RESUMEN
The hippocampus is characterized by the presence of life-long neurogenesis. To elucidate the molecular mechanism regulating hippocampal neurogenesis, we studied the functions of the chemorepellent Draxin in neuronal proliferation and differentiation in the postnatal dentate gyrus. The present in vivo cell labeling and fate tracking analyses revealed enhanced differentiation of hippocampal neural stem and progenitor cells (hNSPCs) in the subgranular zone (SGZ) of Draxin-deficient mice. We observed a reduction in the number of BrdU-pulse labeled or Ki-67 immunopositive SGZ cells in the mutant mice. However, Draxin deficiency did not affect cell cycle duration of SGZ cells. In situ hybridization analysis indicated that the receptor component of the canonical Wnt pathway, Lrp6, is expressed in SGZ cells, including Nestin and Sox2 double-positive hNSPCs. Taken together with the previous finding that Draxin interacts physically with Lrp6, we postulate that Draxin plays a pivotal role in the regulation of Wnt-driven hNSPC differentiation to modulate the rate of neuronal differentiation in the progenitor population.
Asunto(s)
Hipocampo , Células-Madre Neurales , Animales , Diferenciación Celular , Proliferación Celular , Giro Dentado , Péptidos y Proteínas de Señalización Intercelular , Ratones , NeurogénesisRESUMEN
Hippocampal neurogenesis in the dentate gyrus (DG) is controlled by diffusible molecules that modulate neurogenic processes, including cell proliferation, differentiation and survival. To elucidate the mechanisms underlying hippocampal neurogenesis, we investigated the function of draxin, originally identified as a neural chemorepellent, in the regulation of neuronal survival in the DG. Draxin was expressed in Tbr2 (+) late progenitors and NeuroD1 (+) neuroblasts in the dentate granule cell lineage, whereas expression of its receptor DCC (deleted in colorectal cancer) was mainly detectable in neuroblasts. Our phenotypic analysis revealed that draxin deficiency led to enhanced apoptosis of DCC-expressing neuroblasts in the neurogenic areas. Furthermore, in vitro assays using a hippocampal neural stem/progenitor cell (HNSPC) line indicated that draxin inhibited apoptosis in differentiating HNSPCs, which express DCC. Taken together, we postulate that draxin plays a pivotal role in postnatal DG neurogenesis as a dependence receptor ligand for DCC to maintain and promote survival of neuroblasts.
Asunto(s)
Apoptosis , Receptor DCC/metabolismo , Giro Dentado/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Neurogénesis , Animales , Caspasas/metabolismo , Diferenciación Celular , Células Cultivadas , Receptor DCC/antagonistas & inhibidores , Receptor DCC/genética , Hipocampo/metabolismo , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Mutagénesis Sitio-Dirigida , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , RatasRESUMEN
The RING finger protein 213 (RNF213) is an important susceptibility gene for moyamoya disease (MMD) and is also implicated in other types of intracranial major artery stenosis/occlusion (ICAS); however, the role of RNF213 in the development of ICAS including MMD is unclear. The constitutive expression of the RNF213 gene is relatively weak in brain tissue, while information regarding the expression patterns of the RNF213 gene under cerebral ischemia, which is one of characteristic pathologies associated with ICAS, is currently limited. Our objective was to address this critical issue, and we investigated Rnf213 mRNA expression in rat brains after 5 minutes of transient global cerebral ischemia (tGCI) by occluding the common carotid arteries coupled with severe hypotension. Rnf213 gene expression patterns were investigated with in situ RNA hybridization and a real-time polymerase chain reaction (PCR) from 1 to 72 hours after tGCI. In situ RNA hybridization revealed a significant increase in Rnf213 mRNA levels in the hippocampus CA1 sub-region 48 hours after tGCI. The significant induction of the Rnf213 gene was also evident in the ischemic cortex. Double staining of Rnf213 mRNA with NeuN immunohistochemistry revealed Rnf213 hybridization signal expression exclusively in neurons. The real-time PCR analysis confirmed the induction of the Rnf213 gene after tGCI. The up-regulation of the Rnf213 gene in vulnerable neurons in the hippocampus CA1 after tGCI suggests its involvement in forebrain ischemia, which is an underlying pathology of MMD. Further investigations are needed to elucidate its exact role in the pathophysiology of ICAS including MMD.
