MRGPRX1, a human mas-related G protein-coupled receptor (MRGPR), detects itchy stimuli in the skin. But in new research, MRGPRX1 activation in the spinal cord dampens pain in mice, confirming the receptor as a viable target for drugs to relieve chronic pain in people.
Intrathecal delivery of an MRGPRX1 agonist inhibited evoked pain hypersensitivity and ongoing pain in several mouse models of neuropathic and inflammatory pain. Importantly, a positive allosteric modulator (PAM) of MRGPRX1 was analgesic even without a direct ligand for the receptor, suggesting that such an agent might boost endogenous activation of MRGPRX1.
The research, made possible through generation of a “humanized” mouse that expresses MRGPRX1 in place of its own orthologous MRGPR, was led by Xinzhong Dong, Johns Hopkins University, Baltimore, US.
Diana Bautista, a neuroscientist at University of California, Berkeley, US, who was not involved in the work, said the researchers “used the mice to investigate MRGPRX1 at the cellular, circuit, and behavioral levels, and that’s pretty amazing.” Between previous in vitro data and the new work, she said, “this looks like a really good candidate to move forward with” toward clinical development.
“It’s a complicated story, but this one receptor has two functions: In the periphery, it controls itch, and in central terminals, it controls pain,” Dong said. Dong also contributed to the increasingly intricate story of itch and pain processing in the spinal cord with a recent paper published in Neuron (see PRF related news).
The new work was published February 21 in Proceedings of the National Academy of Sciences.
Creating a humanized mouse
The MRGPRs, a large family of diverse “orphan” receptors, were first described by Dong together with David Anderson at California Institute of Technology, Pasadena, US, in 2001 (Dong et al., 2001). In humans and rodents, several MRGPR subtypes are found only in subsets of sensory neurons, and multiple studies have established the receptors’ roles in itch and pain (see Tiwari et al., 2016; Liu and Dong, 2015). In mice, those receptors include MRGPRD, MRGPRA3, and MRGPRC11. In humans, MRGPRX1 is thought to be the orthologue to MRGPRC11 because these two receptors bear similar expression patterns in dorsal root ganglia (DRG), and they share over 50 percent amino acid sequence identity.
Mice lacking MRGPRs exhibit enhanced inflammatory pain and prolonged neuropathic pain compared to normal mice, suggesting that MRGPRs could potentially act to inhibit pain. Activation of MRGPRC11 at central terminals in mice was previously found to inhibit neuropathic pain (He et al., 2014). But although MRGPRC11 and MRGPRX1 are thought to be orthologous, the receptors respond differently to some pharmacological agents, so drugs aimed at MRGPRX1 could not be reliably tested in animals in vivo. In the new study, Dong and colleagues set out to solve that problem by creating mice that express the human MRGPRX1.
To do that, co-first authors Zhe Li, Pang-Yen Tseng, and Vinod Tiwari generated a transgenic mouse line that expressed human MrgprX1 driven by the promoter for MrgprC11. Then, they crossed those mice with a line of mice bred to express green fluorescent protein (GFP) under control of the promoter for MrgprA3, which the authors write is expressed with MrgprC11 in “largely overlapping subsets of DRG neurons.” These mice in turn were crossed with animals lacking 12 Mrgpr genes, including MrgprC11 and MrgprA3. The end result was a line of “humanized” mice—called MrgprX1 mice—that expressed only the human receptor in fluorescently labeled sensory neurons normally containing MRGPRC11.
“In other fields, humanized mice have provided a really nice preclinical approach, but it’s not been widely used in the pain field,” Bautista said. This is the first time that a humanized mouse has been created by manipulating a pain-related gene, “which is kind of crazy, considering how desperate we are for new drug targets.” The new mice, she said, “will be a really nice boon to bridging basic research and human studies.”
BAM8-22 relieves pain
Bovine adrenal medulla (BAM) peptide is a 22-amino acid endogenous opioid peptide found in humans and rodents as well as cows. A truncated version, BAM8-22, lacks the seven amino acids required to activate all three subtypes of opioid receptors but still activates MRGPRX1 and MRGPRC11. (Only those two MRGPRs respond to BAM8-22—another argument for the proteins’ homology).
Following chronic constriction injury (CCI) to the sciatic nerve, the humanized MrgprX1 mice developed heat hypersensitivity that was alleviated by intrathecal treatment with BAM8-22. Importantly, treatment also appeared to reduce ongoing pain, because mice in a conditioned place preference (CPP) test preferred the chamber where they received BAM8-22 over one where they received saline. Mice lacking MRGPRs altogether did not display pain relief in either test.
