Time-dependent and sequential modulation of signaling, CREB activation and BDNF expression induced by antidepressants

Although cellular processes involved in depression and antidepressants mechanisms are still not well characterized, recent studies showed that intracellular pathways modulating gene transcription are involved in the pathophysiology of depression and in antidepressant mechanisms [1]. The effects of antidepressant on cAMPresponsive element binding protein (CREB) function, a transcription factor regulating expression of several genes involved in neuroplasticity, cell survival, and cognition, has been extensively studied. CREB is activated by several physiological stimuli; in neurons activitydependent phosphorylation of CREB at Ser133 is induced by activation of the calcium/calmodulin-dependent kinase (CaMK) and the mitogen-activated kinase (ERK1/2) cascades [2]. These pathways are involved in both activity-dependent regulation of gene expression and antidepressant mechanisms. Although there is general agreement that chronic antidepressants stimulate CREB function, conflicting results suggest that different effects may depend on drug type, way of administration and different time window of measurement. In this study we analyzed the time-course of the treatment with the antidepressants fluoxetine (SSRI) and reboxetine (NaRI), by measuring the temporal effects of antidepressants on CaMKIV, CaMKII, ERK1/2 activation in nuclear fraction from hippocampus (HC) and prefrontal/frontal cortex (P/FC). Furthermore, we measured the timecourse of nuclear CREB activation in the same areas and the expression of brain-derived neurotrophic factor (BDNF), a CREB-regulated gene, implicated in both pathophysiology and pharmacology of mood disorders. Male rats were treated with fluoxetine (10 mg/kg) or reboxetine (10 mg/kg) administered in drinking water. Rats were divided in 5 groups following this schedule: control, one-week treatment, two-week treatment, three-week treatment and three-week treatment plus one-week drug wash-out. Nuclei from HC and P/FC were prepared by differential centrifugation. Phosphorylation and expression levels of proteins were measured by SDS-PAGE, Western Blot and immunostaining P/FC, while both drugs persistently activated ERK2 in HC. Finaly, BDNF (14kDa) expression showed a profile similar to CREB activation, with an increase of expressionat 2 weeks followed by a reduction. Our results suggest a transient profile of CREB activation by antidepressants, with a peak of phosphorylation in the first 1-2 weeks and subsequent return to basal levels. This profile is consistent with t. Pharmacological treatments did not alter CREB expression, but fluoxetine for 1 and 2 weeks increased CREB phosphorylation in nuclear fraction from HC, while one-week treatment with reboxetine induced CREB activation in nuclei from P/FC. With both drugs CREB phosphorylation returned to basal levels during three-week treatment and the wash out week. CaMKIV activation followed a temporal profile similar to CREB time-course in HC, while in P/FC phosphorylation was sustained up to the wash-out week. CaMKII activation showed a down-regulation in HC from rats treated with fluoxetine. Regarding ERK1/2 activation, three-week treatment reduced ERK1/2 phosphorylation in he activation of CaMKIV in HC and P/FC and ERK2 in P/FC. Furthermore, BDNF expression shows a similar biphasic profile, with the increase following one week after the peak of CREB activation. These results suggest that time-dependent activation of signalling and CREB by antidepressants is a rather early event, followed by sequential activation of the CREB-dependent gene BDNF. Reference(s) [1] Tardito D, Perez J, Tiraboschi E, et al., 2006, Signaling pathways regulating gene expression, neuroplasticity, and neurotrophic mechanisms in the action of antidepressants: a critical overview. Pharmacol Rev 58(1): 115-34. [2] Kasahara J, Fukunaga K, Miyamoto E, 2001, Activation of calcium/calmodulin-dependent protein kinase IV in long term potentiation in the rat hippocampal CA1 region. J Biol Chem 276(26): 24044-50.