armed with the ribosome inactivating protein, saporin. In vitro 192-saporin was highly toxic to neurons expressing p75NGFr. Intraventricular injections of 192-saporin destroyed the CBF and impaired passive avoidance learning. These results indicate that 192-saporin treated rats can be used to model a key feature of Alzheimer's disease and that anti-neuronal immunotoxins are a powerful approach to selective neural lesioning.


Asunto(s)
Enfermedad de Alzheimer , Anticuerpos Monoclonales/toxicidad , Ganglios Basales/efectos de los fármacos , Conducta Animal/fisiología , Fibras Colinérgicas/efectos de los fármacos , Modelos Animales de Enfermedad , Inmunotoxinas/toxicidad , Glicoproteínas de Membrana/efectos de los fármacos , N-Glicosil Hidrolasas , Proteínas de Plantas/toxicidad , Receptores de Factor de Crecimiento Nervioso/efectos de los fármacos , Tabique Pelúcido/efectos de los fármacos , Sustancia Innominada/efectos de los fármacos , Animales , Anticuerpos Monoclonales/administración & dosificación , Reacción de Prevención/fisiología , Ganglios Basales/patología , Ganglios Basales/fisiopatología , Recuento de Células , Células Cultivadas , Fibras Colinérgicas/patología , Ganglios Simpáticos/citología , Ganglios Simpáticos/efectos de los fármacos , Humanos , Inmunoconjugados , Inmunoglobulina G/inmunología , Inmunotoxinas/administración & dosificación , Inyecciones Intraventriculares , Discapacidades para el Aprendizaje/inducido químicamente , Masculino , Glicoproteínas de Membrana/inmunología , Neuronas/efectos de los fármacos , Proteínas de Plantas/administración & dosificación , Ratas , Ratas Sprague-Dawley , Receptor de Factor de Crecimiento Nervioso , Receptores de Factor de Crecimiento Nervioso/inmunología , Proteínas Inactivadoras de Ribosomas Tipo 1 , Saporinas , Tabique Pelúcido/patología , Tabique Pelúcido/fisiopatología , Sustancia Innominada/patología , Sustancia Innominada/fisiopatología
12.
Dev Biol ; 165(1): 63-72, 1994 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-8088451

RESUMEN

During the development of the nervous system extensive programmed neuronal death occurs that is regulated by neurotrophic factors. Invariably, degeneration and death of the neuronal soma as a result of trophic factor deprivation is accompanied by concurrent degeneration of the neurites. By examining the degeneration of sympathetic neurons after deprivation of their physiological trophic factor nerve growth factor, we show that the "slow Wallerian degeneration" allele (Wld6) expressed by homozygous mutant C57BL/Ola mice alters the normal time course of programmed neuronal death by selectively and dramatically delaying the onset of neurite disintegration. In contrast, degenerative events affecting the neuronal soma are not altered: Atrophy of the soma, apoptotic disintegration of the nucleus, commitment to die, and loss of viability occur normally. The enucleate neurites remaining after death of the soma have an intact plasma membrane, are metabolically active, and require an active metabolism for physical integrity. We suggest that the degeneration of neurites during developmentally occurring neuronal death is controlled by events confined to the neurites and occurs autonomously from the neuronal soma. Furthermore, programmed neuronal death of the soma proceeds independent from any influence exerted by degenerating neurites.


Asunto(s)
Apoptosis , Neuritas/fisiología , Neuronas/citología , Animales , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Degeneración Nerviosa , Factores de Crecimiento Nervioso/metabolismo , Sistema Nervioso Simpático/citología , Sistema Nervioso Simpático/metabolismo
13.
Annu Rev Neurosci ; 16: 31-46, 1993.
Artículo en Inglés | MEDLINE | ID: mdl-8460896

