RESUMEN
Adenine phosphoribosyltransferase (APRT) deficiency is a rare autosomal recessive enzyme defect of purine metabolism that usually manifests as 2,8-dihydroxyadenine (2,8-DHA) nephrolithiasis and more rarely chronic kidney disease. The disease is most often misdiagnosed and can recur in the renal allograft. We analyzed nine patients with recurrent 2,8-DHA crystalline nephropathy, in all of whom the diagnosis had been missed prior to renal transplantation. The diagnosis was established at a median of 5 (range 1.5-312) weeks following the transplant procedure. Patients had delayed graft function (n=2), acute-on-chronic (n=5) or acute (n=1) allograft dysfunction, whereas one patient had normal graft function at the time of diagnosis. Analysis of allograft biopsies showed birefringent 2,8-DHA crystals in renal tubular lumens, within tubular epithelial cells and interstitium. Fourier transformed infrared microscopy confirmed the diagnosis in all cases, which was further supported by 2,8-DHA crystalluria, undetectable erythrocyte APRT enzyme activity, and genetic testing. With allopurinol therapy, the allograft function improved (n=7), remained stable (n=1) or worsened (n=1). At last follow-up, two patients had experienced allograft loss and five had persistent chronic allograft dysfunction. 2,8-DHA nephropathy is a rare but underdiagnosed and preventable disorder that can recur in the renal allograft and may lead to allograft loss.
Asunto(s)
Adenina Fosforribosiltransferasa/deficiencia , Rechazo de Injerto , Trasplante de Riñón , Errores Innatos del Metabolismo/etiología , Urolitiasis/etiología , Adulto , Anciano , Femenino , Humanos , Masculino , Errores Innatos del Metabolismo/fisiopatología , Persona de Mediana Edad , Recurrencia , Urolitiasis/fisiopatologíaAsunto(s)
Adenina Fosforribosiltransferasa/genética , Cromosomas Humanos Par 16/genética , Disomía Uniparental/genética , Urolitiasis/genética , Adenina Fosforribosiltransferasa/deficiencia , Secuencia de Bases , Femenino , Humanos , Lactante , Errores Innatos del Metabolismo , Datos de Secuencia Molecular , Linaje , Disomía Uniparental/diagnóstico , Urolitiasis/diagnósticoRESUMEN
A unique sensitivity to specific biochemical processes is responsible for selective vulnerability of midbrain dopamine neurons in several diseases. Prior studies have shown these neurons are susceptible to energy failure and mitochondrial dysfunction, oxidative stress, and impaired disposal of misfolded proteins. These neurons also are especially vulnerable to the loss of purine recycling. In the brains of humans or mice with inherited defects of the purine recycling enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT), the most prominent defect is loss of basal ganglia dopamine. To investigate the nature of the relationship between HPRT deficiency and dopamine, the mouse MN9D dopaminergic neuronal cell line was used to prepare 10 sublines lacking HPRT. The mutant sublines grew more slowly than the parent line, but without morphological signs of impaired viability. As a group, the mutant sublines had significantly lower dopamine than the parent line. The loss of dopamine in the mutants did not reflect impaired energy status, as judged by ATP levels or vulnerability to inhibitors of energy production. Indeed, the mutant lines as a group appeared energetically more robust than the parent line. The loss of dopamine also was not accompanied by enhanced susceptibility to oxidative stress or proteasome inhibitors. Instead, the loss of dopamine reflected only one aspect of a broad change in the molecular phenotype of the cells affecting mRNAs encoding tyrosine hydroxylase, the dopamine transporter, the vesicular monoamine transporter, monoamine oxidase B, catechol-O-methyltransferase, and GTP-cyclohydrolase. These changes were selective for the dopamine phenotype, since multiple control mRNAs were normal. These studies suggest purine recycling is an intrinsic metabolic process of particular importance to the molecular phenotype of dopaminergic neurons independent of previously established mechanisms involving energy failure, oxidative stress, or proteasome dysfunction.
