Small G-Protein Rheb gates mTOR signaling to regulate morphine tolerance in mice

Background Analgesic tolerance due to long-term use of morphine remains a challenge for pain management. Morphine acts on μ-opioid receptors and downstream of the phosphatidylinositol-3-kinase signaling pathway to activate the mammalian target of rapamycin (mTOR) pathway. Rheb is an important regulator of growth and cell-cycle progression in the central nervous system owing to its critical role in the activation of mTOR. We hypothesized that signaling via the GTP-binding protein Rheb in the dorsal horn of the spinal cord is involved in morphine-induced tolerance. Methods Male and female wild-type C57BL/6J mice or transgenic mice (6–8 weeks old) were injected intrathecally with saline or morphine twice daily at 12-h intervals for five consecutive days to establish a tolerance model. Analgesia was assessed 60 min later using the tail-flick assay. After five days, the spine was harvested for western blot or immunofluorescence analysis. Results Chronic morphine administration resulted in the upregulation of spinal Rheb by 4.27±0.195 fold (P=0.0036, n=6), in turn activating mTOR by targeting rapamycin complex 1 (mTORC1). Genetic overexpression of Rheb impaired morphine analgesia, resulting in a tail-flick latency of 4.65±1.10 s (P<0.0001, n=7) in Rheb KI mice compared to 10 s in control mice (10±0 s). Additionally, Rheb overexpression in spinal excitatory neurons led to mTORC1 signaling overactivation. Genetic knockout of Rheb or inhibition of mTORC1 signaling by rapamycin potentiated morphine-induced tolerance (MPE: 52.60±9.56% in the morphine + rapamycin group vs 16.60±8.54% in the morphine group, P<0.0001). Moreover, activation of endogenous adenosine 5‘-monophosphate-activated protein kinase inhibited Rheb upregulation and retarded the development of morphine-dependent tolerance (MPE: 39.51±7.40% in morphine + metformin group vs 15.58±5.79% in morphine group, P<0.0001). Conclusion This study suggests spinal Rheb as a key molecular factor for regulating mTOR signaling.