Persistent Rheb-induced mTORC1 activation in spinal cord neurons induces hypersensitivity in neuropathic pain

Abstract

The small GTPase Ras homolog enriched in the brain (Rheb) can activate mammalian target of rapamycin (mTOR) and regulate the growth and cell cycle progression. We investigated the role of Rheb-mediated mTORC1 signaling in neuropathic pain. A chronic constriction injury (CCI) model was dopted. CCI induced obvious spinal Rheb expression and phosphorylation of mTOR, S6, and 4-E-BP1. Blocking mTORC1 signal with rapamycin alleviated the neuropathic pain and restored morphine efficacy in CCI model. Immunofluoresence showed a neuronal co-localization of CCI-induced Rheb and pS6. Rheb knockin mouse showed a similar behavioral phenotype as CCI. In spinal slice recording, CCI increased the firing frequency of neurons expressing HCN channels; inhibition of mTORC1 with rapamycin could reverse the increased spinal neuronal activity in neuropathic pain. Spinal Rheb is induced in neuropathic pain, which in turn active the mTORC1 signaling in CCI. Spinal Rheb-mTOR signal plays an important role in regulation of spinal sensitization in neuropathic pain, and targeting mTOR may give a new strategy for pain management.

Fig. 1. Neuropathic pain induces the expression of spinal Rheb.

a Daily nociceptive behavior before CCI injury (BL) and on day 1, 3, 5, 7 after CCI injury with vehicle or rapamycin intrathecal injection once daily; b statistical analysis of nociceptive behavior of all these groups on baseline, day 3, 5 and 7 after CCI injury; c CCI significantly increase the expression of Rheb in spinal dorsal compared with sham group; d, e CCI significantly increase the immunofluorescence of Rheb in the spinal dorsal horn; f CCI-induced expression of Rheb mostly co-immunostaining with neuron (left, NeuN in green), but not astroglia (middli, GFAP in green), or microglia (right, Ibal in green). Repeated Measures Two-way ANOVA + Bonferroni (b), Student’s t test, two-tailed (c, e). n = 6 for all groups; *P < 0.05. Error bars are mean ± SEM. Overlaid points are individual subject.

Fig. 2. Rheb-induced activation of mTORC1 in Neuropathic pain and block the mTORC1 signal with rapamycin alleviate the development of neuropathic pain.

a, b, c Western blot analysis of spinal cord to assess the activation of mTORC1 signal in sham mice and mice with CCI of the sciatic nerve after 7 days, CCI obviously increase the phosphorylation of spinal mTOR, S6 and 4-EBP1 (n = 6 for all groups); d CCI-induced phosphorylation of spinal S6 in the dorsal horn (n = 5); e CCI-induced phosphorylation of S6 co-immunostaining with neuron (left, NeuN in green), but nont astroglia (middle, GFAP in green), or microglia (right, CD11b in green) in the spinal dorsal horn, scale bars = 50 μm. Student’s t test (a, b, c, d). *P < 0.05. Error bars are mean ± SEM. Overlaid points are individual animal scores.

Fig. 3. Bolus rapamycin restores aucte morphine efficacy in Neuropathic pain mice and CCI-induced Rheb co-localized with pS6 in the spinal dorsal horn.

a Nociceptive behavior on day 7 after CCI injury and morphine antinociception while co-treatment with vehicle or rapamycin (n = 6); b intrathecal rapamycin (red) restores acute morphine efficacy in CCI model as compared with vehicle group (blue); c CCI-induced Rheb (red) co-localized with phosphorylated S6 (green) in the spinal dorsal horn in neuropathic pain mice. Repeated measures two-way ANOVA + Bonferroni (b). *P < 0.05. Error bars are mean ± SEM. Overlaid points are individual subject.

Fig. 4. Rheb KI mice mimics the impaired acute morphine efficacy and increased tolerance phenotype of morphine-induced antinociception in CCI model.

a Rheb knockin mice showed an impaired morphine-induced antinociception after acute intrathecal injection (10 µg, i.t.; n = 7 for littermate control group, n = 9 for CKI group); b antinociceptive efficacy of morphine (10 µg, i.t.) in mice with Chronic Constriction Injury (CCI) of the sciatic nerve after 7 days (n = 6 for sham group, n = 6 for CCI group); c Daily nociceptive behavior and opioid antinociception throughout a 5 day chronic morphine schedule (10 μg i.t., twice daily) in CCI mice; Nociceptive behavior (pre-morphine BL timepoints only): tail immersion; Antinociception (post-morphine 1 h timepoints only): tail immersion; antinociceptive tolerance: d maximal possible effect (MPE) for morphine antinociception from the first administration on day 1 (Day 1: +1 h) compared to the first administration on day 3 (Day 3: +1 h) (tail immersion), and e the percent change between day 1 and 3 of each subject. sham, n = 6; CCI, n = 6; f Daily nociceptive behavior and opioid antinociception throughout a 5 day chronic morphine schedule (10ug i.t., twice daily) in Rheb CKI mice and littermate control group; Nociceptive behavior (pre-morphine BL timepoints only): tail immersion; antinociception (post-morphine 1 h timepoints only): tail immersion; antinociceptive tolerance: g Maximal possible effect (MPE) for morphine antinociception from the first administration on day 1 (Day 1: +1 h) compared to the first administration on day 3 (Day 3: +1 h) (tail immersion), and h the percent change between day 1 and 3 of each subject. Littermate control group, n = 12; Rheb CKI, n = 10.

Fig. 5. Rapamycin suppressed the dorsal horn neuron’s spike firing in morphine-induced tolerance and neuropathic pain models.

a Representative traces showing dorsal horn neuron’s voltage responses evoked by current injections (−100 pA, 160 pA) before (left) and immediately after (right) bath application of 10 μM ZD7288, n = 40; b Sag ratio of the same neuron before and after bath application of 10 μM ZD7288; representative traces showing dorsal horn neuron’s voltage responses evoked by current injections (−100 pA, 160 pA) in control mice after 5 day continuously application of saline (c) or rapamycin (d), summary of data showing the effect of current injection evoked spike firing after application of saline or rapamycin (e), n = 8. Representative traces showing dorsal horn neuron’s voltage responses evoked by current injections (−100 pA, 160 pA) in CCI induced neuropathic pain model after 5 day continuously application of saline (f) or rapamycin (g), summary of data showing the effect of current injection evoked spike firing after application of saline or rapamycin (h), n = 10 in CCI + saline and 13 in CCI + rapamycin. The paired Student’s t test was performed for the data in (b) and Kolmogorov–Smirnov test for the data in (e, h). n.s., not significant; *P < 0.05, ***P < 0.001. Data are represented as mean ± SEM.