Epigenetic mechanisms impact gene function without affecting the underlying DNA sequence, and are one way in which the environment can have long-lasting effects on the genome. Epigenetics has been slow to catch on in the pain field compared to many other branches of science and medicine, but that may be about to change. Emerging research is beginning to show how epigenetics can help explain why particular individuals are more vulnerable to chronic pain than others, reveal the mechanisms underlying the transition from acute to chronic pain, and identify new targets for pain therapeutics. So argued co-presenters Stephen McMahon and Franziska Denk, King’s College London, UK, in a PRF webinar titled Pain Epigenetics: Current Research and Future Challenges, which took place on April 16. The talk was followed by a panel discussion with Chas Bountra, University of Oxford, UK, and Santina Chiechio, University of Catania, Italy. Laura Stone, McGill University, Montreal, Canada, moderated the event.
The webinar was the seventh in a new series of webinars, Challenges in Pain Drug Development, supported by unrestricted educational grants from Genentech and MedImmune. A recording of the session is available here.
What is epigenetics?
“Epigenetics is a relatively new field for pain scientists,” McMahon said at the start of the presentation. To illustrate the point, he noted that just before the webinar began, he searched the term “genetics and pain” on PubMed and found close to 15,000 hits. “Epigenetics and pain” returned only 71. “And just to rub salt into the wound, I followed up and asked how many of those 71 were original articles, and nearly half of them were reviews,” McMahon added.
To help orient the audience to the epigenetics field, Denk provided a primer on basic epigenetic mechanisms, including DNA methylation and post-translational modifications of histones, the proteins around which DNA wraps (for a review, see Borrelli et al., 2008). “Many of these epigenetic mechanisms are fixed during development, but they are also thought to remain plastic throughout the lifetime of an organism,” Denk said. “These mechanisms are thought to be ways in which the environment can influence gene function.”
Denk first turned to DNA methylation, a process in which DNA methyltransferase enzymes add methyl groups to DNA. Though methylation can take place anywhere along the DNA molecule, it occurs most often on those cytosines that are located across from guanines on the phosphate backbone—so-called CpG methylation. Denk noted that not all CpGs are methylated. In fact, promoter regions of genes classically have many unmethylated CpGs and are referred to as CpG islands.
Next, Denk discussed post-translational histone modifications including methylation, acetylation, and phosphorylation. These modifications help determine how DNA wraps around histones; when DNA wraps tightly, gene transcription is impeded, and when it wraps more loosely, transcription is facilitated.
Proteins known as epigenetic writers, readers, and erasers modulate the various types of epigenetic modifications of which Denk spoke. Histone acetyltransferases (HATs), histone methyltransferases (HMTs), and kinases are "writers," which acetylate, methylate, or phosphorylate histones, respectively. “Readers” interpret these epigenetic marks, while “erasers,” including histone deacetylases (HDACs), DNA methyltransferases (DNMTs), and phosphatases, remove them.
Denk emphasized that DNA methylation and post-translational modification of histones (along with the substitution of major histones with histone variants) are crucial processes for normal development and for cell lineage, helping to determine whether cells turn into neurons, microglia, or any other cell type (Ziller et al., 2013). Also, epigenetic modifications are specific to the cells in which they occur and are often stable during the lifetime of the cell.
Epigenetic variation occurs most often at regulatory regions of DNA, Denk also stressed. Moreover, it tends to increase over time, as indicated by twin studies showing that the epigenomes of identical twins, who share the same DNA, are similar early in life, but then diverge over time, possibly due to the twins accumulating different environmental experiences (Fraga et al, 2005). “We are very interested in epigenetic variation because it is one way in which the environment can impact our genes, and this is what we really want to study,” she said.
Why pain researchers should care about epigenetics
Is epigenetics relevant to chronic pain research? It was this question McMahon addressed in the second half of the webinar presentation.
One way in which a focus on epigenetics could benefit pain research, McMahon said, is that it could be a better strategy than the candidate gene approach to identifying novel pain therapeutic targets. To illustrate this point, McMahon cited a study from his group using a rodent model of spinal nerve ligation (SNL) to study patterns of gene expression in dorsal root ganglia. The group discovered over 2,000 dysregulated genes in the cells, making it daunting to pinpoint which ones merit the most attention.
