Morphine-induced RACK1-dependent autophagy in immortalized neuronal cell lines.

Autophagy is a critical cellular catabolic process in cell homoeostasis and brain function. Recent studies indicate that receptor for activated C kinase 1 (RACK1) is involved in autophagosome formation in Drosophila and mice, and that it plays an essential role in morphine-associated memory. However, the exact mechanism of the role of RACK1 in morphine-induced autophagy is not fully understood. SH-SY5Y cells were cultured and morphine, rapamycin, 3-methyladenine and RACK1 siRNA were used to evaluate the regulation of RACK1 protein in autophagy. Western blotting and immunofluorescence were used to assess protein expression. Activation of autophagy (i.e. autophagosome accumulation and an increase in the LC3-II/LC3-I ratio) induced by morphine contributes to the maintenance of conditioned place preference (CPP) memory in mice. Moreover, morphine treatment significantly increased Beclin-1 expression and decreased the p-mTOR/mTOR and SQSTM1/p62 levels, whereas knockdown of RACK1 prevented morphine-induced autophagy in vitro. Furthermore, we found that in the mouse hippocampus, knockdown of RACK1 also markedly suppressed morphine-induced autophagy (decreased LC3-II/LC3-I ratio and increased p-mTOR/mTOR ratio). Importantly, morphine-induced autophagy in a RACK1-dependent manner. Conversely, morphine-induced RACK1 upregulation in vitro is partially inhibited by autophagy feedback. Our findings revealed a critical role for RACK1-dependent autophagy in morphine-promoted maintenance of CPP memory in mice and supported the notion that control of RACK1-dependent autophagic pathways may become an important target for novel therapeutics for morphine-associated memory.