Asunto(s)
Isquemia Encefálica/metabolismo , Región CA1 Hipocampal/metabolismo , Proteínas Portadoras/metabolismo , Enfermedad de Moyamoya/metabolismo , Neuronas/metabolismo , Animales , Antígenos Nucleares/metabolismo , Isquemia Encefálica/genética , Isquemia Encefálica/patología , Región CA1 Hipocampal/patología , Proteínas Portadoras/genética , Modelos Animales de Enfermedad , Masculino , Enfermedad de Moyamoya/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Although recent genome-wide and locus-specific association studies revealed that the RING finger protein 213 (RNF213) gene is an important susceptibility gene for moyamoya disease (MMD), the exact mechanism by which the genetic alteration of RNF213 contributes to the development of MMD has not yet been elucidated. A quantitative reverse transcription polymerase chain reaction (PCR) analysis revealed that the constitutive expression of the RNF213 gene was very low in adult and embryonic brain tissue. However, information regarding the temporal and spatial expression patterns of the RNF213 gene under the condition of cerebral ischemia, which is one of characteristic pathologies associated with MMD, is currently limited. In order to address this critical issue, Rnf213 mRNA expression was investigated in mouse brains subjected to 60 min of transient middle cerebral artery occlusion (tMCAO). Male C57BL6/j mice underwent tMCAO through the intraluminal blockade of MCA. Expression of the Rnf213 gene in the tMCAO brain was investigated with in situ RNA hybridization and a real-time PCR analysis from 1 to 72 h after tMCAO. In situ RNA hybridization revealed a significant increase in Rnf213 mRNA levels in the cerebral cortex supplied by the affected MCA, especially at the penumbra area, as early as 6h after tMCAO, and these levels had increased further by 24 h. Rnf213 gene expression remained unchanged in the non-ischemic hemisphere or control specimens. Double staining of Rnf213 mRNA with NeuN immunohistochemistry revealed Rnf213 hybridization signal expression mostly in neurons. The real-time PCR analysis confirmed induction of the Rnf213 gene after tMCAO. Therefore, the Rnf213 gene was up-regulated in the ischemic brain, especially at the penumbra area, 6 h after tMCAO. Early increases in RNF213 gene expression in neurons after tMCAO indicate its involvement in cerebral ischemia, which is an underlying pathology of MMD. Further investigation is required to clarify its exact role in the pathophysiology of MMD.
Asunto(s)
Encéfalo/metabolismo , Infarto de la Arteria Cerebral Media/metabolismo , Enfermedad de Moyamoya/metabolismo , Neuronas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Adenosina Trifosfatasas , Animales , Encéfalo/patología , Proteínas de Unión al ADN , Modelos Animales de Enfermedad , Predisposición Genética a la Enfermedad , Inmunohistoquímica , Hibridación in Situ , Infarto de la Arteria Cerebral Media/patología , Masculino , Ratones Endogámicos C57BL , Enfermedad de Moyamoya/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/patología , Proteínas Nucleares/metabolismo , ARN Mensajero , Distribución Aleatoria , Reacción en Cadena en Tiempo Real de la Polimerasa , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Despite apparent resection of tumors, breast cancer patients often suffer relapse due to remnant dormant tumor cells. Although quiescence of cancer stem cells is thought as one of the mechanisms regulating dormancy, the mechanism underlying quiescence is unclear. Since ΔNp63α, an isoform of p51/p63, is crucial in the maintenance of stem cells within mammary epithelium, we investigated its roles in the regulation of dormancy in normal and malignant breast cells. Inducible expression of ΔNp63α in MCF7 estrogen receptor positive (ER+) luminal breast cancer cells led to quiescence and acquisition of progenitor-like properties. Judging from mRNA-microRNA microarray analysis, activation of bone morphogenetic protein (BMP) signaling and inhibition of Wnt signaling emerged as prominent mechanisms underlying ΔNp63α-dependent induction of quiescence and acquisition of stemness in MCF7. More interestingly, through Ingenuity Pathway analysis, we found for the first time that BRCA1 pathway was the most significantly downregulated pathway by ΔNp63α expression in quiescent MCF7 cells, where miR-205 was a downstream mediator. Furthermore, ΔNp63α-expressing MCF7 cells exhibited resistance to paclitaxel and doxorubicin. Expression of ΔNp63α in normal MCF10A basal cells increased proliferation and stemness, but did not affect more aggressive luminal (T47D) and basal (MDA-MB-231) cells with p53 mutation. Gene expression datasets analyses suggested that ΔNp63 expression is associated with relapse-free survival of luminal A/B-type patients, but not of the other subtypes. Our results established a cell type-specific function of ΔNp63α in induction of quiescence and downregulation of the BRCA1 pathway which suggested a role of ΔNp63α in the dormancy of luminal breast cancers.