Asunto(s)
Dopamina/deficiencia , Hipoxantina Fosforribosiltransferasa/genética , Mesencéfalo/metabolismo , Neuronas/metabolismo , Purinas/metabolismo , Adenosina Trifosfato/biosíntesis , Animales , Línea Celular Transformada , Dopamina/biosíntesis , Metabolismo Energético/genética , Enzimas/genética , Enzimas/metabolismo , Regulación Enzimológica de la Expresión Génica/genética , Mesencéfalo/patología , Mesencéfalo/fisiopatología , Ratones , Neuronas/patología , Estrés Oxidativo/genética , Complejo de la Endopetidasa Proteasomal/genética , ARN Mensajero/metabolismoRESUMEN
Ovine placental lactogen (oPL) is produced by the conceptus trophectoderm and is secreted into both the maternal and fetal circulations. The present study was designed to examine in vivo the luteotropic effect of recombinant oPL (roPL), as determined by monitoring progesterone concentration and cycle length (experiment 1), and the antioxidative and antiapoptotic effects of roPL, as determined respectively by monitoring antioxidant enzymatic activity and apoptosis in the corpus luteum (CL) of cyclic ewes (experiment 2). We also studied whether roPL is capable of stimulating progesterone secretion in vitro by cultured luteal tissue of functionally active CL obtained from day-10 cyclic ewes (experiment 3) and day-60 pregnant ewes (experiment 4). Circulating concentrations of progesterone and cycle length were not affected by treatment of ewes with 80 microg/kg body weight per day of roPL (n = 4 ewes) for 10 days beginning on day 11 post-estrus, as compared with saline-treated ewes (n = 4 ewes). Luteolysis occurred between days 15 and 16 post-estrus in the four saline-treated ewes and in 3/4 roPL-treated ewes. The activities of the key antioxidant enzymes copper-zinc superoxide dismutase (Cu,Zn-SOD), manganese SOD (Mn-SOD), glutathione peroxidase (GPX), glutathione reductase (GSR) and glutathione S-transferase (GST) were unaffected by treatment of ewes with 80 microg/kg per day of roPL (n = 4 ewes) for 3 days, between days 11 and 14 post-estrus, as compared with saline-treated ewes (n = 4 ewes). In situ TUNEL method revealed that the number of apoptotic cells was not different between the two groups of ewes. There was no significant change in progesterone secretion by explants from day-10 estrous cycle (n = 3 ewes) or day-60 pregnancy (n = 3 ewes) CL cultured with different concentrations (10, 100 and 1000 ng/ml) of roPL, whereas treatment with oLH at the concentration of 100 or 1000 ng/ml caused a significant increase in progesterone secretion by explants from day-10 estrous cycle CL (P < 0.05) and by explants from day-60 pregnancy CL (P < 0.01). In conclusion, our results demonstrate that oPL has no luteotropic and/or luteoprotective actions in sheep, either in vivo or in vitro.
Asunto(s)
Cuerpo Lúteo/efectos de los fármacos , Infertilidad/metabolismo , Lactógeno Placentario/farmacología , Ovinos/fisiología , Animales , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Cuerpo Lúteo/metabolismo , Ciclo Estral , Femenino , Etiquetado Corte-Fin in Situ , Embarazo , Progesterona/sangre , Progesterona/metabolismo , Proteínas Recombinantes/farmacología , Técnicas de Cultivo de TejidosRESUMEN
A patient with hyperuricaemia and gouty arthritis due to a new variant of hypoxanthine-guanine phosphoribosyltransferase is described. The mutation (I136T, HPRT Marseille) is in the phosphoribosylpyrophosphate-binding region of the gene and leads to almost total loss of enzyme activity in erythrocytes, with 5% in lymphocytes. Nevertheless, the patient showed no neurological abnormality.
Asunto(s)
Hipoxantina Fosforribosiltransferasa/genética , Mutación , Artritis Gotosa/enzimología , Sitios de Unión/genética , Eritrocitos/enzimología , Francia , Humanos , Hiperuricemia/enzimología , Hipoxantina Fosforribosiltransferasa/deficiencia , Linfocitos/enzimología , Masculino , Persona de Mediana Edad , SíndromeRESUMEN
There are numerous studies describing the neuroprotective effects of Ginkgo biloba extract EGb 761 on patients with disturbances of vigilance, memory and cognitive functions associated with aging and senility. Describing the pattern of gene expression in EGb 761-treated human hNT neurons may elucidate the molecular pathways leading to the neuroprotection. We used cDNA macroarrays including genes implicated in the antioxidant and stress responses to define the transcriptional effects of EGb 761 (250 microg/ml, 24 hr) on human hNT neurons. Seven genes were identified whose expression was strongly modified by the EGb 761 treatment. Three groups are distinguished: genes encoding transcription factors (increase of NF-kappaB p65 subunit and zinc finger protein 91 mRNAs, and decrease of c-myc transcripts), genes involved in antioxidant defenses (increase of the CuZn SOD mRNAs, and decrease of glutathione reductase and glutathione S-transferase pi mRNAs) and genes involved in stress responses (up-regulation of HSP70 transcripts). Consistent with the modulation of mRNAs by EGb 761, the enzymatic activities of glutathione reductase and glutathione S-transferase were decreased. Surprisingly, CuZn SOD activity was decreased despite increased abundance of the mRNAs; furthermore MnSOD activity was unmodified, and thus the effect of EGb 761 was specific to CuZn SOD. These results support the idea that modulation of target genes and transcription factors may be involved in the neuroprotective action of EGb 761.
