Wnt Signaling Linked to Neuropathic Pain

The idea that reawakening of dormant developmental pathways, in mature sensory and spinal neurons after injury or insult, contributes to neuropathic pain has gained traction in recent years. Two new papers bolster that concept by implicating the Wnt family of signaling peptides, key players in nervous system development, in neuropathic pain in animal models, and in people.

In one study, researchers led by Xue-Jun Song at Parker University in Dallas, Texas, US, demonstrated that Wnt signaling pathways contribute to the production of hyperalgesia and allodynia in a nerve injury model of neuropathic pain in rats and in a model of bone cancer pain in mice. In a second study, Shao-Jun Tang and colleagues at the University of Texas Medical Branch, Galveston, US, showed that Wnt proteins are upregulated in the spinal cord of patients with human immunodeficiency virus (HIV)-related pain, but not in HIV patients without pain. Together, the findings point to the Wnt signaling pathway as a potential therapeutic target for neuropathic pain.

Despite extensive research over the past 70 years, Song said, the mechanisms behind neuropathic pain remain elusive, and effective treatments are limited. Michael Salter, who studies neuropathic pain at the Hospital for Sick Children, Toronto, Canada, and was not involved in either study, agreed. “Neuropathic pain is a big global problem, and it is not going away despite our best efforts. We need to keep working, and [these two new studies] give us another potential clue,” Salter said.

The study from Song and colleagues was published May 1 in the Journal of Clinical Investigation, while the report from Tang and co-investigators appeared June 5 in the Journal of Neuroimmune Pharmacology.

Evidence from animal models

The 19 members of the Wnt protein family are secreted signaling proteins most often associated with developmental processes including cell proliferation, differentiation, migration, and establishment of cell polarity. Wnts bind to G protein-coupled “Frizzled” receptors and control cellular levels of β-catenin, a protein that acts in concert with transcription factors to regulate gene expression.

Song, co-first authors Yan-Kai Zhang, Zhi-Jiang Huang, Su Liu, and Yue-Peng Liu, and coauthor Angela Song used several techniques, including immunohistochemistry and RT-PCR, to show that a number of Wnt proteins, particularly the Wnt3a family member, were rapidly upregulated in spinal cord neurons and astrocytes, and in dorsal root ganglia (DRG) neurons of rats after chronic constriction injury (CCI) to the sciatic nerve. The swift and lasting upregulation (from day one through 21 after injury) of Wnts both in nociceptive small- and medium-sized neurons as well as larger, mechanically sensitive neurons of the DRG supports the authors’ hypothesis that the proteins contribute to both hyperalgesia, which is thought to arise from the small- and medium-sized neurons, and allodynia, believed to arise from the larger sensory neurons. Several Frizzled proteins were also upregulated in the spinal cord, where the investigators also observed the accumulation of β-catenin in the cytoplasm of neurons and astrocytes. The group saw increased levels of active β-catenin in the nucleus of dorsal horn neurons that specifically receive synaptic input from primary sensory DRG neurons.

The researchers reported similar findings in a model of bone cancer pain in mice, where injection of tumor cells into the bone also resulted in a sustained increase in Wnt signaling proteins. Expression levels rose beginning five days after tumor cell injection (TCI), an increase that preceded by two days the development of hyperalgesia and allodynia. Meanwhile, injection of complete Freund’s adjuvant (CFA), a model of inflammatory pain, also resulted in upregulation of Wnt3a and related signaling proteins, but only after the onset of inflammatory pain. That suggested that Wnt signaling does not play a role in the establishment of inflammatory pain but rather may be a downstream consequence of inflammatory signaling.

Having established a correlation between Wnt signaling and neuropathic pain, the investigators then asked whether blocking that signaling could block pain. They found that each of a number of inhibitors directed at different targets in the Wnt signaling pathway delayed the onset of neuropathic pain behaviors when administered intrathecally in the first few days following CCI or TCI. Treatment later, after injury, produced transient inhibition of pain, suggesting that blocking Wnt activity will have some effect on established pain. CFA-induced inflammatory pain was unaffected by the blockers.

