RESUMEN
The temporal and spatial patterning involved in the specification, differentiation, and myelination by oligodendroglia is coordinated in part by the activation and repression of various transcriptional programs. Olig2 is a basic helix-loop-helix transcription factor necessary for oligodendroglial development and expressed continuously throughout the lineage. Despite evidence for the critical role of Olig2 in oligodendroglial specification and differentiation, the function for Olig2 during later stages of oligodendroglial development, namely, the transition into mature oligodendrocytes (OLs) and the formation of the myelin sheath, remains unclear. To address the possibility for a stage-specific role, we deleted Olig2 in oligodendrocyte precursor cells (OPCs) under the control of the CNPase-promoter or in immature OLs under the inducible proteolipid protein promoter. As expected, ablation of Olig2 in OPCs significantly inhibits differentiation, resulting in hypomyelination. However, deletion of the Olig2 gene in immature OLs significantly enhances the maturation process and accelerates the kinetics of myelination/remyelination. Underlying the stage-specific roles for Olig2 is the compensatory expression and function of Olig1, a transcription factor that promotes OL maturation and (re)myelination. Olig1 expression is significantly reduced upon Olig2 deletion in OPCs but is dramatically increased by nearly threefold when deleted in immature OLs. By enforcing expression of Olig1 into OPCs in a null Olig2 background, we demonstrate that overexpression of Olig1 is sufficient to rescue the differentiation phenotype and partially compensates for the Olig2 deletion in vitro. Our results suggest a stage-specific regulatory role for Olig2, mediated by Olig1 that conveys opposing functions on the differentiation and maturation of oligodendrocytes.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/deficiencia , Diferenciación Celular/fisiología , Proteínas del Tejido Nervioso/deficiencia , Oligodendroglía/fisiología , 2',3'-Nucleótido Cíclico Fosfodiesterasas/genética , 2',3'-Nucleótido Cíclico Fosfodiesterasas/metabolismo , Animales , Animales Recién Nacidos , Proteínas de Arabidopsis/metabolismo , Proteínas Relacionadas con la Autofagia , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/ultraestructura , Bromodesoxiuridina/metabolismo , Diferenciación Celular/efectos de los fármacos , Cuprizona/toxicidad , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Etiquetado Corte-Fin in Situ , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Transferasas Intramoleculares/metabolismo , Masculino , Ratones , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Inhibidores de la Monoaminooxidasa/toxicidad , Proteína Básica de Mielina/metabolismo , Proteína Proteolipídica de la Mielina/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/ultraestructura , Factor de Transcripción 2 de los Oligodendrocitos , Oligodendroglía/efectos de los fármacos , Transfección , Proteína de Unión al GTP ran/metabolismoRESUMEN
This paper aimed at investigating the alterations in interstitial cells of Cajal (ICC) in the proximal, middle and distal colon of mice from 0-day to 56-day post-partum (P0-P56) by immunohistochemistry. The Kit(+) ICC, which situated around myenteric nerve plexus (ICC-MY) were prominent at birth, meanwhile those cells within the smooth muscle layers (ICC-IM) and in the connective tissue beneath serosa (ICC-SS) began to appear. ICC-SM, which located at the submucosal border of circular muscle layer emerged at P6 in the proximal colon and subsequently in the distal colon at P8, and ICC in the oral side of colon revealed an earlier development in morphology and a higher density than that in the anal side. The density of ICC altered obviously during postnatal period, and the estimated total amount of ICC increased approximately 30 folds at P56 than that at P0. Some Kit(+)/Ki67(+) and Kit(+)/BrdU(+) cells were observed in ICC-MY, ICC-IM and ICC-SS, but not in ICC-SM from P0 to P24. Our result indicates a proximal to distal and transmural gradient development of ICC in the postnatal colon along with a dramatic increase of ICC cell number from neonatal to adult life, and an age-dependent proliferation of ICC is also involved.
