New findings in mice show that CGRP activates Schwann cells to sensitize trigeminal neurons, leading to migraine-like pain.
How does calcitonin gene-related peptide (CGRP) lead to migraine pain, and which cell types does it affect? These questions are of particular interest for the migraine field, considering the use of anti-CGRP drugs for migraine treatment. A recent study in mice now provides some answers, pointing the finger at a non-neuronal cell as a key player.
Work from the groups of Pierangelo Geppetti at the University of Florence in Italy, and Nigel Bunnett at New York University in the US, shows that CGRP from trigeminal nerve fibers activates surrounding Schwann cells; these non-neuronal cells are found in the peripheral nervous system where they surround and protect axons.
The activation of the Schwann cells triggers a complicated molecular cascade, ultimately leading to activation of nociceptive neurons and mechanical allodynia (a phenomenon where normally innocuous mechanical stimuli become painful), in the periorbital area of mice.
The authors also show that nanoparticles containing a drug that dampens CGRP signaling in endosomes – vesicles that bud off from the cell membrane – reduce migraine-like pain in the animals.
“This incredibly detailed study connects the dots of what many in the field have been suspecting for some time. It really drives home how important Schwann cells are in migraine pain,” said Paul Durham, a migraine researcher at Missouri State University who was not part of the study.
The research appeared online February 3, 2022, in Nature Communications.
CGRP recruits Schwann cells to provoke migraine pain
When the team started the project, their central question was how and where CGRP acts to cause migraine pain. The scientists began by investigating where CGRP receptors are present in the trigeminal ganglia.
The CGRP receptor is a protein made up of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1), collectively called CLR/RAMP1. Using immunohistochemistry techniques, the group found CLR/RAMP1 in cultured human Schwann cells, as well as in mouse Schwann cells, taken from the sciatic or trigeminal nerve. They also detected CLR/RAMP1 in Schwann cells in nerve bundles from mouse and human skin.
With Schwann cells now in their sights as a possible contributor to the workings of CGRP, the team set out to provide functional evidence for that idea. Here, they used genetically modified mice missing RAMP1 in Schwann cells of the periorbital region. Then they tested whether the animals still developed periorbital mechanical allodynia, in a mouse model of migraine.
In this model, CGRP or capsaicin (the component of chili peppers that makes them “hot” and which elevates CGRP levels) injected into the periorbital area causes robust migraine pain. However, CGRP failed to do so in mice lacking RAMP1.
The animals were also prevented from developing migraine pain after systemic injection of glyceryl trinitrate, a substance that normally causes such pain.
“From these data, it was clear that CGRP acts to cause migraine pain via activation of Schwann cells. We think that it’s acting in a very localized area between the trigeminal nerve terminals and the surrounding Schwann cells,” Geppetti told Migraine Science Collaborative.
“It’s really impressive how they overwhelmingly demonstrate the mechanism of how CGRP works,” said Durham, who added that he would have liked the authors to “discuss a dual role with satellite glial cells, as there’s plenty of evidence showing that satellite glial cells are also involved in neuroinflammation in migraine.” (Satellite glial cells wrap around the cell bodies of neurons.)
A painful and complex molecular cascade
Next, the team wanted to know which signaling molecules in Schwann cells spurred the pain from CGRP. The researchers already knew that CLR/RAMP1, once activated, becomes engulfed into endosomes (a process known as endocytosis). Previous research in mice had also shown that CLR/RAMP1 can signal from endosomes to drive prolonged pain by regulating neurons in the spinal cord.
Which specific signaling molecules following endosome formation come into play to drive migraine pain? A series of experiments from the current study provided a complicated answer.
First, CLR/RAMP1 signaling in endosomes leads to the formation of cyclic adenosine monophosphate (cAMP; this signaling molecule is known as a “second messenger” and plays a role in many biological processes).
cAMP then activates protein kinase A (PKA), an enzyme that activates another enzyme called endothelial nitric oxide synthase (eNOS), which makes nitric oxide (NO). Nitric oxide is a molecule that causes molecular damage called oxidative stress and contributes to migraine.
NO then activates TRPA1, a protein known as an ion channel, in Schwann cells. Ultimately, this causes the Schwann cells to release reactive oxygen species (ROS). These cell-signaling molecules then activate TRPA1 on trigeminal neurons. The end result is migraine pain.
Targeting the pathway with antagonists to reduce migraine pain
The team next used drugs to inhibit the different components of the molecular pathway to see how that affected the response of mice to CGRP.
At first, the researchers found that treatment with CLR/RAMP1 antagonists only decreased migraine pain when the mice received the antagonists before injection of capsaicin or CGRP, but not after. Inhibiting other components of the pathway, including PKA, eNOS, or molecules required for endocytosis produced the same result.
Those findings suggest that endocytosis and NO initiate pain in response to CGRP. But these processes are not sufficient to sustain the pain.
However, the results were different with TRPA1 antagonists. This approach, whether the mice received the antagonists before or after CGRP, blocked migraine pain in the animals. Post-CGRP treatment with an ROS scavenger, a molecule that reduces oxidative stress from ROS, similarly reduced the pain.
All eyes on the endosome
Together, the findings suggest that migraine pain from CGRP is initiated by NO. The pain is then sustained by persistent ROS release from Schwann cells and subsequent activation of TRPA1.
But how best to target these mechanisms to treat migraine pain?
Based on their data, the team highlighted persistent G protein-coupled receptor (GPCR) signaling from endosomes as the target with the most therapeutic potential to treat migraine pain; GPCRs are a family of receptors to which CLR/RAMP1 belongs.
Nanoparticles have been used to deliver drugs via endosomal pathways, to target tumor cells, for example. With that in mind, the team developed nanoparticles that would target their GPCR of interest, CLR/RAMP1, in Schwann cells.
The group created nanoparticles containing a potent CLR/RAMP1 antagonist. The nanoparticles enter cells by endocytosis, and then deliver the antagonist to the cells.
Cell culture experiments with human Schwann cells showed that the nanoparticles reduced CGRP signaling. The team also tested whether the nanoparticles could inhibit migraine pain in mice. When injected into the periorbital region before CGRP treatment, the nanoparticles indeed decreased the animals’ pain.
“Endosomes are a key subcellular particle that is full of therapeutic options for migraine pain. Our nanoparticles show early preclinical success, so we’re looking to work with the tool for clinical drug development. The main challenge is to have drugs that target CLR/RAMP1 endosomes selectively in Schwann cells in the trigeminal nerve fibers,” Geppetti said.
Geppetti’s concerns about the selectivity of the nanoparticles for Schwann cells were echoed by Durham, who is skeptical of their clinical potential.
“The endosome-targeting nanoparticles are intriguing. However, it would be hard to control their access to trigeminal ganglion Schwann cells in patients to treat their migraines,” he said.
“Looking at the bigger picture, this is an incredible basic science paper,” Durham continued. “But do I think it’ll be a game changer for the pharmaceutical industry and clinicians? I’m not convinced. We already know that NO inhibitors and TRP inhibitors aren’t successful at treating migraine pain, and I’m not so hopeful about the selectivity of the nanoparticles.”
Nonetheless, the new research adds several missing pieces to the puzzle of CGRP signaling, with Schwann cells looking more and more like the bad guys of migraine pain.
Fred Schwaller, PhD, is a freelance science writer based in Germany.
Schwann cell endosome CGRP signals elicit periorbital mechanical allodynia in mice. De Logu et al. Nat Commun. 2022 Feb 3;13(1):646.
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
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