“That’s really what the pain field has been doing for the last 10 or 15 years—looking for candidate genes,” McMahon said. “And we’ve asked which of those genes are important for specific aspects of abnormal pain signaling. It’s a lot of work to do.” Rather than seek candidate genes, it may be fruitful to take a more holistic approach that “examines the epigenetic mechanisms that coordinate and drive these different patterns of gene expression in chronic painful conditions,” he stressed. These epigenetic mechanisms, he argued, could be more tractable therapeutic targets, since there are a manageable number of them. “By targeting the processes that lead to different patterns of gene expression rather than individual genes, we may have a greater effect on the outcome of the whole system,” he added.
To show the feasibility of this approach, McMahon pointed to studies from his group investigating the effects of histone deacetylase (HDAC) inhibitors on pain behaviors in rats. HDACs remove acetyl groups from histones by putting chromatin into a more relaxed state that favors gene transcription, and inhibitors of these epigenetic modifiers have already been used clinically to treat certain types of cancer. Using a number of animal models of neuropathic pain, McMahon and colleagues administered various numbers and types of HDAC inhibitors continuously for one week in rats before inducing a spinal cord nerve injury in the animals. The drugs led to hyperacetylation within the spinal cord but did not cause any apparent behavioral changes before the onset of injury, suggesting that basal pain sensitivity was unaffected. However, after nerve injury, animals receiving HDAC inhibitors showed a 40-50 percent decrease in pain hypersensitivity compared to vehicle-treated controls (Denk et al., 2013).
McMahon thinks it is unlikely that the HDAC inhibitors used in the experiments had non-specific effects such as sedation, since such changes were not seen in control animals without nerve injury. Furthermore, while current HDAC inhibitors are unable to target individual HDAC subtypes, more selective compounds that target particular HDAC isoforms are in development, making HDAC inhibition an even more desirable therapeutic possibility.
In further support of the relevance of epigenetics to pain research, McMahon’s group has also used mouse genetics to identify specific epigenetic readers and writers that could be tractable targets for pain drug development. Of particular interest is HDAC4, which is highly expressed in nervous tissue, may affect synaptic function and neuron survival, and appears to play a role in pain. For instance, people with HDAC4 insufficiency exhibit decreased pain sensitivity and self-injurious behavior. Furthermore, in animals, pathway analysis revealed that HDAC4 (and HDAC1) had unexpected connections to many of the genes that McMahon and Denk found in their earlier studies to be dysregulated in neuropathic pain. “This gave us a clue that these HDACs might be coordinators of gene transcription changes,” he said.
In recently published work, McMahon’s group studied the role of HDAC4 in pain using a Cre recombinase strategy to create two different lines of transgenic mice (Crow et al., 2015). In one line, Cre recombinase is expressed under the control of the Nav1.8 promoter, allowing for knockout of HDAC4 in peripheral nociceptive neurons late in development. The other line was an inducible conditional knockout in which Cre recombinase is expressed under the control of the Advillin promoter. In this line, administration of tamoxifen results in HDAC4 knockout in all primary sensory neurons. “In both of these lines, we are able to eliminate HDAC4 from most, if not all, nociceptors,” McMahon said.
Results revealed no behavioral changes at baseline. “The basal phenotype of both of these lines is remarkably unimpressive; they have normal responses to acute noxious stimuli,” McMahon noted. But both knockout lines exhibited significantly reduced thermal hypersensitivity in the complete Freund’s adjuvant (CFA) model of inflammatory pain; no alterations were observed in neuropathic pain models. In addition, the team found that HDAC4 was required for an appropriate injury-induced transcription of a number of genes involved in inflammatory pain, including calcitonin gene-related peptide (Calca) and transient receptor potential cation channel subfamily V member 1 (TRPV1), both of which were significantly downregulated in the knockout animals.