RESUMEN

Data derived from several experimental approaches demonstrate that naturally occurring neuronal death during development has many parallels with the physiologically appropriate death seen in nonneuronal cells. Physiologically appropriate death in different cell types may share some common mechanisms. These general notions must remain vague and tentative, because details of the mechanisms by which cells die in response to physiological positive or negative signals are poorly understood in any cell type. Current thinking focuses on the idea that cells possess a mechanism, which involves specific gene products, that are designed to kill the cell in response to appropriate physiological signals. Genetic studies of cell death in C. elegans and the demonstrations of increased expression of specific genes temporally associated with death in nonneuronal cells are consistent with this view. However, in the latter studies, there is no direct evidence that such temporally related genes are critical to the process of cell death or whether such gene expression may be related to somE other aspect of the response to the hormonal manipulations that produce the death of the cell under study. Therefore, the mechanism of death of any cell type is not understood, and whether neuronal death during development or after experimental manipulation results from the same mechanism is unknown. Several approaches are currently being pursued in a number of laboratories to address this general problem. These include pharmacological studies, such as described above, and studies aimed at analyzing biochemical and morphological changes associated with death. Attempts to find mRNAs or proteins whose increased expression is associated with neuronal death can be addressed by subtractive and differential hybridization strategies, by two-dimensional protein gel electrophoresis, and by examining genes whose increased expression is temporally correlated with cell death. Success in these various strategies will provide an understanding of neuronal death and relate to it cell death in other cell types. If future work provides direct evidence for a genetic program acting physiologically to produce death in the developing nervous system, an obvious question becomes the possible role that loss of transcriptional control of such a program plays in the adult in responses to mechanical or chemical trauma, neurodegenerative disease, or neuronal attrition associated with aging. Studies addressing the basic developmental process of trophic factor deprivation-induced death should provide molecular markers of and pharmacological approaches to these pathological processes in the adult.


Asunto(s)
Envejecimiento/fisiología , Muerte Celular , Neuronas/fisiología , Animales , Calcio/metabolismo , Supervivencia Celular , Invertebrados , Neuronas/citología , Vertebrados
14.
EMBO J ; 9(5): 1465-9, 1990 May.
Artículo en Inglés | MEDLINE | ID: mdl-1970294

RESUMEN

Active accumulation of neurotransmitters by synaptic vesicles is an essential component of the synaptic transmission cycle. Isolated vesicles show energy-dependent uptake of several transmitters by processes which are apparently mediated by a proton electrochemical potential across the vesicle membrane. Although this energy gradient is probably generated by a proton ATPase, the functional separation of ATP cleavage and transmitter uptake activity has only been shown clearly for monoamine transport. We report here that the light-driven proton pump, bacteriorhodopsin, can replace the endogenous proton ATPase in proteoliposomes reconstituted from vesicular detergent extracts. The system shows light-dependent uptake of glutamate with properties very similar to those observed in intact vesicles, e.g. chloride dependence or stimulation by NH4+. Our experiments show that the proton pump and the glutamate transporter are separate entities and provide a powerful tool for further characterization of the glutamate carrier.


Asunto(s)
Bacteriorodopsinas/farmacología , Glutamatos/metabolismo , Glicoproteínas/metabolismo , Vesículas Sinápticas/metabolismo , Sistema de Transporte de Aminoácidos X-AG , Animales , Transporte Biológico Activo/efectos de los fármacos , Ácido Glutámico , Técnicas In Vitro , Liposomas , Ratas , Vesículas Sinápticas/efectos de los fármacos , Tritio
15.
J Biol Chem ; 263(30): 15423-8, 1988 Oct 25.
Artículo en Inglés | MEDLINE | ID: mdl-2902091

RESUMEN

The dependence of glutamate uptake on ATP-generated proton electrochemical potential was studied in a highly purified preparation of synaptic vesicles from rat brain. At low chloride concentration (4 mM), the proton pump present in synaptic vesicles generated a large membrane potential (inside-positive), associated with only minor acidification. Under these conditions, the rate of L-[3H]glutamate uptake was maximal. In addition, L-glutamate induced acidification of the vesicle interior. D-Glutamate produced only 40% of the effect, and L-aspartate or gamma-aminobutyric acid produced less than 5%. The initial rate of glutamate-induced acidification increased with increasing glutamate concentration. It was saturable and showed first-order kinetics (KM = 0.32 mM). Correspondingly, L-glutamate induced a small reduction in the membrane potential. The rate of ATP hydrolysis was unaffected. In comparison, glutamate had no effect on acidification or membrane potential in resealed membranes of chromaffin granules. At high chloride concentration (150 mM), the vesicular proton pump generated a large pH difference, associated with a small change in membrane potential. Under these conditions, uptake of L-[3H]glutamate by synaptic vesicles was low. For reconstitution, vesicle proteins were solubilized with the detergent sodium cholate, supplemented with brain phospholipids, and incorporated into liposomes. Proton pump and glutamate uptake activities of the proteoliposomes showed properties similar to those of intact vesicles indicating that the carrier was reconstituted in a functionally active form. It is concluded that glutamate uptake by synaptic vesicles is dependent on the membrane potential and that all components required for uptake are integral parts of the vesicle membrane.