Asunto(s)
Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Extractos Vegetales/farmacología , Antioxidantes/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Ginkgo biloba , Proteínas de Choque Térmico/metabolismo , Humanos , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Beside its role in electron transfer in the mitochondrial respiratory chain, ubiquinone is known to prevent lipid peroxidation and DNA damage by trapping cellular free radicals. Thanks to its antioxidant properties, ubiquinone may represent an important factor controlling both necrotic and apoptotic processes. We have investigated the consequences of a profound inherited ubiquinone depletion on cultured skin fibroblasts of a patient presenting with encephalomyopathy. Interestingly, cell respiration, mitochondrial oxidation of various substrates, and cell growth of ubiquinone-deficient fibroblasts were only partially decreased. Moreover, these cells did not apparently overproduce superoxide anions or lipoperoxides. Finally, apoptosis did not increase as compared to control, even after serum deprivation. These observations suggest that ubiquinone may not play a major role in the antioxidant defenses of cultured fibroblasts and that its role in controlling oxidative stress and apoptosis may greatly vary across cell types, especially as not all tissues were equally affected in the patient despite the widespread ubiquinone depletion in vivo.
Asunto(s)
Antioxidantes/metabolismo , Citoprotección/fisiología , Fibroblastos/citología , Encefalomiopatías Mitocondriales/metabolismo , Piel/citología , Ubiquinona/análogos & derivados , Ubiquinona/fisiología , Antioxidantes/uso terapéutico , Supervivencia Celular/fisiología , Células Cultivadas , Niño , Coenzimas , Femenino , Humanos , Peróxido de Hidrógeno/farmacología , Hierro/farmacología , Peroxidación de Lípido , Masculino , Potenciales de la Membrana , Encefalomiopatías Mitocondriales/tratamiento farmacológico , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismo , Ubiquinona/uso terapéuticoRESUMEN
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the extracellular deposition of amyloid beta-peptide (Abeta) in the brain. Abeta is involved in the pathogenesis of AD but the molecular mechanisms of its neurotoxicity are unknown. Here, we report that Abeta exposure on human preneuronal NT2 cells provoked a strong and early up-regulation of growth arrest and DNA damage inducible gene (Gadd45 mRNA), an indicator of DNA damage and DNA excision-repair processes, strongly suggesting that Abeta causes an early DNA strand breakage leading to a cellular DNA repair response. Comet assay clearly demonstrated that both full-length Abeta (1-42), and its minimal cytotoxic fragment Abeta (25-35), caused DNA breakage as early as 3h after the start of Abeta exposure. This extensive DNA damage provoked by Abeta constitutes an early event in the pathogenic cascade leading to neuronal death which could contribute to the neuropathogenesis of AD.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , ADN/efectos de los fármacos , Neuronas/efectos de los fármacos , Fragmentos de Péptidos/toxicidad , Biosíntesis de Proteínas , Proteínas , Northern Blotting , Células Cultivadas , Ensayo Cometa , Daño del ADN , Reparación del ADN , Humanos , Péptidos y Proteínas de Señalización Intracelular , Plásmidos/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factores de Tiempo , Regulación hacia Arriba , Proteína bcl-X , Proteinas GADD45RESUMEN
Using adenoviruses encoding reporter genes as retrograde tracers, we assessed the capacity of motoneurons to take up and retrogradely transport adenoviral particles injected into the muscles of transgenic mice expressing the G93A human superoxide dismutase mutation, a model of amyotrophic lateral sclerosis. Surprisingly, transgene expression in the motoneurons was significantly higher in symptomatic mice than in control or presymptomatic mice. Using botulinum toxin to induce nerve sprouting at neuromuscular junctions, we showed that the unexpectedly high level of motoneurons retrograde transduction results, at least in part, from newly acquired uptake properties of the sprouts. These findings demonstrate the remarkable uptake properties of amyotrophic lateral sclerosis motoneurons in response to denervation and the rationale of using intramuscular injections of adenoviruses to overexpress therapeutic proteins in motor neuron diseases.
