Epigenetic Downregulation of GABA Signaling Perpetuates Pain

Experience can produce lasting changes in gene expression via the modification of chromatin structure, in a mechanism known as epigenetic regulation. So how does the experience of pain—surely one of the strongest forces shaping the nervous system—leave its mark on our genes? A new study in mice and rats finds evidence that epigenetic changes near the Gad2 gene (encoding glutamic acid decarboxylase 65, GAD65) in response to inflammation or nerve injury lead to persistent pain by altering pain-modulating pathways in the brainstem nucleus raphe magnus (NRM). Researchers led by Zhizhong Pan at the University of Texas MD Anderson Cancer Center in Houston demonstrated that changes in histone acetylation drive down GAD65 expression, which diminishes inhibitory γ-aminobutyric acid (GABA) neurotransmission and heightens pain.

The findings, published online October 9 in Nature Medicine, support the notion that inhibitors of histone deacetylases (HDACs) could be useful for combating chronic pain.

In the last several years, evidence has gathered to suggest that chromatin remodeling is involved in pain (for a recent review, see Doehring et al., 2011). Modifications to chromatin, either on DNA itself (methylation) or on the histone proteins that package it (acetylation and other changes), govern gene expression by determining how easily genes can be transcribed. In particular, higher levels of histone acetylation are associated with a more open conformation of DNA and more active transcription.

In the new study, Pan and colleagues zero in on the GABA-producing enzyme GAD65 as a specific target for epigenetic regulation in pain. GABA works primarily as an inhibitory neurotransmitter, putting the brakes on pain-facilitating neurons. “One of the hypotheses for [what causes] chronic pain is the loss of GABA inhibition in the central nervous system,” Pan told PRF.

Consistent with that idea, when first author Zhi Zhang and coworkers injected complete Freund’s adjuvant (CFA) into the hindpaw of rats to induce inflammatory pain, GABAergic synaptic activity of NRM neurons decreased. Looking for the basis of the dropoff in GABA neurotransmission, they investigated histone acetylation around Gad2. The researchers found that, overall, acetylation of histones H3 and H4 in NRM tissue increased following CFA injection. Near Gad2, however, H3 acetylation decreased. Likewise, levels of GAD65 mRNA and protein decreased.

Treating the animals with small-molecule HDAC inhibitors restored the levels of Gad2 histone acetylation, GAD65 expression, and GABA synaptic function, indicating that histone deacetylation was responsible for the changes triggered by inflammation. Further, the HDAC inhibitors reduced the animals’ hypersensitivity to mechanical pain.

The correlations among GAD65 expression, GABA synaptic function, and hyperalgesia indicated epigenetic regulation of Gad2 in the production of pain. To nail down the case, the researchers investigated Gad2 knockout mice. The mutant animals showed heightened pain behaviors compared to wild-type, both under basal conditions and in response to CFA, and GABA neurotransmission in NRM neurons was decreased. HDAC inhibitors were powerless to restore GABA synaptic function or to dial back pain. All together, the data indicate that GAD65 downregulation via histone deacetylation drives CFA-induced pain.

In line with the results from CFA-induced inflammation in mice, the team found that spinal nerve ligation (SNL) in rats, a model of neuropathic pain, also reduced histone acetylation upstream of Gad2 and downregulated GAD65 expression. Importantly, the changes in histone acetylation, GAD65 expression, and GABA synaptic function were measured days after CFA injection or SNL surgery; none of the effects were seen early on, even though pain hypersensitivity set in quickly. Thus, the changes in histone acetylation seem to be involved in triggering later stages of pain. A key feature of epigenetic changes observed in the nervous system, Pan said, is that they are activity-dependent—they kick in only after repeated stimulation. In the case of pain, he said, “Epigenetic modulation is probably involved in the transition from acute pain to chronic pain.”

Other groups have found that HDAC inhibitors produce analgesia in additional mouse models, and that the compounds normalize expression of a variety of potential protein mediators (Chiechio et al., 2009; Lu et al., 2010). The new study from Pan and colleagues identifies a specific protein whose expression is boosted by the inhibitors to relieve pain.

The next order of business, Pan said, is to investigate the efficacy of HDAC inhibitors in relieving chronic pain, and to see which compounds can be used therapeutically. Some have already been tested clinically as anti-cancer agents (Prince et al., 2009), and one, suberoylanilide hydroxamic acid (SAHA or vorinostat), a nonspecific HDAC inhibitor, has been approved for the treatment of cutaneous T cell lymphoma. One challenge, however, is that HDACs—and thus the compounds that inhibit them—have a broad range of targets, so side effects are a concern. Drugs that target specific HDAC subtypes may help. One study has narrowed the field a bit by suggesting that inhibitors of class IIa HDACs (HDAC4, 5, 7, 9) could be good targets for pain therapy (Bai et al., 2010).

Image: Schematic representation of a nucleosome—a segment of DNA spooled around histone proteins. Credit: R. Wheeler, modified by Rekymanto, Wikimedia Commons.