Asunto(s)
Glutamatos/farmacocinética , Proteolípidos/metabolismo , Vesículas Sinápticas/metabolismo , Naranja de Acridina , Adenosina Trifosfato/metabolismo , Animales , Encéfalo/citología , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Ácido Glutámico , Potenciales de la Membrana , Ratas
16.
J Neurosci ; 17(24): 9656-66, 1997 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-9391020

RESUMEN

During development, sympathetic neurons are critically dependent on nerve growth factor (NGF) for survival. Neurons isolated from the superior cervical ganglia (SCG) of embryonic rodents and maintained for 1 week in vitro undergo programmed cell death in response to NGF deprivation. As the cells mature in vitro and in vivo, however, these neurons develop a resistance to NGF deprivation and become much less acutely dependent on NGF for survival. Using an in vitro model of neuronal maturation, we confirmed that SCG neurons maintained in culture for 3-4 weeks did not experience a dramatic loss in viability after NGF removal, yet they did undergo the initial biochemical and genetic changes elicited by NGF deprivation of young neurons. NGF deprivation of mature neurons produced rapid decreases in glucose uptake and protein and RNA synthesis rates, increased phosphorylation of c-Jun, and an increase in c-jun mRNA. Mature neurons, however, experienced a block in the cell death program before the final stages of the pathway activated in young neurons, which includes the induction of c-fos mRNA and characteristic apoptotic nuclear changes. This maturation-induced block was indistinguishable by these criteria from the block produced by Bax deficiency. Expression of Bax in mature neurons restored the apoptotic pathway, such that after NGF removal, Bax-overexpressing mature neurons resumed the apoptotic program, including the induction of c-Fos and passage through a caspase checkpoint. Thus, a block in the apoptotic program at or near the BAX checkpoint accounts for the decreased dependence of mature neurons on neurotrophic factor to maintain survival.


Asunto(s)
Factores de Crecimiento Nervioso/farmacología , Neuronas/citología , Proteínas Proto-Oncogénicas/genética , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Senescencia Celular/fisiología , Femenino , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica/fisiología , Mutagénesis/fisiología , Degeneración Nerviosa/metabolismo , Neuronas/química , Neuronas/metabolismo , Isomerasa de Peptidilprolil/metabolismo , Fenotipo , Fosforilación , Embarazo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Ratas , Ratas Sprague-Dawley , Sistema Nervioso Simpático/citología , Proteína X Asociada a bcl-2 , Proteína bcl-X
17.
J Neurosci ; 15(1 Pt 2): 643-64, 1995 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-7823169

RESUMEN

Continuous exposure of many types of neurons in cell culture to elevated concentrations of K+ greatly enhances their survival. This effect has been reported to be mediated by a sustained rise of cytoplasmic free Ca2+ concentration caused by influx of Ca2+ through voltage-gated channels activated by K(+)-induced chronic depolarization. In this report we investigate the effects of elevated K+ on the programmed death that embryonic rat sympathetic neurons undergo in culture when deprived of NGF. Elevated K+ in the culture medium did not significantly prevent death of NGF-deprived cells until after the third day following plating of embryonic day 21 neurons. On the fifth day after plating, incrementally increasing K+ concentrations in the culture medium from 5 to 100 mM caused chronic depolarization of neurons and had a biphasic effect on survival of NGF-deprived cells. Enhanced survival was steeply related to membrane potential, increasing from no enhanced survival in cells held at potentials between -51 and -34 mV to 90-100% of control survival at about -21 mV. At potentials positive to -21 mV, survival decreased. Associated with the chronic depolarization was a sustained rise of steady-state free Ca2+ concentration that showed a biphasic relationship to membrane potential roughly similar to that exhibited by survival. Steady-state Ca2+ concentration increased with increasingly lower membrane potentials to a peak at about -23 mV (to approximately 240 nM from approximately 40 nM at about -51 mV) and then decreased at more positive potentials. The elevation of intracellular Ca2+ was largely blocked by dihydropyridine and phenylalkylamine Ca2+ channel antagonists and was potentiated by a dihydropyridine Ca2+ channel agonist. Neither the rise of Ca2+, or survival was affected by the Ca2+ channel antagonist, omega-conotoxin. Therefore, the Ca2+ elevation was probably caused by Ca2+ influx through L-type, but not N-type, channels. Antagonists of L channels blocked both survival and the sustained increase of steady-state free Ca2+ at similar concentrations, suggesting that the relevant factor determining survival of depolarized cells was Ca2+ influx rather than some other effect of depolarization. Surprisingly, however, there was no clear correlation between the sustained rise of Ca2+ and survival. Some membrane potentials that induced similar increases of Ca2+ concentration produced widely different levels of survival. While chronic depolarization promoted survival of neurons in the absence of NGF, cells supported in this manner showed little growth as measured by neurite extension, total cellular protein, and mean somal diameter.(ABSTRACT TRUNCATED AT 400 WORDS)


