Unraveling Analgesic Cell Signaling and Circuit Mechanisms of Compounds Derived from Natural Products

Abstract

Chronic pain is a debilitating condition that affects one in five people in the United States adult population. Limited efficacy and/or harsh side effects limit many pain drugs, driving the need for alternate analgesics. In our past research, we have shown that terpenes, small hydrocarbons found in Cannabis sativa, are capable of reducing nociception in mouse models via multiple receptor targets. Furthermore, we showed the terpenes geraniol, linalool, β-pinene, α-humulene, and β caryophyllene are efficacious in the treatment of mouse chemotherapy-induced peripheral neuropathy (CIPN). By utilizing CRISPR knockdown and the small molecule inhibitor istradefylline, we showed that terpenes act in CIPN through the activation of the Adenosine A2a Receptor (A2aR) in the spinal cord. However, the cell type and spinal circuitry for A2aR-induced antinociception by terpenes are unknown. For our model, we used male and female CD-1 mice treated with paclitaxel (2 mg/kg) over 7 days to induce CIPN. Here we show that the terpenes geraniol and β-caryophyllene produce comparable antinociception to morphine in cold allodynia in the acetone drop assay, mechanical pain, in the percent withdrawal assay, and were significantly antihypertensive in heat-induced pain, in the hot plate assay, when compared to vehicle at at least one time point. However, these terpenes were not antinociceptive in cold, mechanical, or heat pain assays in uninjured mice. Using immunofluorescence, we found that the A2aR was strongly upregulated in the spinal cord by CIPN to determine if this upregulation is the result of a pain-relieving feedback mechanism we induced CIPN then treated mice with istradefylline (3.2 mg/kg). Istradefylline significantly increased the nociception in the CIPN paw withdrawal assay, suggesting that this increase results as a feedback mechanism downregulating pain transmission. Given the known role of the A2aR as a stimulatory GPCR, we hypothesized that the A2aR is localized in inhibitory interneurons in the spinal dorsal horn and act by inhibiting neuronal pain transmission. Using immunofluorescence, We further observed that this A2aR expression was found in 95% of neurons, as measured by colocalization with the neuronal marker NeuN. Following this, we found that the A2aR is expressed in 74% of inhibitory interneurons, as measured by colocalization with the marker Pax2. We then used Pax2+ cell-selective CRISPR knockdown of the A2aR by using the Pax2 promoter to drive Cas9 expression along with a gRNA targeted to the A2aR. This resulted in a complete loss of terpene antinociception in CIPN, strongly suggesting that terpenes work through the A2aR in this specific cell population to relieve pain. To further confirm these findings, we used Fos staining in spinal cord, showing that Fos activation was increased by CIPN and repressed by terpene treatment. We showed similar results with pERK where treated with CIPN along had a significant increase compared to vehicle and geraniol-treated CIPN spinal cord had signal similar to vehicle-treated mice. Future experiments will include measurement of evoked GABA release in response to terpenes as an activity analysis of ascending nociceptive neurons. Taken together, we show that terpenes have antinociceptive effects in neuropathic pain via the A2aR on Pax2+ inhibitory interneurons in mouse spinal cord. With this work, we aim to identify the detailed mechanism by which terpenes relieve neuropathic pain, and establish a scientific basis for their use as a novel non-opioid pain therapeutic. Finally, we sought to link Src kinase signaling in microglia to Heat shock protein 90 (Hsp90) induced increased antinociception. Previously, our lab discovered that inhibiting Hsp90 in the spinal cord through intrathecal administration of 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), in mice, led to an increase in morphine anti-nociception, which could enable an opioid dose-reduction strategy. Here, 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 anti-nociception seen with 17-AAG was completely abolished in the Src 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 ERK in this signaling cascade. Immunofluorescence confirmed the upregulation of Src in the spinal dorsal horn and co-localized activated Src to 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 anti-nociception 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.