Asunto(s)
Envejecimiento/fisiología , Colon/citología , Células Intersticiales de Cajal/citología , Animales , Animales Recién Nacidos , Proliferación Celular , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB CRESUMEN
Increasing evidence suggests that white matter disorders based on myelin sheath impairment may underlie the neuropathological changes in schizophrenia. But it is unknown whether enhancing remyelination is a beneficial approach to schizophrenia. To investigate this hypothesis, we used clemastine, an FDA-approved drug with high potency in promoting oligodendroglial differentiation and myelination, on a cuprizone-induced mouse model of demyelination. The mice exposed to cuprizone (0.2% in chow) for 6 weeks displayed schizophrenia-like behavioral changes, including decreased exploration of the center in the open field test and increased entries into the arms of the Y-maze, as well as evident demyelination in the cortex and corpus callosum. Clemastine treatment was initiated upon cuprizone withdrawal at 10 mg/kg per day for 3 weeks. As expected, myelin repair was greatly enhanced in the demyelinated regions with increased mature oligodendrocytes (APC-positive) and myelin basic protein. More importantly, the clemastine treatment rescued the schizophrenia-like behavioral changes in the open field test and the Y-maze compared to vehicle, suggesting a beneficial effect via promoting myelin repair. Our findings indicate that enhancing remyelination may be a potential therapy for schizophrenia.
Asunto(s)
Conducta Animal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Clemastina/administración & dosificación , Enfermedades Desmielinizantes/patología , Vaina de Mielina/efectos de los fármacos , Esquizofrenia/patología , Animales , Diferenciación Celular/efectos de los fármacos , Cuprizona/toxicidad , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/complicaciones , Enfermedades Desmielinizantes/prevención & control , Modelos Animales de Enfermedad , Conducta Exploratoria/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Actividad Motora/efectos de los fármacos , Proteína Básica de Mielina/metabolismo , Vaina de Mielina/metabolismo , Vaina de Mielina/fisiología , Oligodendroglía/metabolismo , Esquizofrenia/inducido químicamente , Esquizofrenia/complicaciones , Esquizofrenia/prevención & controlRESUMEN
Microglial activation has been considered as a crucial process in the pathogenesis of neuroinflammation and psychiatric disorders. Several antipsychotic drugs (APDs) have been shown to display inhibitory effects on microglial activation in vitro, possibly through the suppression of elevated intracellular calcium (Ca(2+)) concentration. However, the exact underlying mechanisms still remain elusive. In this study, we aimed to investigate the inhibitory effects of quetiapine (Que), an atypical APD, on microglial activation. We utilized a chronic cuprizone (CPZ)-induced demyelination mouse model to determine the direct effect of Que on microglial activation. Our results showed that treatment with Que significantly reduced recruitment and activation of microglia/macrophage in the lesion of corpus callosum and promoted remyelination after CPZ withdrawal. Our in vitro studies also confirmed the direct effect of Que on lipopolysaccharide (LPS)-induced activation of microglial N9 cells, whereby Que significantly inhibited the release of nitric oxide (NO) and tumor necrosis factor α (TNF-α). Moreover, we demonstrated that pretreatment with Que, neutralized the up-regulation of STIM1 induced by LPS and declined both LPS and thapsigargin (Tg)-induced store-operated Ca(2+) entry (SOCE). Finally, we found that pretreatment with Que significantly reduced the translocation of nuclear factor kappa B (NF-κB) p65 subunit from cytoplasm to nuclei in LPS-activated primary microglial cells. Overall, our data suggested that Que may inhibit microglial activation by neutralization of the LPS-induced abnormal STIM1-mediated intercellular calcium homeostasis.
