One hour after OGD in the primary cortical cultures, CREB was phosphorylated, and the level of phosphorylation reached a maximum at 3–6 hr after reoxygenation, then declined to basal levels after 12 hr post-reoxygenation (Figure 1A and S1A available online). To measure CRE activity, neurons were infected with an adenovirus as a CRE reporter. We found a significant enhancement of CRE activity after 3–12 hr post-reoxygenation (Figure 1B). During OGD and early (1 hr) after reoxygenation, CREB phosphorylation was not followed by an increase in CRE activity, www.selleckchem.com/products/nu7441.html but late (12 hr) after reoxygenation, CRE activity remained high, regardless of the basal level

of CREB phosphorylation. The results of a reporter assay using Gal4-fusion Ser133-disrupted CREB suggested the presence of the Ser133-independent activation of CREB following OGD (Figure S1B). To examine Ser133-independent regulation in cortical neurons, we performed CRE-reporter assays in the presence of cAMP agonists and a kinase inhibitor. Staurosporine at low dose (10 nM), a nonspecific kinase inhibitor, decreased the level of phospho-Ser133, but it induced the activity of a CRE reporter to comparable levels as the phospho-Ser133-inducer db-cAMP or forskolin (Figure S1C), suggesting the involvement of another

type of CREB coactivator, TORC. It has been shown that CREB activity is blocked by the find more inhibition of calcineurin. Indeed, we found this to Terminal deoxynucleotidyl transferase be true in neurons after OGD because the calcineurin inhibitor cyclosporine A (CsA) and FK 506 suppressed CRE activity (Figure 1C), despite the upregulation of CREB phosphorylation by FK 506 (Figure 1C). The mechanism by which calcineurin activates CREB could be the dephosphorylation-dependent nuclear entry of CREB-coactivator TORCs. To examine the activity of endogenous TORCs after OGD, the activity of Gal4-fusion full-length CREB (TORC

activatable) and bZIP-less CREB (TORC silent) was monitored. The activity of the full-length CREB, but not bZIP-less CREB, was enhanced after OGD (Figure 1D), suggesting that TORCs may contribute to the Ser 133-independent activation of CREB after OGD reoxygenation. A high level of TORC1 protein and a moderate level of TORC2 protein were detected in primary cortical cultures (Figure S2A). Under basal conditions, endogenous TORC1 was predominantly localized in the cytoplasm of cortical neurons (Figure 2A, control). Although the phosphorylation levels of CREB at Ser133 were increased during OGD (Figure S1A), TORC1 remained in the cytoplasm before reoxygenation but quickly translocated into the nucleus after reoxygenation (Figure 2A). Indeed, GFP-TORC1 translocated into the nucleus in response to OGD (Figure S2B). Next, we examined the intracellular distribution of TORC1 in response to OGD by separately isolating nuclei from cytoplasmic compartments. OGD followed by reoxygenation induced the nuclear localization of TORC1 (Figure S2C).