Asunto(s)
Proteína BRCA1/biosíntesis , Neoplasias de la Mama/metabolismo , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Receptores de Estrógenos/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteína BRCA1/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proliferación Celular/genética , Femenino , Humanos , Células MCF-7 , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Estrógenos/genética , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
Moyamoya disease (MMD) is a chronic, occlusive cerebrovascular disease with an unknown etiology. Recent genome-wide and locus-specific association studies identified the RNF213 gene (RNF213) as an important susceptibility gene of MMD among East Asian populations; however, the mechanism by which an abnormality in RNF213 leads to MMD has not yet been elucidated. Therefore, we herein generated Rnf213-knock-in mice (RNF213-KI) expressing a missense mutation in mouse Rnf213, p. R4828K, on Exon 61, corresponding to human RNF213, p. R4859K, on Exon 60, in MMD patients, and investigated whether they developed MMD. We assessed the temporal profile of intracranial arteries by 9.4-T magnetic resonance angiography (MRA) continuously in the same mouse up to 64 weeks of age. The ratios of the outer diameter of the internal carotid artery (ICA)/basilar artery (BA) and middle cerebral artery (MCA)/BA were evaluated histopathologically. The common carotid arteries (CCA) were sectioned and arterial wall thickness/thinness was evaluated by Elastica-Masson staining before and after CCA ligation, which selectively induced vascular hyperplasia. The results obtained showed that RNF213-KI grew normally, with no significant difference being observed in MRA findings or the anatomy of the circle of Willis between homozygous RNF213-KI and wild-type (Wt) littermates. Furthermore, no significant difference was noted in the diameter of the intracranial vasculature (ICA/BA; p=0.82, MCA/BA; p=0.27) or in vascular remodeling after CCA ligation. Therefore, RNF213-KI did not spontaneously develop MMD. Multiple secondary insults such as environmental factors may contribute to the onset of MMD in addition to genetic factors.
Asunto(s)
Vasos Sanguíneos/patología , Encéfalo/patología , Angiografía por Resonancia Magnética , Enfermedad de Moyamoya/patología , Mutación/genética , Ubiquitina-Proteína Ligasas/genética , Adenosina Trifosfatasas , Análisis de Varianza , Animales , Arginina/genética , Modelos Animales de Enfermedad , Humanos , Hiperplasia/etiología , Hiperplasia/patología , Procesamiento de Imagen Asistido por Computador , Lisina/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Enfermedad de Moyamoya/genética , Factores de TiempoRESUMEN
Moyamoya disease (MMD) is a chronic occlusive cerebrovascular disease with unknown etiology that is characterized by the development of abnormal vascular networks at the base of the brain. Recent genome-wide studies identified RNF213 as an important MMD susceptibility gene. However, the exact mechanism by which the RNF213 abnormality leads to MMD remains unknown. Thus, we sought to clarify the role of RNF213 in angiogenesis under ischemic conditions using conventional RNF213 knockout mice. We assessed the infarction volume, cerebral edema, and vascular density in the ischemic brain after transient middle cerebral artery occlusion (tMCAO). To further evaluate systemic angiogenesis following chronic ischemia, we investigated blood flow recovery using laser speckle flowmetry, the severity of ambulatory impairments, and vascular density in the hind-limb after permanent femoral artery ligation. Results were compared between homozygous RNF213 knockout mice (RNF213 -/-) and wild-type littermates (Wt). No significant differences were observed in infarction volume or the formation of edema following tMCAO, or in vascular density 28 days after tMCAO between RNF213 -/- and Wt. Blood flow recovery was significantly improved in RNF213 -/- from 3 to 28 days after femoral artery ligation, and angiogenesis as shown by vascular density in the hind-limb was significantly enhanced in RNF213 -/- at 28 days. The amelioration of ambulatory impairments was also evident in RNF213 -/-. Angiogenesis was enhanced in mice lacking RNF213 after chronic hind-limb ischemia, which suggested the potential role of the RNF213 abnormality in the development of pathological vascular networks in chronic ischemia.
