Stress Regulation Gene Drives Development and Maintenance of Chronic Pain in Mice
Spinal FKBP51, a gene known to play a role in the brain’s stress response, is important for the development and maintenance of chronic pain, reports a new mouse study published February 10 in Science Translational Medicine. Researchers led by Sandrine Géranton, University College London, UK, demonstrated a reduction in mechanical hypersensitivity following blockade of FKBP51 activity in a model of ankle joint inflammation, suggesting that FKBP51 may be a new target for therapies aimed at alleviating chronic pain. The new work also demonstrates that FKBP51 exerts its pro-nociceptive effects via modulation of the glucocorticoid system.
“This is a very thorough and very exciting paper. [Based on these results], I think it’s definitely worthwhile to see if blocking FKBP51 can reduce chronic pain in humans,” said Moshe Szyf, McGill University, Montreal, Canada, who was not involved in the new study. “What’s unique about this paper is that it focuses on one critical regulator of stress, showing that it is altered by pain, which causes a change in the way it works that could be driving the development of chronic pain,” he added.
Géranton and her lab are interested in epigenetic modifications—changes that alter gene functioning without altering the underlying DNA sequence—that occur in the dorsal horn following peripheral nerve injury. These epigenetic changes are thought to explain in part why some individuals are more susceptible to chronic pain than others (see PRF related webinar summary).
Previously, Géranton’s team performed a microarray study that examined gene expression changes in the dorsal horn during chronic pain states (see PRF related news story; Géranton et al., 2007). They identified a small number of epigenetically regulated genes, including FKBP51, whose messenger RNA (mRNA) was highly upregulated within two hours of initiating inflammation of the rat ankle joint. In the new study, the researchers went beyond the initial correlation to see whether FKBP51 could contribute to the development and maintenance of chronic pain.
A role for FKBP51 in pain
After confirming their previous finding of elevated FKBP51 mRNA in the mouse dorsal horn (but not the dorsal root ganglion or cervical spinal cord) following ankle joint inflammation, first author Maria Maiarù and colleagues examined the development of pain in mice with a global deletion of FKBP51. “Right away, we could clearly see that the knockout animals were not as sensitive as their wild-type littermates under chronic pain conditions,” Géranton told PRF.
Compared to wild-type animals, the knockout mice had significantly less mechanical hypersensitivity following inflammation induced by injection of complete Freund’s adjuvant (CFA) into either the ankle joint or hindpaw. In addition, in the spared nerve injury model of neuropathic pain, knockout animals showed less mechanical hypersensitivity than wild-type mice, beginning five days after surgery, and performed better on the rotarod test of motor ability.
Importantly, deletion of FKBP51 did not appear to affect acute nociception in the knockouts, as thresholds for cutaneous mechanical sensory, and for thermal heat and cold stimulation, did not differ from wild-type animals. In addition, injection of the short-term inflammatory agents formalin or interleukin-6 did not affect nociceptive sensitivity. Taken together, these results suggested that FKBP51 is important for the development of chronic but not acute pain states, said Géranton.
To tease out whether the nociceptive effects of FKBP51 were mediated in the brain through the stress response or in the spinal cord, the researchers used intrathecal injections of small interfering RNA (siRNA) to selectively silence FKBP51 in the dorsal horn of wild-type animals 48 hours prior to CFA injection in the ankle joint. Consistent with a dorsal horn mechanism, the researchers found that siRNA-treated animals displayed a reduction in mechanical hypersensitivity following inflammation of the ankle joint similar to that found in the global FKBP51 knockout mice. “That’s when we knew that FKBP51 was acting at the spinal level to regulate the pain state,” Géranton told PRF. Also consistent with the findings in the global knockouts, baseline mechanical thresholds and motor performance were unaffected in siRNA-treated mice.
Interestingly, an siRNA injection given three days after CFA injection significantly increased the animals’ mechanical thresholds, indicating that FKBP51 is important not only for the development but also the maintenance of chronic pain. A similar reduction in mechanical hypersensitivity was achieved when FKBP51 activity was blocked by intrathecal injection of a recently developed, highly specific FKBP51 antagonist called SAFit2, also administered three days post-CFA. The fact that SAFit2 reduced pain is exciting, said Szyf. “[The beneficial effect of blocking FKBP51] is not just theoretical; it might have an impact on chronic pain in humans,” he added.
