RESUMEN
Fe65 is a binding partner of the Alzheimer's beta-amyloid precursor protein APP. The possible involvement of this protein in the cellular response to DNA damage was suggested by the observation that Fe65 null mice are more sensitive to genotoxic stress than WT counterpart. Fe65 associated with chromatin under basal conditions and its involvement in DNA damage repair requires this association. A known partner of Fe65 is the histone acetyltransferase Tip60. Considering the crucial role of Tip60 in DNA repair, we explored the hypothesis that the phenotype of Fe65 null cells depended on its interaction with Tip60. We demonstrated that Fe65 knockdown impaired recruitment of Tip60-TRRAP complex to DNA double strand breaks and decreased histone H4 acetylation. Accordingly, the efficiency of DNA repair was decreased upon Fe65 suppression. To explore whether APP has a role in this mechanism, we analyzed a Fe65 mutant unable to bind to APP. This mutant failed to rescue the phenotypes of Fe65 null cells; furthermore, APP/APLP2 suppression results in the impairment of recruitment of Tip60-TRRAP complex to DNA double strand breaks, decreased histone H4 acetylation and repair efficiency. On these bases, we propose that Fe65 and its interaction with APP play an important role in the response to DNA damage by assisting the recruitment of Tip60-TRRAP to DNA damage sites.
Asunto(s)
Roturas del ADN , Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Proteínas del Tejido Nervioso/fisiología , Proteínas Nucleares/fisiología , Acetilación , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Reparación del ADN , Lisina Acetiltransferasa 5 , Ratones , Proteínas Nucleares/metabolismo , Nexinas de Proteasas , Transporte de Proteínas , Receptores de Superficie Celular/metabolismo , TransactivadoresRESUMEN
Notch proteins are definitely recognized as key regulators of the neuronal fate during embryo development, but their function in the adult brain is still largely unknown. We have previously demonstrated that Notch pathway stimulation increases microtubules stability followed by the remodeling of neuronal morphology with neurite varicosities loss, thicker neuritis, and enlarged growth cones. Here we show that the neurite remodeling is a dynamic event, dependent on transcription and translation, and with functional implications. Exposure of differentiated human SH-SY5Y neuroblastoma cells to the Notch ligand Jagged1 induces varicosities loss all along the neurites, accompanied by the redistribution of presynaptic vesicles and the decrease in neurotransmitters release. As evaluated by time lapse digital imaging, dynamic changes in neurite morphology were rapidly reversible and dependent on the activation of the Notch signaling pathway. In fact, it was prevented by the inhibition of the proteolytic gamma-secretase enzyme or the transcription machinery, and was mimicked by the transfection of the intracellular domain of Notch. One hour after treatment with Jagged1, several genes were downregulated. Many of these genes encode proteins that are known to be involved in protein synthesis. These data suggest that in adult neurons, Notch pathway activates a transcriptional program that regulates the equilibrium between varicosities formation and varicosities loss in the neuronal presynaptic compartment involving the expression and redistribution of both structural and functional proteins.
Asunto(s)
Diferenciación Celular/fisiología , Neuritas/fisiología , Neuronas/citología , Receptor Notch1/metabolismo , Actinas/genética , Actinas/metabolismo , Análisis de Varianza , Proteínas de Unión al Calcio/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular Transformada , Dactinomicina/farmacología , Inhibidores Enzimáticos/farmacología , Perfilación de la Expresión Génica/métodos , Proteínas Fluorescentes Verdes/genética , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/farmacología , Proteína Jagged-1 , Proteínas de la Membrana/farmacología , Análisis por Micromatrices/métodos , Proteínas Asociadas a Microtúbulos , Neuritas/efectos de los fármacos , Neuroblastoma/patología , Neuronas/efectos de los fármacos , Norepinefrina/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Receptor Notch1/genética , Proteínas Serrate-Jagged , Transducción de Señal/fisiología , Sinapsinas/genética , Sinapsinas/metabolismo , Factores de Tiempo , Transfección , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMEN
Fe65 interacts with the cytosolic domain of the Alzheimer amyloid precursor protein (APP). The functions of the Fe65 are still unknown. To address this point we generated Fe65 knockout (KO) mice. These mice do not show any obvious phenotype; however, when fibroblasts (mouse embryonic fibroblasts), isolated from Fe65 KO embryos, were exposed to low doses of DNA damaging agents, such as etoposide or H2O2, an increased sensitivity to genotoxic stress, compared with wild type animals, clearly emerged. Accordingly, brain extracts from Fe65 KO mice, exposed to non-lethal doses of ionizing radiations, showed high levels of gamma-H2AX and p53, thus demonstrating a higher sensitivity to X-rays than wild type mice. Nuclear Fe65 is necessary to rescue the observed phenotype, and few minutes after the exposure of MEFs to DNA damaging agents, Fe65 undergoes phosphorylation in the nucleus. With a similar timing, the proteolytic processing of APP is rapidly affected by the genotoxic stress: in fact, the cleavage of the APP COOH-terminal fragments by gamma-secretase is induced soon after the exposure of cells to etoposide, in a Fe65-dependent manner. These results demonstrate that Fe65 plays an essential role in the response of the cells to DNA damage.