The same genes and proteins that sculpt the developing nervous system are often called upon again in adulthood, even if for very different reasons. Now, a research team led by Rohini Kuner, Heidelberg University, Germany, has discovered that a signaling pathway that regulates axon guidance and neural connectivity is rekindled during inflammation, fueling persistent pain.
Kuner and her colleagues show that the transmembrane receptor Plexin-B2 is expressed by peripheral sensory neurons in adult mice, as is the receptor’s ligand, semaphorin 4C (Sema4C). Inflammation increased the levels of both proteins in nociceptive neurons, while those of Sema4C also rose in immune and skin cells. And, the Sema4C-Plexin-B2 signaling pathway contributed to the hyperalgesia observed in a mouse inflammatory pain model.
“This is a solid study that characterizes a novel signaling pathway in inflammatory pain,” said Ru-Rong Ji, Duke University, Durham, US. It uncovers an additional line of communication between immune cells and neurons during persistent pain, added Ji, and “may provide new targets for pain treatment.”
The findings were published online August 2 in Nature Communications.
A reawakening
Kuner has been working to piece together the cellular and molecular events responsible for chronic pain (see PRF related news stories here and here, and PRF related interview). In parallel, she has been investigating the signaling pathways that build the nervous system. “I was always interested in semaphorin and plexin signaling, trying to understand their roles in development,” she said.
“I knew that both [Sema4C and Plexin-B2] are expressed in developing dorsal root ganglia neurons and that there are developmental phenotypes in mice missing either protein,” said Kuner. For example, in 2007, Kuner’s lab found that mouse embryos without Plexin-B2 had a neural tube that failed to close, a defect that prevents the peripheral nervous system from forming properly (Deng et al., 2007). But whether Sema4C-Plexin-B2 signaling exists in adult animals was unknown.
To learn more, lead author Eszter Paldy and colleagues used transgenic mice that produce beta-galactosidase (beta-GAL) only in cells that also make Plexin-B2. Immunohistochemical labeling for beta-GAL revealed that adult mice still had dorsal root ganglia (DRG) neurons that carried the receptor, including populations containing isolectin B4, calcitonin gene-related peptide, or neurofilament 200; these are markers of non-peptidergic nociceptors, peptidergic nociceptors, and large-diameter myelinated neurons, respectively. Consistent with this labeling, the researchers could detect Sema4C in adult DRG neurons using a similar approach.
When the researchers injected complete Freund’s adjuvant (CFA) into the paws of adult mice, a model of inflammatory pain, they saw a clear uptick in the levels of both proteins in the DRG. Sema4C levels also increased in macrophages and T cells that infiltrated the inflamed skin, as well as in keratinocytes. “Semaphorins can be secreted by immune cells, and so maybe this is another way of signaling to the sensory system that something is wrong,” said Kuner.
To begin to test this idea, the researchers exposed adult mice genetically modified to lack Plexin-B2 mainly in nociceptors to either intense pressure or intense heat. The knockout animals were not as sensitive to either stimulus compared to their normal counterparts, and they showed less biting and licking, two pain-related behaviors, upon injection of the TRPV1 agonist capsaicin. But the results were confounded by the absence of Plexin-B2 from birth. That is, the knockouts had fewer DRG nociceptive neurons, and fewer pain-related nerve fibers in the spinal cord and skin, making it difficult to know whether these behaviors reflected the receptor’s involvement in pain transmission or developmental abnormalities.
So Kuner and colleagues turned to a strategy that deletes Plexin-B2 receptors in nociceptors only during adulthood. Unlike the first group of knockout mice, these animals showed no change in sensitivity to touch or temperature but did develop less mechanical hyperalgesia after being injected with CFA, compared to controls. Mice missing Sema4C throughout the entire body responded similarly. Conversely, injection of Sema4C into the paws of naïve mice raised the animals’ pain sensitivity.
Kuner thinks the results could lay the foundation for more effective ways of alleviating chronic inflammatory pain. “A large part of inflammatory pain is still being treated with non-steroidal anti-inflammatory drugs [NSAIDs],” which stymie the production of prostaglandins. “And they have value in the treatment of some disorders, but not all.
“What we’re learning is that the types of mediators that are released in chronic inflammatory disorders go way beyond prostaglandins and involve some really new signaling systems that you would not typically imagine in this context,” said Kuner. “We may need to target other mediators apart from the classical ones that are released during acute inflammation.”
Intracellular events
The group went on to identify key events in the intracellular cascade set off by Plexin-B2 activity. To their surprise, in adult neurons the receptor did not recruit the small GTPase that developing neurons depend on, called Ras, instead activating Ras homology gene family, member A (RhoA). Indeed, by genetically disabling RhoA signaling, the researchers could reduce the magnitude and duration of mechanical hyperalgesia induced with CFA. As further experiments indicated, RhoA likely achieved its effect by increasing the number of TRPA1 ion channels in the cell membrane.
The contribution of Sema4C and Plexin-B2 to inflammatory pain is another case of chronic pain repurposing molecules that are important for neural development, said Kuner. For instance, ephrins and their receptors guide the migration of newly born neurons and assemble circuits, but they also promote long-lasting pain later in life through their actions in the adult spinal cord (Battaglia et al., 2003).
It’s unclear, however, if other forms of chronic pain, such as neuropathic pain and cancer pain, similarly reinstate Sema4C-Plexin-B2 signaling, said Ji. “There are different animal models of neuropathic pain, some of which have more inflammation, like the chronic constriction injury [CCI] model, compared to others. In the CCI model, this signaling pathway may be at work, at least in the induction phase” of inflammation.
Because immune cells seem to be a major source of Sema4C, “there is the potential for this pathway to exist in types of neuropathic pain which involve immune cell migration and immune-derived mechanisms,” said Kuner. “This is something we hope to test in the future.”
As for cancer pain, Kuner’s lab has already collected some data that connect it to the newly studied pathway. “Cancer cells secrete semaphorins, so this makes complete sense,” said Kuner. “I think that the findings so far only represent the tip of the iceberg.”
Matthew Soleiman is a science writer currently residing in Nashville, Tennessee. Follow him on Twitter @MatthewSoleiman.
Image credit: Paldy et al., 2017
