Unraveling the Molecular Signaling Pathway Through Which Spinal Hsp90 Inhibition Enhances Opioid Antinociception

Abstract

Research efforts to discover new ways to improve opioid analgesic therapy have led to our lab’s discovery that inhibition of Heat Shock Protein 90 (Hsp90) in the spinal cord leads to enhanced opioid antinociceptive effects in acute pain models. To uncover the mechanism behind this enhancement, we hypothesized that both Src Kinase and Protein Kinase C (PKC) would be increased following administration of the Hsp90 inhibitor, 17-AAG. To test this, mice were administered 17-AAG intrathecally followed 24 hours later with either a Src or PKC inhibitor intrathecally, followed by morphine subcutaneously, and a tail flick assay was performed. We found that inhibition of both Src and PKC were able to block the enhanced opioid antinociception seen with 17-AAG. Further, activation of PKC was able to mimic the Hsp90 inhibitor effects, and Src inhibition was able to block baseline morphine effects. Western blotting and immunohistochemistry (IHC) analysis of spinal cords from mice treated with 17-AAG and the potent mu opioid receptor agonist, DAMGO, confirmed the upregulation of both kinases in the dorsal horn. Using siRNA knockdowns, an isoform specific effect for PK β was identified and confirmed via IHC. Colocalization studies revealed an upregulation of Src in neurons by DAMGO treatment alone, and a further upregulation of Src in microglia when DAMGO was combined with 17-AAG. PKCβ appeared to be upregulated in calcitonin gene related peptide (CGRP) neurons. Further pharmacological manipulation of microglia revealed that treatment with lipopolysaccharide was able to block the enhanced opioid antinociception seen with 17-AAG, while treatment with minocycline was able to mimic it. Custom CRISPR knockdowns were performed targeting Src in microglia and PKCβ in CGRP neurons and both were able to successfully block the effects of 17-AAG, thus confirming a role for both Src and PKCβ activation in the pathway leading from spinal Hsp90 inhibition to the enhanced opioid antinociception seen behaviorally. To further define this pathway, a series of Western blotting and IHC studies were performed to examine the effects that knocking down one signaling molecule would have on other signaling molecules known to play a role in this pathway. Through these efforts, we were able to systematically place downregulation of epidermal growth factor receptor (EGFR) at the beginning of this pathway following Hsp90 inhibition. Colocalization studies showed the downregulation of EGFR also occurred in microglia. This downregulation lies upstream of the activation of Src in microglia cells. DUSP15 activation in CGRP neurons appears downstream of Src activation. A new target, protein kinase B (AKT), was identified as being upregulated by DAMGO treatment alone as well as 17-AAG and DAMGO treatments combined, though this effect seems to occur in males only. Upregulation of AKT appears to be in CGRP neurons in the dorsal horn, and a CRISPR knockdown targeting AKT in CGRP neurons was able to block the effects of both DAMGO and 17-AAG/DAMGO combined treatments. AKT activation lies downstream of DUSP15 upregulation, but upstream of PKCβ activation. Lastly, downregulation of 5’ adenosine monophosphate-activated protein kinase (AMPK) following 17-AAG and DAMGO treatments lies downstream of PKCβ activation, ultimately leading to an activation of ERK and the enhanced opioid antinociception seen in acute pain. Through the unraveling of this pathway, we have improved basic understanding of the µ opioid receptor signaling pathway, as well as increased understanding of microglia signaling in acute pain. We have identified several potential targets that may be manipulated to create new analgesics, or improve the therapeutic index of opioid pharmaceuticals. Lastly, by identifying the order of the pathway, we can target these signaling molecules in a way that better predicts downstream and off target effects.