Development of a genetically encoded sensor for probing endogenous nociceptin opioid peptide release.

Nociceptin/orphanin-FQ (N/OFQ) is a recently appreciated critical opioid peptide with key regulatory functions in several central behavioral processes including motivation, stress, feeding, and sleep. The functional relevance of N/OFQ action in the mammalian brain remains unclear due to a lack of high-resolution approaches to detect this neuropeptide with appropriate spatial and temporal resolution. Here we develop and characterize NOPLight, a genetically encoded sensor that sensitively reports changes in endogenous N/OFQ release. We characterized the affinity, pharmacological profile, spectral properties, kinetics, ligand selectivity, and potential interaction with intracellular signal transducers of NOPLight in vitro. Its functionality was established in acute brain slices by exogeneous N/OFQ application and chemogenetic induction of endogenous N/OFQ release from PNOC neurons. In vivo studies with fibre photometry enabled direct recording of NOPLight binding to exogenous N/OFQ receptor ligands, as well as detection of endogenous N/OFQ release within the paranigral ventral tegmental area (pnVTA) during natural behaviors and chemogenetic activation of PNOC neurons. In summary, we show here that NOPLight can be used to detect N/OFQ opioid peptide signal dynamics in tissue and freely behaving animals.

Developmental and adult striatal patterning of nociceptin ligand marks striosomal population with direct dopamine projections.

Circuit influences on the midbrain dopamine system are crucial to adaptive behavior and cognition. Recent developments in the study of neuropeptide systems have enabled high-resolution investigations of the intersection of neuromodulatory signals with basal ganglia circuitry, identifying the nociceptin/orphanin FQ (N/OFQ) endogenous opioid peptide system as a prospective regulator of striatal dopamine signaling. Using a prepronociceptin-Cre reporter mouse line, we characterized highly selective striosomal patterning of Pnoc mRNA expression in mouse dorsal striatum, reflecting early developmental expression of Pnoc . In the ventral striatum, Pnoc expression was was clustered across the nucleus accumbens core and medial shell, including in adult striatum. We found that Pnoc tdTomato reporter cells largely comprise a population of dopamine receptor D1 ( Drd1 ) expressing medium spiny projection neurons localized in dorsal striosomes, known to be unique among striatal projections neurons for their direct innervation of midbrain dopamine neurons. These findings provide new understanding of the intersection of the N/OFQ system among basal ganglia circuits with particular implications for developmental regulation or wiring of striatal-nigral circuits.

Development of a genetically-encoded sensor for probing endogenous nociceptin opioid peptide release.

Nociceptin/orphanin-FQ (N/OFQ) is a recently appreciated critical opioid peptide with key regulatory functions in several central behavioral processes including motivation, stress, feeding, and sleep. The functional relevance of N/OFQ action in the mammalian brain remains unclear due to a lack of high-resolution approaches to detect this neuropeptide with appropriate spatial and temporal resolution. Here we develop and characterize NOPLight, a genetically encoded sensor that sensitively reports changes in endogenous N/OFQ release. We characterized the affinity, pharmacological profile, spectral properties, kinetics, ligand selectivity, and potential interaction with intracellular signal transducers of NOPLight in vitro. Its functionality was established in acute brain slices by exogeneous N/OFQ application and chemogenetic induction of endogenous N/OFQ release from PNOC neurons. In vivo studies with fiber photometry enabled a direct recording of binding by N/OFQ receptor ligands, as well as the detection of natural or chemogenetically-evoked endogenous N/OFQ release within the paranigral ventral tegmental area (pnVTA). In summary, we show that NOPLight can be used to detect N/OFQ opioid peptide signal dynamics in tissue and freely-behaving animals.

Heat shock protein 90 inhibitors block the antinociceptive effects of opioids in mouse chemotherapy-induced neuropathy and cancer bone pain models.

Heat shock protein 90 (Hsp90) is a ubiquitous signal transduction regulator, and Hsp90 inhibitors are in clinical development as cancer therapeutics. However, there have been very few studies on the impact of Hsp90 inhibitors on pain or analgesia, a serious concern for cancer patients. We previously found that Hsp90 inhibitors injected into the brain block opioid-induced antinociception in tail flick, paw incision, and HIV neuropathy pain. This study extended from that initial work to test the cancer-related clinical impact of Hsp90 inhibitors on opioid antinociception in cancer-induced bone pain in female BALB/c mice and chemotherapy-induced peripheral neuropathy in male and female CD-1 mice. Mice were treated with Hsp90 inhibitors (17-AAG, KU-32) by the intracerebroventricular, intrathecal, or intraperitoneal routes, and after 24 hours, pain behaviors were evaluated after analgesic drug treatment. Heat shock protein 90 inhibition in the brain or systemically completely blocked morphine and oxymorphone antinociception in chemotherapy-induced peripheral neuropathy; this effect was partly mediated by decreased ERK and JNK MAPK activation and by increased protein translation, was not altered by chronic treatment, and Hsp90 inhibition had no effect on gabapentin antinociception. We also found that the Hsp90 isoform Hsp90α and the cochaperone Cdc37 were responsible for the observed changes in opioid antinociception. By contrast, Hsp90 inhibition in the spinal cord or systemically partially reduced opioid antinociception in cancer-induced bone pain. These results demonstrate that Hsp90 inhibitors block opioid antinociception in cancer-related pain, suggesting that Hsp90 inhibitors for cancer therapy could decrease opioid treatment efficacy.

Inhibition of Hsp90 in the spinal cord enhances the antinociceptive effects of morphine by activating an ERK-RSK pathway.

Morphine and other opioids are commonly used to treat pain despite their numerous adverse side effects. Modulating µ-opioid receptor (MOR) signaling is one way to potentially improve opioid therapy. In mice, the chaperone protein Hsp90 mediates MOR signaling within the brain. Here, we found that inhibiting Hsp90 specifically in the spinal cord enhanced the antinociceptive effects of morphine in mice. Intrathecal, but not systemic, administration of the Hsp90 inhibitors 17-AAG or KU-32 amplified the effects of morphine in suppressing sensitivity to both thermal and mechanical stimuli in mice. Hsp90 inhibition enabled opioid-induced phosphorylation of the kinase ERK and increased abundance of the kinase RSK in the dorsal horns of the spinal cord, which are heavily populated with primary afferent sensory neurons. The additive effects of Hsp90 inhibition were abolished upon intrathecal inhibition of ERK, RSK, or protein synthesis. This mechanism downstream of MOR, localized to the spinal cord and repressed by Hsp90, may potentially be used to enhance the efficacy and presumably decrease the side effects of opioid therapy.

The Alpha Isoform of Heat Shock Protein 90 and the Co-chaperones p23 and Cdc37 Promote Opioid Anti-nociception in the Brain.

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/ß, 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.