Asunto(s)
Enfermedad de la Neurona Motora/fisiopatología , Neuronas Motoras/fisiología , Músculo Esquelético/fisiopatología , Unión Neuromuscular/fisiopatología , Superóxido Dismutasa/genética , Sinapsis/fisiología , Adenoviridae , Animales , Tronco Encefálico/enzimología , Tronco Encefálico/fisiopatología , Modelos Animales de Enfermedad , Técnicas de Transferencia de Gen , Vectores Genéticos , Humanos , Luciferasas/genética , Ratones , Ratones Transgénicos , Enfermedad de la Neurona Motora/enzimología , Enfermedad de la Neurona Motora/genética , Neuronas Motoras/enzimología , Desnervación Muscular , Músculo Esquelético/inervación , Mutación Missense , Unión Neuromuscular/fisiología , Valores de Referencia , Superóxido Dismutasa/metabolismo , Lengua/inervación , beta-Galactosidasa/análisis , beta-Galactosidasa/genéticaRESUMEN
Alzheimer's disease is a neurodegenerative disorder characterized by the extracellular deposition in the brain of amyloid beta-peptide (A beta), presumed to play a pathogenic role. However, the precise molecular mechanisms of its neurotoxicity are not fully understood. Recent studies have suggested that it may exert its toxic effect via activation of transcription factors. We investigated A beta-responsive genes in human preneuron NT2 cells, at early stages of A beta (25-35) exposure, by RNA differential display. A beta induced the expression of (i) the growth arrest and DNA damage-inducible gene (gadd45) implicated in the DNA excision-repair process; (ii) a stress-signaling kinase gene encoding the mitogen-activated protein kinase/Erk kinase kinase-1 (MEKK1); (iii) a new growth factor-inducible immediate-early gene, CYR61, the product of which functions as an extracellular matrix signaling molecule; (iv) other immediate-early genes, such as c-jun and c-fos; (v) the gene encoding the basic fibroblast growth factor (bFGF); (vi) a gene encoding a constituent of the mitochondrial pyruvate dehydrogenase complex, the dihydrolipoamide dehydrogenase-binding protein (E3-BP); and (vii) an unidentified human gene (KIAA0099). A beta not only activates but also respresses genes: (i) the gene encoding "hinge" protein, a subunit of the mitochondrial cytochrome-c reductase and (ii) the SRp55 gene encoding a splicing factor involved in constitutive pre-mRNA splicing and alternative splice site selection. Our results underscored A beta-responsive genes that play key roles in the response (damage/recovery) of neuron cells to A beta exposure. In particular, the strong upregulation of gadd45, indicating DNA damage, was detected early in A beta cytotoxicity. This suggests that DNA strand breaks occurred rapidly in cells exposed to A beta, which may be a critical event in A beta neurotoxicity.
Asunto(s)
Péptidos beta-Amiloides/fisiología , Daño del ADN/genética , Expresión Génica/genética , Proteínas/genética , ARN/genética , Enfermedad de Alzheimer/genética , Línea Celular Transformada , Genes Relacionados con las Neoplasias/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Transcripción Genética/genética , Regulación hacia Arriba/genética , Proteinas GADD45RESUMEN
If permanent focal ischemia is induced by middle cerebral artery occlusion (MCAO), neurons within the infarcted territory die by necrosis and apoptosis (or programmed cell death). We have previously shown, using a mouse strain transgenic (tg) for the nerve growth factor (NGF) gene, that tg mice have consistently smaller infarcted areas than wild-type (wt) animals, correlated with upregulated NGF synthesis and impaired apoptotic cell death. We studied, in wt and tg mice subjected to MCAO, the activities of several antioxidant enzymes and the synthesis of the proteins of the Bcl-2 family. Our results show that the antiapoptotic Bcl-2 protein and glutathione peroxidase are recruited after MCAO. NGF-tg mice also had an intrinsic resistance to oxidative stress because their basal copper zinc superoxide dismutase (SOD) and glutathione transferase activities were high. Additionally, manganese SOD activity increased in NGF-tg mice after MCAO, correlating strongly with the resistance of these mice to apoptosis.
