Abstract
Opioid activation of the mu opioid receptor (MOR) promotes signaling cascades that evoke both analgesic responses to pain and side effects like addiction and dependence. Manipulation of these cascades, such as by biased agonism, has great promise to improve opioid therapy. However, the signaling cascades of the MOR are in general poorly understood, providing few targets for drug development. In our earlier work, we identified Heat shock protein 90 (Hsp90) as a novel and crucial regulator of opioid anti-nociception in the brain by promoting ERK MAPK activation. In this study, we sought to identify the molecular isoforms and co-chaperones by which Hsp90 carried out this role, which could provide specific targets for future clinical intervention. We used novel selective small molecule inhibitors as well as CRISPR/Cas9 gene editing constructs delivered by the intracerebroventricular (icv) route to the brains of adult CD-1 mice to target Hsp90 isoforms (Hsp90α/β, Grp94) and co-chaperones (p23, Cdc37, Aha1). We found that inhibition of the isoform Hsp90α fully blocked morphine anti-nociception in a model of post-surgical paw incision pain, while blocking ERK and JNK MAPK activation, suggesting Hsp90α as the main regulator of opioid response in the brain. We further found that inhibition of the co-chaperones p23 and Cdc37 blocked morphine anti-nociception, suggesting that these co-chaperones assist Hsp90α in promoting opioid anti-nociception. Lastly, we used cycloheximide treatment in the brain to demonstrate that rapid protein translation within 30 min of opioid treatment is required for Hsp90 regulation of opioid response. Together these studies provide insight into the molecular mechanisms by which Hsp90 promotes opioid anti-nociception. These findings thus both improve our basic science knowledge of MOR signal transduction and could provide future targets for clinical intervention to improve opioid therapy.
Highlights
The mu opioid receptor (MOR) evokes complex signal transduction cascades upon activation by opioid ligands like morphine
Over a full morphine dose range of 1–10 mg/kg, we found that KUNA115 strongly blocked morphine anti-nociception in paw incision pain, suggesting the involvement of the isoform Hsp90α (Figure 1A)
We have identified the isoform Hsp90α as a key player in promoting opioid anti-nociception and signaling in the brain using both selective small molecule inhibitors (Figures 1, 2) and CRISPR/Cas9 gene editing in the brains of adult mice (Figure 3)
Summary
The mu opioid receptor (MOR) evokes complex signal transduction cascades upon activation by opioid ligands like morphine. It is clear that signaling regulators beyond this simple cascade have a strong impact on opioid anti-nociception and side effects, including other G proteins, ERK MAPK (Macey et al, 2009), Src (Zhang et al, 2017), CaMKII (Li et al, 2016), RSK2 (Darcq et al, 2012), and others. These signaling regulators could provide important targets for opioid drug development; for instance βarrestin was shown to reduce opioid anti-nociception while promoting side effects like tolerance and dependence, leading to the development of βarrestin biased agonists with reduced side effects (Bohn et al, 1999; Raehal et al, 2005; Dewire et al, 2013; Manglik et al, 2016; Schmid et al, 2017). This gap illustrates the need for investigation into the signalosome of the MOR and the mechanisms by which these regulators impact opioid physiology
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