Dong said there are multiple parallels between the MRGPRs and opioid receptors—not least that they are both G protein-coupled receptors activated by endogenous opioid peptides and seem to inhibit pain. “But the advantage here,” Dong said, “is that MRGPR expression is much more restricted to DRG neurons” compared to opioid receptors, which might limit side effects associated with MRGPR activation.
Next, the researchers wanted to determine how MRGPRX1 activation led to pain relief. Activation of G protein-coupled receptors including opioid receptors and MRGPRC11 has previously been shown to inhibit high-voltage-activated (HVA) calcium currents. “If calcium influx is blocked, then release of the neurotransmitter glutamate is also blocked from central terminals to spinal cord neurons,” Dong said.
BAM8-22 rapidly inhibited HVA calcium currents in dissociated DRG neurons from MrgprX1 mice. When the team pre-applied blockers of N-, P/Q-, or L-type voltage-gated calcium channels, they found that only the N-type channel inhibitor ω-conotoxin GVIA prevented HVA inhibition by BAM8-22, indicating that MRGPRX1 targeted N-type channels. The inhibition was also prevented by pertussis toxin, signifying that so-called Gi/o downstream signaling that occurs upon receptor activation was at least partly responsible for the calcium channel inhibition.
Boosting natural signals with the positive allosteric modulator ML382
Having shown that BAM8-22 alleviated pain when delivered to the spinal cord in injured MrgprX1 mice, the authors next turned to immunostaining experiments to study the effect of injury on BAM expression. They showed that BAM22, the full-length endogenous opioid peptide, increased in the dorsal horn of the spinal cord two days after injection with complete Freund’s adjuvant (CFA) in the hindpaw, a model of chronic inflammatory pain. In addition, a series of mass spectrometry (MS) experiments confirmed that BAM22 was increased in the spinal cord after injury.
Positive allosteric modulators bind receptors at non-classical binding sites to enhance receptor activation by classical ligands, but they do not activate receptors by themselves. The researchers wondered whether a PAM acting at MRGPRX1 could enhance signaling by naturally occurring BAM22 to reduce pain. Accordingly, ML382, a previously identified MRGPRX1 PAM, enhanced the inhibition of calcium currents in dissociated DRG neurons by low but not high doses of BAM8-22, and had no effect on currents on its own.
In order to more closely examine the role of MRGPRX1 in synaptic transmission, the researchers used a spinal cord slice preparation. They made patch-clamp recordings from neurons in lamina II of the dorsal horn while electrically stimulating the intact dorsal root, the nerve input to the spinal cord from the DRG. Using an elegant series of electrophysiological experiments, the authors showed that BAM8-22 inhibited evoked excitatory postsynaptic currents (eEPSCs), and that co-application of ML382 enhanced the inhibition. Further, the evidence suggested the inhibition worked by reducing presynaptic neurotransmitter release.
Strikingly, intrathecal injection of ML382 a half-hour before injection of formalin to the hindpaw of MrgprX1 mice had no effect on immediate pain but significantly attenuated animals’ later pain response to the inflammatory substance. In addition, heat hypersensitivity was reduced by intrathecal injection of ML382 in MrgprX1 mice with CFA-induced inflammatory pain or CCI-induced neuropathic pain. No such effects were seen in Mrgpr-deficient mice. Ongoing pain was also attenuated by ML382 according to the conditioned place preference test. “The fact that they used three different models of pain and consistently saw the same thing suggests that targeting MRGPRX1 may represent a good strategy for treating the many types of pain observed in the clinic,” Bautista said.
In a final experiment, the researchers showed that the dual nature of MRGPRX1 in itch and pain should not preclude it as a pain target. As expected, subcutaneous injection of BAM8-22 elicited scratching in MrgprX1 mice, but subcutaneous injection of ML382 did not. Intrathecal delivery of BAM8-22 or ML382 produced no behavioral signs of itch. That indicates that “MRGPRX1 signaling in the skin is really distinct from what happens in the spinal cord,” Bautista said. The separation of MRGPRX1 function at peripheral and central terminals allows it to fulfill two different roles—a phenomenon not uncommon in sensory physiology, she added.
As a caveat, Dong said there are still many hurdles to developing ML382 into a therapeutic agent. “It’s not ideal because it’s not that stable, and it doesn't cross the blood-brain barrier, so it’s not orally bioavailable.” Dong is collaborating with biochemists to produce a modified small molecule PAM that would not require intrathecal injection. “There’s a long way to go, but now we have the humanized mouse to work out species specificity, and hopefully to develop novel drugs targeting MRGPRX1,” he said.
Stephani Sutherland, PhD, is a neuroscientist, yogi, and freelance writer in Southern California.