The glucocorticoid connection
Human studies indicate that FKBP51 is not only involved in pain, but also in the stress response. For instance, variants in the gene affect the severity of musculoskeletal pain following a car crash or sexual assault, and are also associated with depression and post-traumatic stress disorder, as well as with the extent of stress hormone dysregulation in depression (Klengel et al., 2013; Menke et al., 2013). Consistent with those findings, FKBP51 knockout mice displayed better coping behavior during social defeat stress than wild-type animals.
In the next series of experiments, the researchers zeroed in on the glucocorticoid receptor (GR), which is also involved in the stress response and is antagonized by FKBP51. “Because FKBP51 is known to contribute to the stress response by manipulating the sensitivity of the GR, and because other groups have shown that glucocorticoid signaling is also important for the regulation of pain states, we hypothesized that the role of FKBP51 in the modulation of pain states occurs via a modulation of glucocorticoid signaling,” Géranton explained.
Typical images of FKBP51 and GR immunoreactivity in the superficial dorsal horn of WT sham mice and mice after CFA injection in the ankle joint. Green, FKBP51; red, GR; blue, NeuN [a neuronal marker]. Arrows, neurons expressing both FKBP51 and GR. Arrowheads, GR-positive neurons that do not express FKBP51. Scale bar, 20 μm. Credit: From Maiarù et al. Reprinted with permission from AAAS.
Consistent with this view, both FKBP51 and GR were co-expressed in dorsal horn neurons, and global deletion of FKBP51 appeared to reduce glucocorticoid signaling. Inflammation of the ankle joint did not affect total GR mRNA or protein levels in the spinal cord, or mRNA levels of the GRα isoform, in the knockouts or in wild-type animals. However, compared to the side contralateral to CFA injection, both genotypes showed increased levels of the GRβ isoform on the ipsilateral side. Across all conditions, the knockouts had lower GRβ levels than the wild-type mice.
Elevated GRβ levels are associated with glucocorticoid resistance in inflammatory diseases (Lewis-Tuffin and Cidlowski, 2006), so the researchers reasoned that during inflammatory pain, FKBP51 knockouts, with their lower levels of GRβ, would be more sensitive to glucocorticoids than wild-type mice. Consistent with this interpretation, knockout mice had lower levels of corticosterone, a corticosteroid hormone that binds to the GR, in both naïve and chronic pain states compared to wild-type animals. This finding is consistent with a stronger suppression of corticosterone release, as previously reported (Touma et al., 2011).
In a final set of experiments, the researchers injected the GR antagonist mifepristone intrathecally into both naïve wild-type and FKBP51 knockout mice, finding that the drug increased mechanical hypersensitivity in both groups. This suggested that GR is anti-nociceptive in the naïve state. However, administration of the antagonist three days after CFA injection into the ankle joint produced genotype-specific results, eliminating the CFA-induced mechanical hypersensitivity in wild-types but aggravating it in FKBP51 knockouts, suggesting that FKBP51 is needed for GR signaling to contribute to pain after injury.
The researchers were amazed that both genotypes behaved the same way in the naïve state, but after injury, responded in opposite ways, said Géranton. “The results suggest that without FKBP51, the GR cannot switch from an anti-inflammatory state during non-injury to a pro-inflammatory or pro-nociceptive state after injury,” she explained.
The new research highlights that there is an immediate (within two hours) epigenetic change (shown in the study to be an alteration in DNA methylation) in response to pain in the dorsal horn that causes the deregulation of a stress-controlled gene, FKBP51, that seems to be very critical to the development of chronic pain, Szyf said. The findings also point to FKBP51 as a potential new drug target for the treatment of chronic pain.
Géranton emphasized that blocking FKBP51 leads to two separate responses. If the blocker is given systemically, it acts at the spinal cord level to block pain, but also at the level of the brain to reduce the chronic stress associated with long-term pain states. “Since we know that both of these conditions exacerbate each other, I think that blocking FKBP51 will be able to alleviate the chronic pain much quicker [than existing therapies],” Géranton concluded.
In the future, the researchers plan to use animal models that recapitulate more clinically relevant pain states such as those induced by chemotherapy or diabetes, and to study how chronic pain and stress interact with each other.
See related plain language summary on PRF’s sister site RELIEF.
Allison Marin, PhD, is a neuroscientist-turned-science writer who resides in Pittsburgh, Pennsylvania, US.
Image credit: Maiarùet al., 2016, with permission from AAAS.
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