Asunto(s)
Apoptosis/fisiología , Calcio/metabolismo , Ganglios Simpáticos/fisiología , Neuronas/fisiología , Proteínas Tirosina Quinasas/fisiología , Animales , Calmodulina/antagonistas & inhibidores , División Celular/fisiología , Electrofisiología , Ganglios Simpáticos/citología , Homeostasis , Membranas Intracelulares/metabolismo , Neuronas/citología , Concentración Osmolar , Fosforilación , Potasio/metabolismo , Ratas , Factores de Tiempo
18.
Am J Pathol ; 155(5): 1459-66, 1999 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-10550301

RESUMEN

Neuronal loss is prominent in Alzheimer's disease (AD), and its mechanisms remain unresolved. Apoptotic cell death has been implicated on the basis of studies demonstrating DNA fragmentation and an up-regulation of proapoptotic proteins in the AD brain. However, DNA fragmentation in neurons is too frequent to account for the continuous neuronal loss in a degenerative disease extending over many years. Furthermore, the typical apoptotic morphology has not been convincingly documented in AD neurons with fragmented DNA. We report the detection of the activated form of caspase-3, the central effector enzyme of the apoptotic cascade, in AD and Down's syndrome (DS) brain using an affinity-purified antiserum. In AD and DS, single neurons with apoptotic morphology showed cytoplasmic immunoreactivity for activated caspase-3, whereas no neurons were labeled in age-matched controls. Apoptotic neurons were identified at an approximate frequency of 1 in 1100 to 5000 neurons in the cases examined. Furthermore, caspase-3 immunoreactivity was detected in granules of granulovacuolar degeneration. Our results provide direct evidence for apoptotic neuronal death in AD with a frequency compatible with the progression of neuronal degeneration in this chronic disease and identify autophagic vacuoles of granulovacuolar degeneration as possible means for the protective segregation of early apoptotic alterations in the neuronal cytoplasm.


Asunto(s)
Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/patología , Apoptosis , Caspasas/metabolismo , Anciano , Anciano de 80 o más Años , Caspasa 3 , Gránulos Citoplasmáticos/enzimología , Gránulos Citoplasmáticos/patología , Fragmentación del ADN , Activación Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neuronas/enzimología , Neuronas/patología , Neuronas/ultraestructura
19.
Exp Neurol ; 152(1): 150-62, 1998 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-9682022

RESUMEN

The BCL2 family member BAX is required for the induction of apoptosis in neonatal sympathetic neurons after NGF withdrawal. Bax-deficient sympathetic neurons are NGF-independent for survival. To characterize the physiological state of neurons protected by BAX deficiency and to place BAX within the death pathway, we determine which of the molecular changes induced by NGF deprivation depend on BAX and compare the results with those for neurons protected by caspase inhibition. We find that neurons deficient in both Bax and Bcl2 resist NGF-deprivation similar to Bax-deficient neurons discounting a role for BCL2 in the mechanism by which Bax deficiency causes trophic factor independence. We identify two new molecular changes, phosphorylation of c-Jun on Ser63 and alpha-spectrin proteolysis, which precede and accompany apoptosis, respectively. Early reversible changes induced by NGF withdrawal, such as decreased protein synthesis and glucose uptake, increased c-Jun phosphorylation, increased steady state c-jun mRNA levels, and cellular atrophy, occur both in wild type and Bax-deficient neurons and thus are BAX-independent. In contrast to neurons protected by caspase inhibition, no c-fos induction occurs in Bax-deficient neurons. Terminal irreversible events of apoptosis such as caspase-mediated alpha-spectrin proteolysis are prevented by both Bax-deficiency and caspase inhibition. This places BAX downstream or in a different pathway of the early changes and upstream of the terminal events such as those leading to c-fos induction and caspase activation. This order indicates that the physiological state of NGF-deprived neurons protected by Bax deficiency may be less perturbed than that of caspase inhibitor-saved neurons.


Asunto(s)
Apoptosis/fisiología , Factores de Crecimiento Nervioso/deficiencia , Proteínas del Tejido Nervioso/deficiencia , Neuronas/fisiología , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Proto-Oncogénicas/fisiología , Sistema Nervioso Simpático/fisiología , Animales , Animales Recién Nacidos , Western Blotting , Células Cultivadas , Metabolismo Energético/fisiología , Técnica del Anticuerpo Fluorescente Directa , Regulación de la Expresión Génica/fisiología , Glucosa/metabolismo , Ratones , Ratones Endogámicos ICR , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/fisiología , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Neuronas/metabolismo , Fosforilación , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Sistema Nervioso Simpático/citología , Sistema Nervioso Simpático/metabolismo , Proteína X Asociada a bcl-2
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