RESUMEN
Quetiapine (Que), a commonly used atypical antipsychotic drug (APD), can prevent myelin from breakdown without immune attack. Multiple sclerosis (MS), an autoimmune reactive inflammation demyelinating disease, is triggered by activated myelin-specific T lymphocytes (T cells). In this study, we investigated the potential efficacy of Que as an immune-modulating therapeutic agent for experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. Que treatment was initiated on the onset of MOG(35-55) peptide induced EAE mice and the efficacy of Que on modulating the immune response was determined by Flow Cytometry through analyzing CD4(+)/CD8(+) populations and the proliferation of effector T cells (CD4(+)CD25(-)) in peripheral immune organs. Our results show that Que dramatically attenuates the severity of EAE symptoms. Que treatment decreases the extent of CD4(+)/CD8(+) T cell infiltration into the spinal cord and suppresses local glial activation, thereby diminishing the loss of mature oligodendrocytes and myelin breakdown in the spinal cord of EAE mice. Our results further demonstrate that Que treatment decreases the CD4(+)/CD8(+) T cell populations in lymph nodes and spleens of EAE mice and inhibits either MOG(35-55) or anti-CD3 induced proliferation as well as IL-2 production of effector T cells (CD4(+)CD25(-)) isolated from EAE mice spleen. Together, these findings suggest that Que displays an immune-modulating role during the course of EAE, and thus may be a promising candidate for treatment of MS.
Asunto(s)
Antipsicóticos/farmacología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/patología , Dibenzotiazepinas/farmacología , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/prevención & control , Animales , Astrocitos/efectos de los fármacos , Astrocitos/patología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Proliferación Celular/efectos de los fármacos , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/patología , Femenino , Inmunomodulación/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Microglía/patología , Vaina de Mielina/efectos de los fármacos , Vaina de Mielina/fisiología , Glicoproteína Mielina-Oligodendrócito/efectos adversos , Fragmentos de Péptidos/efectos adversos , Fumarato de Quetiapina , Médula Espinal/efectos de los fármacos , Médula Espinal/inmunología , Médula Espinal/patología , Médula Espinal/fisiopatologíaRESUMEN
This study aimed at investigating the response of lens epithelial cells in postnatal mice to Imatinib (Glivec®, a potent inhibitor of platelet-derived growth factor receptor (PDGFR)) treatment. Mouse eyes were sampled 10 days after administration of Imatinib (0.5 mg·g(-1)·day(-1)) for 3 days, at either 7, 14, or 21 days postpartum. Structural changes of lens were revealed by routine H.E. staining. Levels of proliferation and apoptosis were revealed by BrdU incorporation and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, respectively, and immunofluorescent staining with anti-PDGFRα antibody was carried out on the sections of eyeball. PDGFRα and p-PDGFRαprotein levels were evaluated by Western blot. Our results indicated that administration of Imatinib led to blockade of PDGFR signaling. Formation of cataracts was found only in those mice where treatment started from 7 days postpartum (P7), but was not observed in those samples from P14 nor P21. Fiber cells were disorganized in cataract lens core as observed histologically, and migration of epithelial cells was also inhibited. No apoptosis was detected with the TUNEL method. Our results indicated blockade of PDGFR at the neonatal stage (P7) would lead to cataracts and lens fiber cells disorganization, suggesting that PDGFR signaling plays a time-specific and crucial role in the postnatal development of lens in the mouse, and also may provide a new approach to produce a congenital cataract animal model.