Asunto(s)
Antioxidantes/metabolismo , Isquemia Encefálica/metabolismo , Isquemia Encefálica/fisiopatología , Factores de Crecimiento Nervioso/genética , Superóxido Dismutasa/metabolismo , Animales , Apoptosis/fisiología , Western Blotting , Isquemia Encefálica/genética , Proteínas Portadoras/análisis , Corteza Cerebral/irrigación sanguínea , Corteza Cerebral/química , Corteza Cerebral/enzimología , Infarto Cerebral/genética , Infarto Cerebral/metabolismo , Infarto Cerebral/fisiopatología , Glutatión Peroxidasa/metabolismo , Glutatión Transferasa/metabolismo , Masculino , Proteínas de la Membrana/análisis , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Proteína Destructora del Antagonista Homólogo bcl-2 , Proteína X Asociada a bcl-2 , Proteína Letal Asociada a bcl , Proteína bcl-XRESUMEN
Recent work has focused attention on the role of oxidative stress in various acute and chronic neurodegenerative diseases. Low concentrations of the powerful antioxidant glutathione (GSH) and impaired brain energy metabolism, particularly in the substantia nigra, are key features of Parkinson's disease (PD). The main goal of this study was to better characterize the deleterious effects of brain GSH depletion on mitochondrial function. We depleted GSH in the brains of newborn wild-type (WT) and transgenic (Tg) mice overproducing either human Cu/Zn-superoxide dismutase (h-CuZnSOD) or human Bcl2 (h-Bcl-2), by subcutaneous injection of l-buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase. GSH was 97% depleted in brain homogenates and 90% depleted in brain mitochondria for both WT and Tg mice. This depletion of brain GSH led to a decrease in the activity of the GSH-dependent antioxidant enzyme glutathione peroxidase, both in WT and in Tg animals. BSO treatment decreased the activities of respiratory complexes I, II, and IV in the brain homogenates of WT mice. BSO-treated h-CuZnSOD or h-Bcl-2 Tg mice had no respiratory chain deficiencies. Thus, brain GSH depletion leads to the impairment of mitochondrial respiratory chain activity. The protection of mitochondrial respiratory function by overproduction of Bcl-2 may result from a decrease in the generation of reactive oxygen species (ROS) or lipid peroxidation. The protection of mitochondria by overproduction of CuZnSOD is consistent with the involvement of superoxide or superoxide-derived ROS in the mitochondrial dysfunction caused by brain GSH depletion. This study demonstrates that the antioxidant balance is critical for maintenance of brain mitochondrial function, and its disruption may contribute to the pathogenesis of PD.
Asunto(s)
Encéfalo/metabolismo , Butionina Sulfoximina/farmacología , Glutatión/metabolismo , Mitocondrias/metabolismo , Consumo de Oxígeno/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Superóxido Dismutasa/genética , Animales , Glutamato-Cisteína Ligasa/antagonistas & inhibidores , Glutatión/antagonistas & inhibidores , Humanos , Peroxidación de Lípido , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Mitocondrias/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
The generation of reactive oxygen species has been implicated in the neurotoxicity of amyloid beta-peptide, the main constituent of the senile plaques that accumulates in the brain of Alzheimer's disease victims. In this study, we have compared the toxicity of amyloid beta-peptide on cultured cortical neurons from control mice and transgenic mice expressing either human copper-zinc superoxide dismutase or human Bcl-2, two proteins that protect cells against oxidative damage. Copper-zinc superoxide dismutase overexpression failed to protect cortical neurons against the toxicity of amyloid beta-peptide(25-35) [the minimal cytotoxic fragment of amyloid beta-peptide(1-42)] as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction and an enzyme-linked immunoabsorbent assay using an antibody directed against microtubule-associated protein-2 (a specific neuronal protein), ruling out a role for superoxide anion and peroxynitrite in amyloid beta-peptide-evoked neurotoxicity. On the contrary, cortical neurons expressing human copper-zinc superoxide dismutase exhibited increased apoptotic nuclei in both untreated and amyloid beta-peptide(25-35)-exposed neurons. Transgenic neurons expressing human Bcl-2 were partially protected against amyloid beta-peptide-induced neuronal death. This neuroprotection appears to be related to the complete inhibition of apoptosis induced by both amyloid beta-peptide(25-35) and amyloid beta-peptide(1-42). This study may be relevant for developing neuroprotective gene therapy to inhibit neuronal apoptosis in Alzheimer's disease.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Corteza Cerebral/metabolismo , Genes bcl-2/genética , Neuronas/metabolismo , Neurotoxinas/toxicidad , Péptidos beta-Amiloides/antagonistas & inhibidores , Animales , Apoptosis/fisiología , Western Blotting , Supervivencia Celular , Células Cultivadas , Corteza Cerebral/citología , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente Directa , Expresión Génica/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/metabolismo , Neurotoxinas/antagonistas & inhibidores , Superóxido Dismutasa/metabolismoRESUMEN
Recent evidence has focused attention on the role of oxidative stress in various acute and chronic neurodegenerative diseases. Particularly, a decrease in the level of the powerful antioxidant glutathione (GSH) and death of dopaminergic neurons in substantia nigra are prominent features in Parkinson's disease. The mode of neuronal death is uncertain; however, apoptosis has been hypothesized to be mediated through the induction of free radicals via oxidative pathways. An approach to determine the role of GSH depletion in neurodegeneration and apoptosis was to create a selective modulation of this antioxidant by metabolic manipulations in a clonal cell line of neuronal origin (mouse neuroblastoma NS20Y). Intracellular GSH levels was lowered by inhibiting its biosynthesis with L-buthionine-(S,R)-sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase. This treatment led to a GSH depletion of 50% after 1 h and 98% after 24 h. A direct cause/effect relationship between GSH depletion and apoptosis was evidenced in this neuronal cell type. GSH depletion induced the death of NS20Y and promoted nuclear alterations of apoptosis as demonstrated by the in situ staining of DNA fragmentation after 5 days of BSO treatment (by terminal-deoxynucleotide transferase-mediated dUTP-nick end labeling), and the appearance of DNA laddering on agarose gel. These results suggested that redox desequilibrium induced by GSH depletion may serve as a general trigger for apoptosis in neuronal cells, and are consistent with the hypothesis that GSH depletion contribute to neuronal death in Parkinson's disease.
Asunto(s)
Apoptosis , Glutatión/fisiología , Neuronas/patología , Animales , Butionina Sulfoximina/farmacología , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Fragmentación del ADN , Electroforesis en Gel de Agar , Glutatión/antagonistas & inhibidores , Ratones , Neuronas/metabolismo , Células Tumorales CultivadasRESUMEN
In Parkinson's disease (PD), dopaminergic cell death in the substantia nigra was associated with a profound glutathione (GSH) decrease and a mitochondrial dysfunction. The fall in GSH concentration seemed to appear before the mitochondrial impairment and the cellular death, suggesting that a link may exist between these events. The relationships between GSH depletion, reactive oxygen species (ROS) production, mitochondrial dysfunction and the mode of cell death in neuronal cells remain to be resolved and will provide important insights into the etiology of Parkinson's disease. An approach to determine the role of GSH in the mitochondrial function and in neurodegeneration was to create a selective depletion of GSH in a neuronal cell line in culture (NS20Y) by inhibiting its biosynthesis with L-buthionine-(S,R)-sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase. This treatment led to a nearly complete GSH depletion after 24 hr and induced cellular death via an apoptotic pathway after 5 days of BSO treatment. By using the reactive oxygen species-sensitive probe 2',7'-dichlorofluorescin, we observed that the rapid GSH depletion was accompanied, early in the process, by a strong and transient intracellular increase in reactive oxygen species evidenced after 1 hr with BSO, culminating after 3 hr when the GSH level decreased to 30% of normal. GSH depletion induced a loss of mitochondrial function after 48 hr of BSO treatment. In particular, the activities of complexes I, II and IV of the respiratory chain were decreased by 32, 70 and 65%, respectively as compared to controls. These results showed the crucial role of GSH for maintaining the integrity of mitochondrial function in neuronal cells. Oxidative stress and mitochondrial impairment, preceding DNA fragmentation, could be early events in the apoptotic process induced by GSH depletion. Our data are consistent with the hypothesis that GSH depletion could contribute to neuronal apoptosis in Parkinson's disease through oxidative stress and mitochondrial dysfunction.