Asunto(s)
Isquemia Encefálica/genética , Enfermedad de Moyamoya/genética , Neovascularización Patológica/genética , Ubiquitina-Proteína Ligasas/deficiencia , Adenosina Trifosfatasas , Animales , Modelos Animales de Enfermedad , Miembro Posterior/irrigación sanguínea , Inmunohistoquímica , Isquemia/genética , Masculino , Ratones , Ratones Noqueados , Enfermedad de Moyamoya/fisiopatología , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Primordial germ cells (PGCs) are undifferentiated germ cells in embryos, the fate of which is to become gametes; however, mouse PGCs can easily be reprogrammed into pluripotent embryonic germ cells (EGCs) in culture in the presence of particular extracellular factors, such as combinations of Steel factor (KITL), LIF and bFGF (FGF2). Early PGCs form EGCs more readily than do later PGCs, and PGCs lose the ability to form EGCs by embryonic day (E) 15.5. Here, we examined the effects of activation of the serine/threonine kinase AKT in PGCs during EGC formation; notably, AKT activation, in combination with LIF and bFGF, enhanced EGC formation and caused â¼60% of E10.5 PGCs to become EGCs. The results indicate that the majority of PGCs at E10.5 could acquire pluripotency with an activated AKT signaling pathway. Importantly, AKT activation did not fully substitute for bFGF and LIF, and AKT activation without both LIF and bFGF did not result in EGC formation. These findings indicate that AKT signal enhances and/or collaborates with signaling pathways of bFGF and of LIF in PGCs for the acquisition of pluripotency.
Asunto(s)
Reprogramación Celular/fisiología , Desarrollo Embrionario/fisiología , Células Germinativas/fisiología , Células Madre Pluripotentes/citología , Transducción de Señal/fisiología , Animales , Quimera/embriología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Genotipo , Factor Inhibidor de Leucemia/metabolismo , Ratones , Oligopéptidos/metabolismo , Células Madre Pluripotentes/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor de Células Madre/metabolismoRESUMEN
Polyisoprenoid alcohols from the livers of temperate sea fish (skipjack tuna, chub mackerel, red sea bream and rainbow trout) were analyzed by using 2D-TLC, electrospray ionization (ESI) mass spectrometry and NMR methods. Dolichols (Dols) were detected in all the fish livers, and they were composed of 19-22 isoprene units with Dol-20 as the predominant prenolog. In addition, Dol-like family compounds were found by using 2D-TLC on skipjack tuna samples. These compounds were found to have a larger molecular mass than the Dol family by 16 mass units. NMR analysis indicated that the Dol-like compounds were consistent with the terminal epoxide structure of Dols (the ω-oxirane derivatives of Dols). ESI analysis also revealed the occurrence of dehydro molecules in both Dols and epoxy Dols (Dol-like) fractions. The occurrence of epoxy Dols in fish is discussed in context with the biosynthesis of Dols, which is responsible for forming Dol phosphate, which lead to Dol-PP-oligosaccharide.
Asunto(s)
Dolicoles/análogos & derivados , Hígado/metabolismo , Atún/metabolismo , Animales , Cromatografía en Capa Delgada , Dolicoles/biosíntesis , Dolicoles/química , Compuestos Epoxi/química , Compuestos Epoxi/metabolismo , Espectroscopía de Resonancia Magnética , Masculino , Redes y Vías Metabólicas , Oncorhynchus mykiss/metabolismo , Perciformes/metabolismo , Dorada/metabolismo , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
The c-ABL non-receptor tyrosine kinase and the p53 tumor suppressor protein are pivotal modulators of cellular responses to DNA damage. However, a comprehensive understanding of the role of c-ABL kinase in p53-dependent transcription of p21(CIP1/WAF1) and ensuing cell fate decision is still obscure. Here, we demonstrate that c-ABL tyrosine kinase regulates p53-dependent induction of p21. As a result, it modulates cell fate decision by p53 in response to DNA damage differently according to the extent of DNA damage. When human cancer cells were treated with DNA damaging agent, adriamycin (0.08 µg/ml), p21 was induced following p53 induction. Owing largely to p21, a substantial fraction of cells treated with adriamycin were blocked at the G2 phase of the cell cycle and most cells eventually became senescent. When these cells were simultaneously treated with a c-ABL kinase inhibitor, STI571, or a c-ABL-specific siRNA along with adriamycin, the p53-dependent p21 induction was dramatically diminished, even though p53 is substantially induced. Accordingly, G2-arrest, and cellular senescence largely dependent on p21 were substantially abrogated. On the contrary, when cells were treated with a relatively high dose of adriamycin (0.4 µg/ml) cells became apoptotic, and the simultaneous presence of a c-ABL kinase inhibitor STI571 augmented the extent of apoptosis. We speculate this is due to abrogation of p53-dependent p21 induction, which leads to elimination of anti-apoptotic function of p21. In summary, c-ABL appears to promote senescence or inhibit apoptosis, depending on the extent of DNA damage. These findings suggest that the combined use of ABL kinase inhibitor and DNA damaging drug in chemotherapy against tumors retaining wild type p53 should be carefully designed.