Asunto(s)
Catarata/inducido químicamente , Células Epiteliales/efectos de los fármacos , Cristalino/efectos de los fármacos , Piperazinas/toxicidad , Inhibidores de Proteínas Quinasas/toxicidad , Pirimidinas/toxicidad , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Benzamidas , Catarata/metabolismo , Catarata/patología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Mesilato de Imatinib , Etiquetado Corte-Fin in Situ , Cristalino/metabolismo , Cristalino/patología , Ratones , Ratones Endogámicos BALB C , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de TiempoRESUMEN
This study investigated the response of interstitial cells of Cajal (ICC) in postnatal mouse colon to treatment with Imatinib (Glivec), a potent inhibitor of Kit receptor). ICC were revealed by immunofluorescent staining on frozen cross-sections and whole-mount preparations by anti-Kit and DOG1 antibodies. Kit and p-Kit protein were also evaluated by Western blot. After administration of Imatinib for 4 days beginning at 8 days post-partum (P8), the mean density of Kit+ ICC, which were localized around the myenteric nerve plexus (ICC-MY), within smooth muscle layers (ICC-IM) and in the connective tissue beneath the serosa (ICC-SS), was dramatically decreased to about 50% when compared with controls, but those Kit+ cells located at the submucosal border of circular smooth muscle layer (ICC-SM) seemed to be unchanged in both cell number and morphology. A small number of DOG1+/Kit(-) cells appeared during Imatinib administration. However, these Kit+ ICC were not changed in mice even after 12 days of Imatinib treatment from P24. When Imatinib was discontinued, the number of ICC recovered to normal within 4 days. Our results indicate that the postnatal development of ICC in the mouse colon is Kit dependent, but ICC-SM are unlikely, and the Kit dependence of ICC development is also age-dependent.
Asunto(s)
Colon/citología , Células Intersticiales de Cajal/metabolismo , Proteínas de la Membrana/metabolismo , Músculo Liso/metabolismo , Plexo Mientérico/metabolismo , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-kit/metabolismo , Pirimidinas/farmacología , Factores de Edad , Animales , Anoctamina-1 , Anticuerpos/inmunología , Antineoplásicos/farmacología , Benzamidas , Diferenciación Celular , Proliferación Celular , Canales de Cloruro/inmunología , Canales de Cloruro/metabolismo , Colon/crecimiento & desarrollo , Tejido Conectivo/metabolismo , Mesilato de Imatinib , Células Intersticiales de Cajal/citología , Células Intersticiales de Cajal/efectos de los fármacos , Proteínas de la Membrana/inmunología , Ratones , Músculo Liso/citología , Plexo Mientérico/citología , Especificidad de Órganos , Proteínas Proto-Oncogénicas c-kit/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-kit/inmunología , Membrana Serosa/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
The platelet-derived growth factor-alpha receptor (PDGFalphaR) has been found specifically expressed in oligodendrocyte precursor cells (OPCs), whereas another membrane protein, NG2, has been widely applied to characterize developing and matured OPCs. In order to investigate whether PDGFalphaR expression is consistent to NG2 in identifying OPCs, we utilized techniques of immunohistochemistry and Western blot to study the PDGFalphaR expression and distribution in rat postnatal brain from a series of ages P0 (postnatal day 0) to P540, and further compared it with NG2. Results showed that PDGFalphaR immunoreactive (PDGFalphaR+) cells existed in both the gray and white matter of the postnatal rat brain, although these cells displayed different features in distinct regions and developmental stages. PDGFalphaR did not express in oligodendrocytes, astrocytes or neurons (indicated by non-co-localization with CC1 and NF200, respectively). Western blot analysis revealed that the expression of PDGFalphaR in the cerebral cortex and hippocampus increased from P0 to P7 and then decreased gradually. PDGFalphaR+ cells displayed similar characteristics as of NG2+ cells in the morphology, distribution and electrophysiology. Like NG2+ cells, the density of PDGFalphaR+ cells had an increase at P7 and a late age-dependent decline, except a lower value from P7 to P540 in cerebral cortex, hippocampus and corpus callosum. PDGFalphaR+ cells exhibited number consistent with NG2+ cells at early developmental stages and were approximately 75% as numerous as NG2+ cells at old age. PDGFalphaR+/NG2- cells were not found. In conclusion, our findings suggest that both PDGFalphaR and NG2 are markers of developing OPCs. PDGFalphaR is specific to the NG2+ OPCs and mainly plays an important role at early developmental stages of OPCs. Aging had an effect on the morphological feature, number and developmental regulation of OPCs in rat CNS. However, further work will be necessary to determine if PDGFalphaR-/NG2+ cells may still maintain the biological characteristics of OPCs or they are other subpopulation of OPCs.