Asunto(s)
Apoptosis/fisiología , Glutatión/metabolismo , Mitocondrias/fisiología , Neuronas/metabolismo , Estrés Oxidativo/fisiología , Enfermedad de Parkinson/metabolismo , Animales , Butionina Sulfoximina/farmacología , Línea Celular , Transporte de Electrón/fisiología , Inhibidores Enzimáticos/farmacología , Glutamato-Cisteína Ligasa/antagonistas & inhibidores , Ratones , Neuronas/patología , Oxidación-Reducción , Enfermedad de Parkinson/patología , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
The neuroprotective potential of the nerve growth factor (NGF) against permanent ischemic brain damage has been investigated in vivo using NGF-transgenic (tg) mice. The expression of the transgene is driven by part of the promoter of the proto-oncogene c-fos, which belongs to the first set of genes activated after brain ischemic insult. Wild-type (wt) mice and tg mice were subjected to permanent focal ischemia induced by electrocoagulation of the middle cerebral artery. Twenty four hours (h) after the ischemic shock, when compared to wt, tg mice displayed a 40% reduction of the infarcted area, which lasted up to 1 week. However, infarcted brain areas were similar in wt and tg mice within the first hours post-occlusion, indicating that NGF acted to block the progression of neuronal damage. Kinetics of NGF synthesis assessed by ELISA was in good agreement with the observed neuroprotective effect, since NGF content peaked 6 h post-ischemia. This was further correlated with the time-course of c-Fos immunoreactivity, detectable only from 6 h post-ischemia. The neuroprotective effect of NGF involved the impairment of apoptotic cell death, as evidenced by a marked decrease of the number of apoptotic profiles inside the ischemic zone in tg mice. These results underline the potential of c-fos-NGF-tg mice to study in vivo the molecular and cellular mechanisms of the NGF-induced neuroprotective effect against ischemic damage.
Asunto(s)
Isquemia Encefálica/complicaciones , Infarto Cerebral/prevención & control , Factores de Crecimiento Nervioso/fisiología , Proteínas del Tejido Nervioso/fisiología , Animales , Apoptosis , Infarto Cerebral/etiología , Infarto Cerebral/patología , Fragmentación del ADN , Masculino , Ratones , Ratones Transgénicos , Factores de Crecimiento Nervioso/genética , Proteínas del Tejido Nervioso/genética , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-fos/análisis , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Recombinantes de Fusión/fisiología , Daño por Reperfusión/prevención & control , TransgenesAsunto(s)
Hipoxantina Fosforribosiltransferasa/deficiencia , Síndrome de Lesch-Nyhan/genética , Secuencia de Bases , Células Cultivadas , Análisis Mutacional de ADN , ADN Complementario/química , ADN Complementario/genética , Salud de la Familia , Mutación del Sistema de Lectura , Francia , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Síndrome de Lesch-Nyhan/enzimología , Mutación , Mutación Missense , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Eliminación de SecuenciaRESUMEN
After an ischemic episode induced by the electrocoagulation of the left middle cerebral artery (MCA) in mouse, neurons within the damaged territory die either by an apoptotic or by a necrotic process. Most of the cortical neurons within the ischemic area display both morphological and biochemical signs of programmed cell death: nuclear condensation, DNA degradation, formation of apoptotic bodies, and glutathione depletion. In fact, apoptosis essentially contributes to the expansion of the ischemic lesion and the maximum of damaged territory is reached 24 h postocclusion. Several potentially neuroprotective pathways have been evidenced in different experimental models of ischemia including the activation of antioxidant enzyme activities and/or the recruitment of neurotrophic as well as antiapoptotic factors. In our model of permanent focal ischemia induced by MCA occlusion, we measured the temporal synthesis of nerve growth factor (NGF) and examined the status of antioxidant enzymes as well as Bcl-2 antiapoptotic product. We detected in both cortices a transient increase of NGF which peaks at 6 h. Moreover, we reported that glutathione peroxidase is recruited with a time course which parallels NGF synthesis. Finally, we observed the induction of Bcl-2 in safe neurons; this may represent a self-protective response against ischemia-induced apoptosis. We provide evidence that in a model of permanent focal ischemia, several neuroprotective pathways could be coactivated.