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Daño del ADN , Proteínas Proto-Oncogénicas c-abl/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Apoptosis/efectos de los fármacos , Benzamidas/farmacología , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Daño del ADN/efectos de los fármacos , Doxorrubicina/toxicidad , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Células HeLa , Humanos , Mesilato de Imatinib , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-abl/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-abl/genética , Pirimidinas/farmacología , Interferencia de ARN , ARN Interferente Pequeño/metabolismoRESUMEN
Primordial germ cells (PGCs) in mouse embryos likely include heterogeneous cells having distinct cellular properties. In the present study, we found that heterogeneity of PGCs can be defined by the expression of integrin α6 and c-Kit. The changes in integrin α6 and c-Kit expression in PGCs were obvious as embryonic development progressed, and the PGCs became a mixture of populations consisting of cells with distinct levels of cell surface protein expression. The changes and heterogeneity of cell surface protein expression mainly reflected asynchronous differentiation of PGCs. Apoptosis of PGCs was biased in populations of c-Kit or integrin α6 negative PGCs at particular developmental stages, suggesting possible linkage between PGC apoptosis and the levels of expression of these cell surface proteins. Histochemical analysis confirmed the heterogeneous expression of c-Kit and integrin α6 in PGCs in embryonic gonads, and revealed that PGCs showing different levels of integrin α6 or c-Kit expression and the apoptotic PGCs were scattered and did not show specific localization within gonads. The present study enables us to analyze and isolate populations of living PGCs showing a distinct status of differentiation, or different properties of proliferation or of cell death in individual embryos, and provides a new strategy to examine the mechanisms of PGC development.
Asunto(s)
Apoptosis , Diferenciación Celular , Proliferación Celular , Células Germinativas/citología , Células Germinativas/fisiología , Integrina alfa6/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Animales , Apoptosis/genética , Bromodesoxiuridina , Ciclo Celular , Diferenciación Celular/genética , Desarrollo Embrionario , Citometría de Flujo , Expresión Génica , Células Germinativas/metabolismo , Gónadas/embriología , Proteínas Fluorescentes Verdes , Integrina alfa6/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-kit/genéticaRESUMEN
Cancer of the esophagus is the seventh leading cause of cancer death worldwide. Esophageal carcinoma cell lines are useful models to study the biological and genetic alterations in these tumors. An important prerequisite of cell line research is the authenticity of the used cell lines because the mistaken identity of a cell line may lead to invalid conclusions. Estimates indicate that up to 36% of the cell lines are of a different origin or species than supposed. The TE series, established in late 1970s and early 1980s by Nishihira et al. in Japan, is one of the first esophageal cancer cell line series that was used throughout the world. Fourteen TE cell lines were derived from human esophageal squamous cell carcinomas and one, TE-7, was derived from a primary esophageal adenocarcinoma. In numerous studies, this TE-7 cell line was used as a model for esophageal adenocarcinoma because it is one of the few esophageal adenocarcinoma cell lines existing. We investigated the authenticity of the esophageal adenocarcinoma cell line TE-7 by xenografting, short tandem repeat profiling, mutation analyses, and array-comparative genomic hybridization and showed that cell line TE-7 shared the same genotype as the esophageal squamous cell carcinoma cell lines TE-2, TE-3, TE-12, and TE-13. In addition, for more than a decade, independent TE-7 cultures from Japan, United States, United Kingdom, France, and the Netherlands had the same genotype. Examination of the TE-7 cell line xenograft revealed the histology of a squamous cell carcinoma. We conclude that the TE-7 cell line, used in several laboratories throughout the world, is not an adenocarcinoma, but a squamous cell carcinoma cell line. Furthermore, the cell lines TE-2, TE-3, TE-7, TE-12, and TE-13 should be regarded as one single squamous cell carcinoma cell line.
