Move Over Alpha4: Nicotinic Receptor Alpha6 Subunit Identified as Potential Pain Target
Dorsal root ganglion (DRG) expression levels of Chrna6, the gene that encodes the nicotinic acetylcholine receptor α6 subunit, are highly correlated with mechanical allodynia in mice, and variability in the gene is also associated with chronic pain in humans, reports a new study published online May 13 in Science Translational Medicine. Researchers led by Jeffrey Mogil, McGill University, Montreal, Canada, also used gain- and loss-of-function mutants to confirm the analgesic role of the α6 subunit in mouse models of neuropathic and inflammatory pain, and to show that this effect is at least partially mediated by direct interaction with and cross-inhibition of DRG P2X2/3 receptors. Furthermore, the analgesic effect of nicotine did not depend on the nicotinic receptor’s α4 subunit, suggesting a possible explanation for a lack of progress in analgesic drug discovery efforts focused on that particular subunit.
“These findings really advance our understanding of the role of nicotinic receptors in pain and provide an entry point for new drug discovery efforts focused on selective activation of the α6 subunit,” wrote Iain Chessell, of AstraZeneca’s MedImmune unit, Cambridge, UK, in an email to PRF.
“Overall, it’s an amazing assembly of work,” said Stephen Arneric, Horizon Pharma Consulting, Shelby, Michigan, US. With 41 authors located at 18 different research institutions, the new study highlights the utility of a global translational research effort, Arneric added.
The authors’ multifaceted approach reinforced the critical role of α4 receptors in higher brain centers, he said. “More importantly, it opens up the door to targeting α6-containing receptors that reside at more accessible peripheral sites to treat both chronic inflammatory and neuropathic pain,” Arneric said.
Nicotinic acetylcholine receptors (nAChRs), which are hetero- or homopentameric ligand-gated ion channels consisting of α and β subunits, have garnered interest as potential analgesic drug targets for many years. Work to date has largely focused on the subunit with the highest central nervous system expression, α4, with little success due to a narrow therapeutic window and significant side effects, said Mogil. However, other α subunits are also known to mediate pain, and the lack of an agonist that can selectively activate α4 but not α6 suggested a potential, as yet undiscovered, role for α6, he added.
The Chrna6 connection
In the new study, first author Jeffrey Wieskopf and colleagues did not set out to look specifically at the role of nAChR subunits in pain. Instead, they took an agnostic approach to survey DRG gene expression associated with mechanical allodynia induced by spared nerve injury (SNI) in 25 inbred mouse strains. A similar strategy has been utilized previously in pain research, though on a much smaller scale, noted Mogil (Li et al., 2010; Nissenbaum et al., 2010).
The two strongest correlations between microarray gene expression and mechanical allodynia (quantified using von Frey filaments) were found with two different probes for Chrna6, with higher gene expression associated with less allodynia. Chrna6 expression levels were also strongly correlated with allodynia in mice following spinal nerve ligation, another model of neuropathic pain, in a second microarray dataset. Quantitative polymerase chain reaction (qPCR) revealed that Chrna6 expression was highest in the DRG, followed by the brain, and then the eye, and was present in various subsets of DRG neurons, including those expressing neurofilament 200, isolectin B4, and calcitonin gene-related peptide.
Chrna6 mRNA expression in a subset of DRG neurons. (A to C) DRG neurons from adult α6*-GFP mice were stained with antibodies against GFP (green) and sensory neuron markers NF200 (A), IB4 (B), and CGRP (C) (all red). Arrows indicate neurons expressing either GFP or the sensory marker. Filled arrowheads indicate neurons co-expressing both markers. Scale bars, 100 μm. Image and caption from Wieskopf et al. Reprinted with permission from AAAS.
Experiments with Chrna6 mutant mice also indicated a role for the gene in neuropathic pain induced by SNI and in chronic inflammatory pain induced by intraplantar injection of complete Freund’s adjuvant (CFA). In both models, mechanical allodynia levels were higher in Chrna6 knockout mice and lower in Chrna6 gain-of-function mutant mice compared to wild-type animals. Chrna6, but not Chrna4, knockout mice also displayed increased mechanical allodynia following a chronic constriction injury (CCI). The effect of the α6 subunit appeared to be specific for mechanical allodynia, as Chrna6 knockouts did not differ from wild-type mice on measures of acute or tonic nociception.
The researchers next examined the role of the α6 receptor in the known anti-allodynic effects of nicotine administration in inflammatory and neuropathic pain models (Vincler, 2005). Using the SNI or CFA model, they found that gain-of-function mutants injected with nicotine via intracerebroventricular (supraspinal), intrathecal (spinal), or intraplantar (peripheral) routes showed similar or elevated reversal of allodynia as wild-type animals also given nicotine. In contrast, nicotine did not produce an anti-allodynic effect in Chrna6 knockouts.
To assess the relative contributions of α6 and α4 to nicotine’s anti-allodynic effects after CCI or CFA, Weiskopf and colleagues directly compared the effects seen via the three different injection routes. Both peripheral and spinal nicotine-induced anti-allodynia were abolished in Chrna6 knockouts but were preserved in Chrna4 knockouts, while anti-allodynia was reduced in Chrna6 knockouts and completely absent in Chrna4 knockouts following supraspinal nicotine.
“The conclusion that we came to was that α4 plays an important role in supraspinal nicotinic analgesia … but outside the brain, it’s all about α6,” said Mogil. “These data suggest that a peripherally restricted α6-specific compound would be a pretty good drug target,” he added.
Jeffrey Mogil
Finally, a number of electrophysiological, fluorescence resonance energy transfer (FRET), and behavioral experiments would show that the mechanism underlying α6’s analgesic effect involved a physical and functional interaction with P2X2/3 receptors, which have been linked to pain in previous studies.
Clinical relevance
The study also examined the association between CHRNA6 single nucleotide polymorphisms (SNPs) and chronic pain in humans. In a cohort of 429 adults who underwent herniotomy, a surgical procedure for hernia repair, and in a cohort of 159 women with temporomandibular disorder, carriers of the minor TT allele (10 out of the combined 588 individuals) in a CHRNA6 promoter region SNP were more likely to develop persistent pain following surgery or have more intense head and orofacial pain, respectively.
“On the one hand, the human data were completely supportive that this gene can play a role in pain,” said Mogil. On the other hand, the number of people with the variant was so small that there’s no way the gene plays any role in chronic pain susceptibility for the vast majority of individuals—it’s simply too rare, he continued. “The irony is that, although we used genetics to find CHRNA6, it turns out that it has almost nothing to do with variability in human pain sensitivity [for most of the population],” Mogil said.
“As with many mediators involved in pain, manipulation of [the α6] system in isolation is unlikely to provide the analgesic panacea that patients need, but the findings provide another important target to investigate in preclinical and clinical settings,” wrote Chessell.
One of the challenges that lies ahead is to build the heterologous expression assays required to screen α6-containing receptors and potentially to co-express other receptor subtypes, said Arneric, who has worked on the development of nicotinic receptor compounds at Eli Lilly, Pfizer, and Abbott Pharmaceuticals.
Since Mogil himself is not a drug developer, he hopes others will follow up on the new findings. “We put this into the world in hopes it will spur rejuvenation of nicotinic drug development for pain by those who do that,” he said.
Allison Marin (Curley), PhD, is a neuroscientist-turned-science writer who resides in Pittsburgh, Pennsylvania, US.
Image credit: Wieskopf et al. Reprinted with permission from AAAS.
Article Contributors:
