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Identification of the Hsp90 Isoforms and Co‐Chaperones that Repress Opioid Anti‐Nociception in the Spinal Cord

The various isoforms of the chaperone protein heat shock protein 90 (Hsp90) in conjunction with specific co‐chaperones are key regulators of signal transduction. We have recently identified a novel role for Hsp90 within downstream mu opioid receptor (MOR) signaling both within the brain and spinal cord. Within the brain, we found that Hsp90α and the co‐chaperones Cdc37 and p23 promote MOR‐induced ERK phosphorylation and subsequent anti‐nociception. However, in the spinal cord Hsp90 represses MOR‐induced ERK phosphorylation and subsequent anti‐nociception, so that Hsp90 inhibitor treatment in the spinal cord results in enhanced opioid pain relief. This study aimed to identify the Hsp90 isoforms and co‐chaperones necessary for this mechanism within the spinal cord, and whether they differ from the brain. We used novel isoform and co‐chaperone selective small molecule inhibitors and in vivo gene knockdown using CRISPR/Cas9 to selectively block each of the Hsp90 isoforms and co‐chaperones in the spinal cords of male and female CD‐1 mice. We then used opioid‐induced anti‐nociception in the tail flick assay as a readout, combined with immunohistochemistry to validate CRISPR/Cas9 knockdown in the spinal cord. In contrast to our earlier brain studies, we found that the isoforms Hsp90α, Hsp90β, and Grp94 all acted to repress opioid anti‐nociception in the spinal cord, in that inhibiting these isoforms resulted in increased opioid anti‐nociception. We similarly found that the co‐chaperones Cdc37, p23, and Aha1 all similarly repressed opioid anti‐nociception in the spinal cord. These findings suggest that Hsp90β, Grp94, and Aha1 all act within the spinal cord but not the brain to regulate opioid anti‐nociception. These findings also suggest, in line with our earlier studies, that blocking these isoforms with systemic selective inhibitors could represent a novel therapeutic approach to enhance opioid therapy, increasing analgesic efficacy while reducing side effects like tolerance and reward/addiction.