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. 2014 Jul 4:10:45.
doi: 10.1186/1744-8069-10-45.

Local translation and retrograde axonal transport of CREB regulates IL-6-induced nociceptive plasticity

Ohannes K Melemedjian, Dipti V Tillu, Jamie K Moy, Marina N Asiedu, Edward K Mandell, Sourav Ghosh, Gregory Dussor, Theodore J Price  1
Affiliations

Local translation and retrograde axonal transport of CREB regulates IL-6-induced nociceptive plasticity

Ohannes K Melemedjian et al. Mol Pain. .

Abstract

Transcriptional regulation of genes by cyclic AMP response element binding protein (CREB) is essential for the maintenance of long-term memory. Moreover, retrograde axonal trafficking of CREB in response to nerve growth factor (NGF) is critical for the survival of developing primary sensory neurons. We have previously demonstrated that hindpaw injection of interleukin-6 (IL-6) induces mechanical hypersensitivity and hyperalgesic priming that is prevented by the local injection of protein synthesis inhibitors. However, proteins that are locally synthesized that might lead to this effect have not been identified. We hypothesized that retrograde axonal trafficking of nascently synthesized CREB might link local, activity-dependent translation to nociceptive plasticity. To test this hypothesis, we determined if IL-6 enhances the expression of CREB and if it subsequently undergoes retrograde axonal transport. IL-6 treatment of sensory neurons in vitro caused an increase in CREB protein and in vivo treatment evoked an increase in CREB in the sciatic nerve consistent with retrograde transport. Importantly, co-injection of IL-6 with the methionine analogue azido-homoalanine (AHA), to assess nascently synthesized proteins, revealed an increase in CREB containing AHA in the sciatic nerve 2 hrs post injection, indicating retrograde transport of nascently synthesized CREB. Behaviorally, blockade of retrograde transport by disruption of microtubules or inhibition of dynein or intrathecal injection of cAMP response element (CRE) consensus sequence DNA oligonucleotides, which act as decoys for CREB DNA binding, prevented the development of IL-6-induced mechanical hypersensitivity and hyperalgesic priming. Consistent with previous studies in inflammatory models, intraplantar IL-6 enhanced the expression of BDNF in dorsal root ganglion (DRG). This effect was blocked by inhibition of retrograde axonal transport and by intrathecal CRE oligonucleotides. Collectively, these findings point to a novel mechanism of axonal translation and retrograde trafficking linking locally-generated signals to long-term nociceptive sensitization.

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Figures

Figure 1
Figure 1
IL-6 induces CREB synthesis in vitro and in vivo. A) Western blot and quantification of CREB following 15 min treatment of DRG cultures with escalating concentrations of IL-6. N = 8 independent culture wells per condition. B) Western blot and quantification of CREB in the ipsilateral sciatic nerve following intraplantar (IPL) injection of IL-6 (0.1 ng) or vehicle. CREB protein is enhanced in the sciatic nerve 2 hrs post-injection but not at 15 min following treatment. N = 6 mice per condition. C) Western blot and quantification for nascently synthesized CREB retrogradely transported to the sciatic nerve. Retrograde axonal trafficking of nascently synthesized CREB was determined by co-injecting IL-6 (0.1 ng) or vehicle with the methionine analogue AHA into the paw and measuring, from the sciatic nerve, the amount of CREB with incorporated AHA, 2 hrs post injection. The input lane contains lysate from sciatic nerve that did not undergo immunoprecipitation and is labeled with biotin-avidin on incorporated AHA. N = 6 mice per condition. * = p < 0.05 and ** = p < 0.01.
Figure 2
Figure 2
Disruption of axonal trafficking in the sciatic nerve prevents IL-6-mediated nociceptive plasticity. A) Disruption of microtubules with the injection of colchicine (100 μg) or nocodazole (10 μg) into the popliteal fossa (PF) of mice prevented the development of mechanical hypersensitivity and hyperalgesic priming caused by intraplantar injection of IL-6 (0.1 ng). N = 6 mice per condition. B) Popliteal fossa injection of the cytoplasmic dynein inhibitor ciliobrevin D (10 μg) prevented the development of mechanical hypersensitivity and hyperalgesic priming caused by Intraplantar injection of IL-6 (0.1 ng). N = 6 mice per condition. C) Right popliteal fossa injection of ciliobrevin D or nocodazole (both at 10 μg) fails to disrupt the effect of IL-6 given into the left hindpaw. N = 6 mice per condition D) Popliteal fossa injection of ciliobrevin D (CBD, 10 μg) at the time of intraplantar injection of IL6 prevented the enhancement of BDNF expression in the L4-L6 DRGs of mice 3 hrs following injections. N = 6 mice per condition. * = p < 0.05 and *** = p < 0.001.
Figure 3
Figure 3
Disruption of CREB action in the DRG attenuates IL-6-induced nociceptive plasticity. A) Intrathecal injection of cAMP response element (CRE) consensus sequence DNA oligonucleotides at the same time as intraplantar IL-6, prevented the development of IL-6-induced mechanical hypersensitivity and hyperalgesic priming. Mutant CRE oligos were not effective. N = 6 mice per condition. B) Intrathecal injection of CRE consensus sequence DNA oligonucleotide (CRE con) at the same time as intraplantar IL-6, prevented enhancement of BDNF expression in L4-L6 DRGs 3 hours following injections. N = 6 mice per condition. C) Intrathecal injection of BDNF (0.1 ng) mechanical hypersensitivity and hyperalgesic priming. Co-injection of CRE consensus sequence DNA oligonucleotides with BDNF did not affect mechanical hypersensitivity and hyperalgesic priming. N = 6 mice per condition. * = p < 0.05, ** = p < 0.01 and *** = p < 0.001.
Figure 4
Figure 4
Popliteal fossa injection of ciliobrevin D does not influence capsaicin-induced flinching but does affect late mechanical hypersensitivity. A) Popliteal fossa injection of vehicle or ciliobrevin D had no effect on capsaicin (0.9%)-induced nocifensive behaviors whereas lidocaine injection completely blocked the effects of capsaicin. N = 4 - 5 mice per condition. B) Lidocaine completely blocked capsaicin-induced mechanical hypersensitivity whereas ciliobrevin D only had an effect at 3 hrs after capsaicin injection. N = 4 – 5 mice per condition. * = p < 0.05 and *** = p < 0.001.
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