Elucidating the biology of chronic pain
Another way in which epigenetics has a bearing on pain research is that it may help to explain why some individuals are particularly vulnerable to chronic pain, as well as to uncover mechanisms underlying the shift from acute to chronic pain. For instance, in a recent study co-authored by McMahon, genomewide analysis of methylation status in identical twins discordant for heat sensitivity revealed that methylation levels of the transient receptor potential ankyrin 1 (TRPA1) promoter were negatively correlated with tolerance to heat pain (Bell et al., 2014). While McMahon emphasized that this result does not imply causation, it does suggest that long-term and stable epigenetic changes may explain why an individual’s pain response evolves over time.
Another way in which epigenetics can further understanding of chronic pain biology comes from the study of latent enhancers. Enhancers are genomic regions to which transcription factors bind and are epigenetically marked by histone methylation. Enhancers have been studied extensively in the immunology field. “In immune cells such as macrophages, it has been shown that while most enhancers are set from development, there are a small but significant number of enhancers that emerge de novo when macrophages are stimulated with different substances,” Denk said. Such latent enhancers may generate a cellular “memory”—for instance, a latent enhancer may emerge and persist when a macrophage is exposed to a particular cytokine, changing the transcriptional profile of the cell and allowing it to respond more vigorously when re-exposed to the cytokine.
Denk presented unpublished work showing the emergence of latent enhancers in microglia, the immune cells of the central nervous system with a well-documented role in pain, in the spinal cord of mice after they had undergone SNL. After nerve injury, novel enhancers marked by increased histone methylation emerged at several genes important for microglial function that were dysregulated after SNL. “We think these changes could be a mechanism explaining how environmental influences such as nerve injuries could leave a lasting mark on the genome,” Denk said.
Future challenges
Much of the panel discussion following the presentation focused on hurdles in the application of knowledge from pain epigenetic studies to the development of new pain therapeutics. Santina Chiechio, who has studied the effects of HDAC inhibitors on pain (Chiechio et al., 2009), raised the question of whether epigenetic therapies could be used alone or in combination with existing drugs used for pain. Combining HDAC inhibitors with other drugs is under investigation in the cancer field, McMahon noted, and a similar approach that combines epigenetic treatments—particularly, Chiechio noted, those whose effects on pain could be examined as secondary outcomes in cancer clinical trials—with traditional pain drugs could be a promising strategy. Considering the difficulty the pain field has seen in developing a single “major bullet” drug that improves pain with minimal side effects, McMahon said, a drug combination approach may be particularly desirable. On the other hand, Chas Bountra noted, epigenetic processes regulate the expression of many genes, which may lessen the need for combination treatments.
Novel compounds targeting epigenetic mechanisms are also needed to fulfill pain drug development hopes. Bountra, chief scientist of the Structual Genomics Consortium (SGC), a public-private partnership between industry and academia with the goal of developing new drugs by sharing research output, is aiming to do just that. The SGC is working with several pharmaceutical companies to generate selective inhibitors targeting epigenetic processes that those in academia can test. Hopefully, “crowdsourcing” science in this way will quicken the pace of developing new treatments, he said.
From a pain therapeutic perspective, another approach may be to harness the “positive” effects of epigenetics, Laura Stone noted. These include beneficial environmental factors that can act through epigenetic mechanisms, such as diet, exercise, and an enriched social environment, to modify the genome (for Stone’s own research in this area, see Tajerian et al., 2013).
While the panel agreed on the promise of epigenetics for pain therapeutics, there is a long road ahead before the pain field sees new treatments—an outcome that is at least a decade or more away, Bountra said. In the meantime, further exploration of epigenetics is warranted. “A lot of us do science because we want to see it applied in some way, while many of us do it because we're just curious,” McMahon said. “I really believe that a deeper understanding of biology will ultimately produce better outcomes for mankind, though not necessarily for the reasons we think. I'm a great believer that it’s good to go digging and see what you find, and that's what's happening in the field of epigenetics—it’s a great opportunity. It's novel, it's different, and who knows where it's going to take us?”
Cynthia McKelvey is a freelance science journalist based in California.
Image credit: molekuul/123RF Stock Photo