Importantly, the inhibitors did not affect normal pain threshold or motor behaviors. Conversely, injection of Wnt signaling agonists produced thermal and mechanical hypersensitivity in naïve (uninjured) rats.

Finally, the research team identified interleukin 18 (IL-18), its receptor (IL-18R) and tumor necrosis factor alpha (TNFα) as key downstream effectors of Wnt signaling in nerve injury-induced pain. Although the cytokines IL-18 and TNFα are known as proinflammatory mediators, they have an established role in neuropathic pain signaling. CCI resulted in rapid upregulation of IL-18, IL-18R, TNFα, and β-catenin in the spinal cord that was blocked by antagonists of Wnt signaling. The investigators used a chromatin immunoprecipitation (ChIP) assay to show that activated nuclear β-catenin formed transcriptional complexes with the promoter for IL-18 and TNFα. On the other hand, IL-1β seemed not to be involved in Wnt signaling that contributed to neuropathic pain.

Viewing neuropathic pain through the lens of development provides an avenue to understand its root mechanism, Song said. And while that perspective is not new, Salter noted, the addition of Wnt proteins to the list of developmental molecules involved in pain fills in a bit more of the picture. Previously, Salter and colleagues have shown that changes in microglial signaling in the spinal cord also contribute to neuropathic pain by altering chloride equilibrium in neurons and increasing excitatory signaling, recapitulating a process that occurs during development of the central nervous system (Coull et al. 2005). Salter described an emerging model in which these developmental signaling cascades are triggered in mature neurons by aberrant activity patterns resulting from insult or injury. “During development, when the nervous system is being built, it is in a hyper-excitable, hyper-plastic state,” he said, which facilitates neural growth and connectivity. One may hypothesize that, after injury, neurons reenter that state in an effort to repair and reconnect damaged nerves.

Wnt signaling inhibitors are currently in early-stage clinical trials as anti-cancer agents. If the inhibitors could target both cancer and its resulting pain, Song said, such agents might “kill two birds with one stone.”

A bit of clinical data

Does Wnt signaling play a role in neuropathic pain in humans? Tang and colleagues present evidence, albeit from a very small number of patients, suggesting that it does.

Recently, Wnt proteins have been recognized to play roles beyond development, and activity-dependent Wnt protein production and signaling have been implicated in synaptic plasticity in mature neurons (Budnik and Salinas, 2011). “We wanted to use this basic finding to study problems with clinical relevance,” Tang said. “We chose pain because it involves a hyperactive excitation state of neurons in the pain transmission pathway in the [spinal cord] dorsal horn.” Tang began that work with the finding that Wnt signaling proteins were present in the naïve mouse spinal cord and upregulated in mouse models of neuropathic pain (Shi et al., 2012).

To extend the work to people, Tang and first author Yuqiang Shi examined postmortem spinal cord tissue from 10 HIV patients, half with pain and half without, and five HIV-negative subjects. The researchers found increased expression of Wnt ligands and downstream effector proteins in the spinal cord dorsal horn in patients with HIV-related neuropathic pain, but not in those without. Interestingly, levels of the Frizzled receptor did not differ between the two groups.

The authors say the association between Wnt proteins and pain supports a role for the Wnt signaling pathway in the pathogenesis of HIV-associated pain in people, though they are cautious to note that their findings do not prove a causal relationship.

Tang is now investigating the utility of small peptide Wnt inhibitors in animal models of HIV-related pain. He is also using genetic mouse models with conditional knockout of Wnt ligands in neurons to further elucidate the role of Wnt signaling in HIV-related pain.

The two studies together suggest that inhibiting the Wnt pathway in the spinal cord might help alleviate intractable forms of neuropathic pain, but “of course, that’s far down the line,” said Tang. The emerging role of Wnt signaling in neuroplasticity suggests that blocking Wnt proteins systemically might have adverse effects, making local delivery a therapeutic requirement.

Stephani Sutherland, PhD, is a freelance neuroscience writer based in Southern California, US.