Molecular Mechanisms by Which HSP90 Inhibition in the Spinal Cord Enhances Opioid Receptor Signaling

Abstract

Our current focus to improve opioid therapy has been studying signal transduction cascades of the mu opioid receptor. Our lab has identified heat shock protein 90 (HSP90) as a key regulator of opioid signaling by demonstrating that when HSP90 is inhibited in spinal cord using 17-AAG, ERK MAPK signaling, and subsequent opioid anti-nociception is enhanced. Quantitative proteomic analysis on the spinal cords of CD-1 mice treated with 17-AAG revealed that both 5' AMP-activated protein kinase subunit beta-1 (AMPK) and Epidermal Growth Factor Receptor (EGFR) were downregulated by 26%, while the Dual Specificity Phosphatase 15 (DUSP15) was upregulated by 35%. This result suggested that a reduction in AMPK and EGFR activity and an increase in DUSP15 activity could lead to enhanced opioid signaling and pain relief. We tested this hypothesis using the AMPK activators AICAR and PT1 (i.t) and EGFR activator EGF (i.t) and found they decreased 17-AAG-enhanced morphine (subcutaneous, SC) antinociception in the tail flick test and paw-incision pain model in both male and female mice. Conversely, the AMPK inhibitor dorsomorphin (i.t) and the EGFR inhibitor Afatinib (i.t) enhanced morphine antinociception greater than 17-AAG alone. This suggests that both AMPK and EGFR work as negative regulators to suppress opioid signaling. Immunohistochemical studies showed opioid treatment increased pAMPK signal in the dorsal horn of the spinal cord; for EGFR there was an upregulation of total receptor expression with a concentrated phosphorylation in the dorsal horn, which are then decreased as a response to 17-AAG. Colocalization studies demonstrated AMPK colocalized with the neuropeptide CGRP and the neuronal marker NeuN, and EGFR colocalized with CGRP. We further used CRISPR gene editing to knockdown AMPK in CGRP neurons, which was sufficient to enhance opioid antinociception without HSP90 inhibition. When we knocked down EGFR in CGRP expressing neurons however, there was no enhancement, even though there appeared to be colocalization with CGRP neurons. These results place AMPK functional activity in CGRP expressing neurons, but not EGFR. Further studies need to be performed to discern location of EGFR’s effect on opioid antinociception. Consequently, we used CRISPR gene editing to knockdown DUSP15 in the spinal cord, and the morphine enhancement with 17-AAG was completely blocked. We also used AMPK CRISPR and small molecule activators/inhibitor to place AMPK upstream of ERK MAPK in the spinal cord anti-nociception signaling cascade. Using DUSP15 CRISPR mice, we treated them with AMPK inhibitor Dorsomorphin (i.t), and the enhanced morphine effect was restored. Similarly, immunohistochemistry of the DUSP15 CRISPR mice showed a decrease in ERK activity placing DUSP15 as a potential deactivator of AMPK which then allows for enhanced ERK activity as a response to HSP90 inhibition. Our data demonstrates that AMPK plays a novel role as an opioid negative feedback loop in spinal cord CGRP neurons, which can be disabled by HSP90 inhibition through DUSP15 activation to enhance opioid anti-nociception. Separately we show that the EGFR also acts as a negative feedback look for opioid anti-nociception in the spinal cord. Together these results improve our understanding of opioid signal transduction in the spinal cord, and also suggest potential targets to improve the therapeutic index of opioids.