Hsp90 inhibition in mouse spinal cord enhances Src kinase signaling in microglia to increase opioid antinociception.

Opioid drugs are the gold standard for pain management, but have many serious drawbacks, which have highlighted the need for new approaches to improve opioid therapy. Previously, our lab discovered that Heat shock protein 90 (Hsp90), when inhibited in the spinal cord through intrathecal administration of 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) in mice, led to an increase in morphine antinociception, which could enable an opioid dose-reduction strategy. In this study, we hypothesized that Src kinase was upregulated by 17-AAG treatment to cause this increase. To test this hypothesis, CD-1 mice were treated with 17-AAG and the Src inhibitor Src-I1 or Src CRISPR knockdown, followed by morphine. The enhanced antinociception seen with 17-AAG was completely abolished in the inhibitor groups in tail-flick and paw-incision pain models, suggesting that Hsp90 inhibition activates Src signaling to lead to enhanced opioid pain relief. Analysis by Western blotting showed upregulation of Src by 17-AAG treatment with the opioid agonist DAMGO, and also suggested that Src is upstream of extracellular signal-regulated kinase in this signaling cascade. Immunohistochemistry confirmed the upregulation of Src in the spinal dorsal horn and colocalization of activated Src with microglia. Inhibition of microglia with minocycline/PLX3397 mimicked the 17-AAG effects, while activation with lipopolysaccharide reversed them, suggesting that microglia could regulate these pain states. Finally, we used microglial-specific CRISPR knockdown to confirm that microglial Src is essential to the enhanced opioid antinociception observed with spinal Hsp90 inhibition. These observations elucidate a key molecular mechanism by which Hsp90 regulates microglia and opioid signaling, creating potential targets to improve opioid treatment.