Downregulation of cAMP response element-binding protein by lentiviral vector-mediated RNAi attenuates morphine withdrawal syndromes in rats

cAMP response element-binding protein(CREB) and the cAMP cascade play a pivotal role in the opiate-dependence. The blockade of this cascade is believed to attenuate signs of physical opiate withdrawal. A lentiviral vector (LV) expressing a small hairpin RNA (shRNA) to silence CREB in vitro and in vivo was used in this study. The effect of the shRNA on the regulation of the relevant protein expression and the signs of opiate withdrawal were subsequently evaluated in rats undergoing chronic morphine treatment. In cultured primary locus coeruleus (LC) neurons, the designed lentiviral vectors were successfully infected into the cells and led to 70% knockdown in CREB expression. In cells treated with chronic morphine, the expression of CREB, adenylyl cyclase (AC) and protein kinase A (PKA) were increased, while in cells infected with LV-CREB3, treated with chronic morphine treatment failed to increase the expressions of CREB and AC. Consistently, in the rat model for chronic morphine treatment, morphine increased the expression of CREB, AC and PKA in LC neurons. However, in rats received bilateral microinjections of LV-CREB3 into the LC, morphine did not alter the levels of these proteins. Moreover, microinjection of LV-CREB3 significantly attenuated the appearance of certain withdrawal behaviors. In conclusion, the lentiviral vectors expressing CREB shRNA inhibited the increase of CREB and AC expression induced by chronic morphine treatment both in vivo and in vitro. This inhibition was associated with the alleviation of some withdrawal behaviors. These findings suggested that lentivirus-mediated RNA interference could be useful for opiate-dependence therapy.

► Lentiviral vectors carrying code for CREB shRNA which effectively downregulate the CREB gene were well established.
► Downregulation of cAMP cascades induced by decreased CREB expression in LC was verified both in vivo and in vitro.
► Downregulation of CREB in LC by lentiviral vector-mediated RNAi attenuates morphine withdrawal syndromes in rats.