Asunto(s)
Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Oligodendroglía/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Células Madre/metabolismo , Envejecimiento/metabolismo , Animales , Animales Recién Nacidos , Antígenos/metabolismo , Biomarcadores/metabolismo , Western Blotting , Encéfalo/citología , Recuento de Células , Diferenciación Celular/fisiología , Proliferación Celular , Corteza Cerebral/citología , Corteza Cerebral/crecimiento & desarrollo , Corteza Cerebral/metabolismo , Hipocampo/citología , Hipocampo/crecimiento & desarrollo , Hipocampo/metabolismo , Inmunohistoquímica , Fibras Nerviosas Mielínicas/metabolismo , Fibras Nerviosas Mielínicas/ultraestructura , Oligodendroglía/citología , Proteoglicanos/metabolismo , Ratas , Ratas Sprague-Dawley , Células Madre/citologíaRESUMEN
This study investigates whether CD44 immunopositive cells truly correspond to the interstitial cells of Cajal (ICCs) in the muscular layers of the gastrointestinal tract in guinea pigs and Balb/c mice using immunohistochemistry with antibodies directed against CD44, Kit, vimentin and neurofilament 200 (NF200). All the sub-groups of ICCs were immunopositive for the anti-Kit antibody in the muscular layers of stomach, small intestine and colon in both cross sections and whole-mount preparations. Kit/CD44/vimentin triple immunolabeling showed that all the ICCs in different segments and muscular layers of the digestive tract were CD44, Kit and vimentin immunopositive. Kit/CD44/NF200 triple immunolabeling revealed that neither enteric nerves nor other major cells were CD44 immunopositive in the muscular layers, apart from the ICCs. CD44 and Kit were co-localized in the same group of cells, apart from a very small number (0.6%) of CD44 immunopositive cells that were not Kit immunopositive. Our results indicate that these CD44 immunopositive cells truly correspond to ICCs, thus immunolocalisation of CD44 can be used as a special marker, in addition to Kit, to identify ICCs in the digestive tract in adult guinea pigs and mice.
Asunto(s)
Tracto Gastrointestinal/citología , Tracto Gastrointestinal/inmunología , Receptores de Hialuranos/inmunología , Receptores de Hialuranos/metabolismo , Animales , Colon/citología , Colon/inmunología , Colon/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Mucosa Gástrica/metabolismo , Tracto Gastrointestinal/metabolismo , Cobayas , Inmunohistoquímica , Intestino Delgado/citología , Intestino Delgado/inmunología , Intestino Delgado/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Proteínas de Neurofilamentos/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Estómago/citología , Estómago/inmunología , Porcinos , Vimentina/metabolismoRESUMEN
This study aimed at evaluating whether apoptosis of interstitial cells of Cajal (ICC), smooth muscle cells (SMC), and enteric neurons was involved in a guinea pig model of intestinal ischemia and reperfusion injury. The small intestinal segments were resected at either 6 (I60/R6h) and 12 h (I60/R12h) or 7 (I60/R7d) to 14 (I60/R14d) days after 60 min intestinal ischemia in the adult guinea pigs and studied by immunohistochemistry with anti-Kit, 5-bromo-2'-deoxyuridine (BrdU), alpha-smooth muscle actin, vimentin, and beta-tublin III antibodies. Also, apoptosis was tested by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. In the I60/R12h injury, there was a approximately 50% decrease of Kit+ cells in cell numbers at the level of myenteric plexus and a number of Kit-/vimentin-positive cells were labeled by TUNEL. Also, a few SMC and enteric neurons were TUNEL positive. The Kit+ ICC recovered to normal and a number of Kit-/BrdU-double-positive cells were observed in the I60/R14d group. Our results indicated that the intestinal I/R injury could lead to apoptosis of ICC, SMC, and enteric neurons which may contribute to the gastrointestinal motility disorders, and proliferation was involved in the recovery of ICC.