RESUMO
The enzymes that determine protein O-GlcNAcylation, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), act on key transcriptional and epigenetic regulators, and both are abundantly expressed in the brain. However, little is known about how alterations in O-GlcNAc cycling affect human embryonic stem cell (hESC) neural differentiation. Here, we studied the effects of perturbing O-GlcNAcylation during neural induction of hESCs using the metabolic inhibitor of OGT, peracetylated 5-thio-N-acetylglucosamine (Ac4-5SGlcNAc). Treatment of hESCs with Ac4-5SGlcNAc during induction limited protein O-GlcNAcylation and also caused a dramatic decrease in global levels of UDP-GlcNAc. Concomitantly, a subpopulation of neural progenitor cells (NPCs) acquired an immature neuronal morphology and expressed early neuronal markers such as ß-III tubulin (TUJ1) and microtubule associated protein 2 (MAP2), phenotypes that took longer to manifest in the absence of OGT inhibition. These data suggest that chemical inhibition of OGT and perturbation of protein O-GlcNAcylation accelerate the differentiation of hESCs along the neuronal lineage, thus providing further insight into the dynamic molecular mechanisms involved in neuronal development.
Assuntos
Azidas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Hexosaminas/farmacologia , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Neurônios/citologia , Acilação/efeitos dos fármacos , Azidas/química , Linhagem Celular , Inibidores Enzimáticos/química , Hexosaminas/química , Humanos , Neurônios/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacosRESUMO
We previously proposed that DNA recombination/repair processes play a role in memory formation. Here, we examined the possible role of the fen-1 gene, encoding a flap structure-specific endonuclease, in memory consolidation of conditioned taste aversion (CTA). Quantitative real-time PCR showed that amygdalar fen-1 mRNA induction was associated to the central processing of the illness experience related to CTA and to CTA itself, but not to the central processing resulting from the presentation of a novel flavor. CTA also increased expression of the Fen-1 protein in the amygdala, but not the insular cortex. In addition, double immunofluorescence analyses showed that amygdalar Fen-1 expression is mostly localized within neurons. Importantly, functional studies demonstrated that amygdalar antisense knockdown of fen-1 expression impaired consolidation, but not short-term memory, of CTA. Overall, these studies define the fen-1 endonuclease as a new DNA recombination/repair factor involved in the formation of long-term memories.