Fig 2: Model for the regulation of mu opioid receptor (MOR) signaling by Hsp90 in the brain. This summary model combines results from this manuscript with our earlier work using non-selective Hsp90 inhibitors in the brain (Lei et al., 2017). Together this data suggests that Hsp90a, p23, and Cdc37 act in concert in the brain to promote the phosphorylation of ERK MAPK by the MOR in response to opioid drugs, thus promoting opioid anti-nociception. Treatment with non-selective or selective inhibitors blocks this role, thus blocking ERK MAPK phosphorylation and blocking anti-nociception in response to opioids.

Fig 3: Identification of the co-chaperones p23 and Cdc37 as promoters of opioid anti-nociception in the brain. Male CD-1 mice used for all experiments, data reported as the mean ± SEM, with sample sizes of mice/group noted in each graph. (A–C) Ten nanomoles of Gedunin (p23, A), 10 nmol of Celastrol (Cdc37, B), or 0.1 nmol of KU177 (Aha1, C) or Vehicle control injected icv concurrently with paw incision surgery with a 24 h recovery, followed by 3.2 mg/kg morphine sc. Experiments performed with two technical replicates for each drug. *,**,****p < 0.05, 0.01, 0.0001 vs. same time point inhibitor treatment group by two-way ANOVA with Fisher’s Least Significant Difference post hoc test. (D) p23, Cdc37, or Negative Control CRISPR-treated mice with icv delivery of constructs analyzed by IHC for target knockdown on day 10. Representative images shown from the PRN. Both targets (p23 – red, Cdc37 – green) have a similar staining pattern to Hsp90α, and both are clearly reduced by CRISPR treatment. (E) Data from (D) for all mice quantitated as described in the “Materials and Methods” section. All mice treated in one technical replicate, with staining and analysis of the resulting tissue performed in more than one technical replicate. *p < 0.05 vs. same target Negative Control group by Unpaired 2-Tailed t-test. CRISPR treatment reduced p23 signal by 36.3% and Cdc37 by 46.0%. (F) CRISPR-treated mice had paw incision surgery performed on day 9, with injection of 3.2 mg/kg morphine sc on day 10. Performed in two technical replicates. *,**,***,****p < 0.05, 0.01, 0.001, 0.0001 vs. both same time point p23/Cdc37 CRISPR groups by two-way ANOVA with Fisher’s Least Significant Difference post hoc test.