Asunto(s)
Adenocarcinoma/diagnóstico , Adenocarcinoma/patología , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/patología , Adenocarcinoma/genética , Animales , Secuencia de Bases , Análisis Mutacional de ADN , Diagnóstico Diferencial , Errores Diagnósticos , Neoplasias Esofágicas/genética , Femenino , Heterogeneidad Genética , Genotipo , Humanos , Ratones , Ratones Desnudos , Análisis de Matrices Tisulares , Trasplante Heterólogo/patologíaRESUMEN
Mitochondria are known to be involved in the early stage of apoptosis by releasing cytochrome c, caspase-9, and the second mitochondria-derived activator of caspases (Smac). We have reported that overexpression of copper/zinc superoxide dismutase (SOD1) reduced superoxide production and ameliorated neuronal injury in the hippocampal CA1 subregion after global ischemia. However, the role of oxygen free radicals produced after ischemia/reperfusion in the mitochondrial signaling pathway has not been clarified. Five minutes of global ischemia was induced in male SOD1-transgenic (Tg) and wild-type (Wt) littermate rats. Cytosolic expression of cytochrome c and Smac and activation of caspases were evaluated by immunohistochemistry, Western blot, and caspase activity assay. Apoptotic cell death was characterized by DNA nick end and single-stranded DNA labeling. In the Wt animals, early superoxide production, mitochondrial release of cytochrome c, Smac, and cleaved caspase-9 were observed after ischemia. Active caspase-3 was subsequently increased, and 85% of the hippocampal CA1 neurons showed apoptotic DNA damage 3 d after ischemia. Tg animals showed less superoxide production and cytochrome c and Smac release. Subsequent active caspase-3 expression was not evident, and only 45% of the neurons showed apoptotic DNA damage. A caspase-3 inhibitor (N-benzyloxycarbonyl-val-ala-asp-fluoromethyl ketone) reduced cell death only in Wt animals. These results suggest that overexpression of SOD1 reduced oxidative stress, thereby attenuating the mitochondrial release of cytochrome c and Smac, resulting in less caspase activation and apoptotic cell death. Oxygen free radicals may play a pivotal role in the mitochondrial signaling pathway of apoptotic cell death in hippocampal CA1 neurons after global ischemia.
Asunto(s)
Isquemia Encefálica/enzimología , Caspasas/metabolismo , Citoprotección/fisiología , Mitocondrias/enzimología , Neuronas/enzimología , Superóxido Dismutasa/biosíntesis , Animales , Animales Modificados Genéticamente , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis , Isquemia Encefálica/patología , Proteínas Portadoras/metabolismo , Caspasa 3 , Caspasa 9 , Inhibidores de Caspasas , Grupo Citocromo c/metabolismo , Daño del ADN/fisiología , Inhibidores Enzimáticos/farmacología , Expresión Génica , Hipocampo/irrigación sanguínea , Hipocampo/enzimología , Hipocampo/patología , Inmunohistoquímica , Masculino , Proteínas Mitocondriales/metabolismo , Neuronas/patología , Ratas , Ratas Sprague-Dawley , Transducción de Señal/fisiología , Superóxido Dismutasa/genética , Superóxido Dismutasa-1 , Superóxidos/metabolismoRESUMEN
Mild hypothermia protects the brain from ischemia, but the underlying mechanisms of this effect are not well known. The authors previously found that hypothermia reduces the density of apoptotic cells, but it is not certain whether temperature alters associated biochemical events. Mitochondrial release of cytochrome c has recently been shown to be a key trigger in caspase activation and apoptosis via the intrinsic pathway. Using a model of transient focal cerebral ischemia, the authors determined whether mild hypothermia altered expression of Bcl-2 family proteins, mitochondrial release of cytochrome c, and caspase activation. Mild hypothermia significantly decreased the amount of cytochrome c release 5 hours after the onset of ischemia, but mitochondrial translocation of Bax was not observed until 24 hours. Mild hypothermia did not alter Bcl-2 and Bax expression, and caspase activation was not observed. The present study provides the first evidence that intraischemic mild hypothermia attenuates the release of cytochrome c in the brain, but does not appear to affect other biochemical aspects of the intrinsic apoptotic pathway. They conclude that necrotic processes may have been interrupted to prevent cytochrome c release, and that the ameliorative effect of mild hypothermia may be a result of maintaining mitochondrial integrity. Furthermore, the authors show it is unlikely that mild hypothermia alters the intrinsic apoptotic pathway.