RESUMEN
Nogo-A is a well-known myelin-enriched inhibitory protein for axonal growth and regeneration in the central nervous system (CNS). Besides oligodendrocytes, our previous data revealed that Nogo-A is also expressed in subpopulations of neurons including retinal ganglion cells, in which it can have a positive role in the neuronal growth response after injury, through an unclear mechanism. In the present study, we analyzed the opposite roles of glial versus neuronal Nogo-A in the injured visual system. To this aim, we created oligodendrocyte (Cnp-Cre(+/-)xRtn4/Nogo-A(flox/flox)) and neuron-specific (Thy1-Cre(tg+)xRtn4(flox/flox)) conditional Nogo-A knock-out (KO) mouse lines. Following complete intraorbital optic nerve crush, both spontaneous and inflammation-mediated axonal outgrowth was increased in the optic nerves of the glia-specific Nogo-A KO mice. In contrast, neuron-specific deletion of Nogo-A in a KO mouse line or after acute gene recombination in retinal ganglion cells mediated by adeno-associated virus serotype 2.Cre virus injection in Rtn4(flox/flox) animals decreased axon sprouting in the injured optic nerve. These results therefore show that selective ablation of Nogo-A in oligodendrocytes and myelin in the optic nerve is more effective at enhancing regrowth of injured axons than what has previously been observed in conventional, complete Nogo-A KO mice. Our data also suggest that neuronal Nogo-A in retinal ganglion cells could participate in enhancing axonal sprouting, possibly by cis-interaction with Nogo receptors at the cell membrane that may counteract trans-Nogo-A signaling. We propose that inactivating Nogo-A in glia while preserving neuronal Nogo-A expression may be a successful strategy to promote axonal regeneration in the CNS.
Asunto(s)
Axones/fisiología , Proteínas de la Mielina/genética , Traumatismos del Nervio Óptico/terapia , Regeneración , Células Ganglionares de la Retina/fisiología , Transducción de Señal , Animales , Dependovirus/genética , Femenino , Vectores Genéticos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Vaina de Mielina/fisiología , Compresión Nerviosa , Neuroglía/citología , Neuronas/citología , Proteínas NogoRESUMEN
Deletions of chromosomal arms 1p and 19q are frequent in oligodendroglial tumours and linked to radio- and chemotherapy response as well as longer survival. The molecular mechanisms underlying this clinically important association are as yet unknown. Here, we studied the peroxiredoxin 1 (PRDX1) gene at 1p34.1 for promoter methylation and expression in primary gliomas and investigated its role in radio- and chemosensitivity of glioma cells in vitro. In total, we screened primary glioma tissues from 93 patients for methylation of the 5'-CpG island of PRDX1 by sodium bisulfite sequencing. PRDX1 mRNA and protein expression levels were determined in subsets of the tumours by quantitative PCR and western blot analysis, respectively. PRDX1 hypermethylation and reduced expression were frequently detected in oligodendroglial tumours and secondary glioblastomas, but not in primary glioblastomas. In oligodendroglial tumours, both PRDX1 hypermethylation and reduced mRNA expression were significantly associated with 1p/19q-deletion. Stable knockdown of PRDX1 by lentiviral transduction of short-hairpin (sh)RNA constructs significantly increased apoptosis and reduced cell viability of Hs683 glioma cells exposed to ionizing irradiation or temozolomide in vitro. Taken together, our findings indicate that epigenetic silencing of PRDX1 is frequent in 1p/19q-deleted oligodendroglial tumours and likely contributes to radio- and chemosensitivity of these tumours.
Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 1/genética , Glioma/patología , Oligodendroglía/metabolismo , Peroxirredoxinas/genética , Regiones Promotoras Genéticas/genética , Tolerancia a Radiación/genética , Adulto , Anciano , Apoptosis/efectos de los fármacos , Apoptosis/genética , Apoptosis/efectos de la radiación , Línea Celular Tumoral , Islas de CpG/genética , Metilación de ADN/efectos de los fármacos , Metilación de ADN/efectos de la radiación , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Regulación hacia Abajo/genética , Femenino , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Glioma/tratamiento farmacológico , Glioma/genética , Glioma/radioterapia , Humanos , Isocitrato Deshidrogenasa/genética , Masculino , Persona de Mediana Edad , Mutación , Oligodendroglía/efectos de los fármacos , Oligodendroglía/patología , Oligodendroglía/efectos de la radiación , Peroxirredoxinas/deficiencia , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/efectos de la radiación , Temozolomida , Adulto JovenRESUMEN
Deletions of chromosomal arms 1p and 19q are frequent in oligodendroglial tumours and have been associated with sensitivity to radio- and chemotherapy as well as favourable prognosis. By using microarray-based expression profiling, we found that oligodendroglial tumours with 1p and 19q losses showed significantly lower expression of the CBP/p300-interacting transactivator with glutamic acid/aspartic acid-rich carboxyl-terminal domain 4 gene (CITED4) at 1p34.2 as compared to tumours without 1p and 19q losses. Mutational analysis showed no CITED4 mutations in gliomas. However, 1p and 19q losses as well as low expression of CITED4 transcripts were significantly associated with hypermethylation of the CITED4-associated CpG island. In line with the latter finding, treatment of CITED4 hypermethylated glioma cell lines with 5-aza-2'-deoxycytidine and trichostatine A resulted in a marked increase of the CITED4 transcript levels. Furthermore, CITED4 hypermethylation was significantly associated with longer recurrence-free and overall survival of patients with oligodendroglial tumours. Taken together, our results indicate that CITED4 is epigenetically silenced in the vast majority of oligodendroglial tumours with 1p and 19q deletions and suggest CITED4 hypermethylation as a novel prognostic marker in oligodendroglioma patients.
Asunto(s)
Neoplasias Encefálicas/genética , Cromosomas Humanos Par 19 , Cromosomas Humanos Par 1 , Pérdida de Heterocigocidad , Oligodendroglioma/genética , Factores de Transcripción/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias Encefálicas/mortalidad , Niño , Islas de CpG , Metilación de ADN , Regulación hacia Abajo , Femenino , Silenciador del Gen , Humanos , Masculino , Persona de Mediana Edad , Oligodendroglioma/mortalidad , Polimorfismo Genético , Análisis de Supervivencia , Factores de Transcripción/biosíntesis , Transcripción GenéticaRESUMEN
Drosophila dosage compensate (equalize X-linked gene products) by doubling the transcription of most X-linked genes in males. The MSL (male-specific lethal) ribonucleoprotein complex consisting of at least five proteins and two non-coding RNAs (roX1 and roX2) is essential for this transcription response. Recently it has been shown that the X-linked roX1 and roX2 genes each contain at least one chromatin entry site for the MSL complex. In this study we show that insertion of either roX1 or roX2 DNA sequences, upstream of an insulated lacZ reporter gene controlled with the constitutive armadillo promoter (arm-lacZ), results in a significant elevation of expression of lacZ in males. However, full compensation, that is a precise doubling of lacZ expression in males relative to females, was only observed in some lines carrying autosomal insertions of either roX1-arm-lacZ or roX2-arm-lacZ transgenes. Furthermore, we found that a 419-base pair fragment of roX1 that contains an MSL binding site was sufficient to cause a modest elevation of expression of lacZ in males, but this response was significantly less than obtained with a full-length roX1 cDNA. This is the first direct demonstration that insertion of an MSL chromatin entry site on an autosome results in elevated expression in males of genes near the entry site.