Two new papers show that Piezo2, the mechanosensitive ion channel that brings about the sensations of gentle touch, vibration, and proprioception, also triggers tactile allodynia in mice and humans, but is not required for sensing other types of noxious mechanical stimuli such as pinch or pinprick.
One paper comes from a group led by Ardem Patapoutian, a Howard Hughes Medical Institute Investigator at The Scripps Research Institute, La Jolla, US, which discovered Piezo2 in 2010 and described its central role in touch sensation in 2014 (Coste et al., 2010; Ranade et al., 2014). In their new work, the researchers show in transgenic mice that while nociceptive neurons contain Piezo2, the channel was not required to sense noxious pinch or pinprick, but it was required for inflammation- or nerve injury-induced mechanical allodynia.
The second paper, conducted by Alex Chesler’s group at the National Center for Complementary and Integrative Health (NCCIH) at the National Institutes of Health (NIH), Bethesda, US, included data from four patients with complete loss-of-function mutations in Piezo2. Like mice lacking Piezo2, these individuals had no sense of gentle touch or vibration and did not develop allodynia, but they felt pain with pinch or pinprick.
“It is not surprising that Piezo2 would have a role in mechanical allodynia, because of its expression in large myelinated sensory neurons, but evidence to date had not been clear, so this work clarifies that question. And the work with the patients shows that it translates to people,” said Rebecca Seal, University of Pittsburgh, US, who was not involved with either of the new studies.
The findings also suggest that a local blocker of Piezo2, potentially applied as a topical cream, might relieve mechanical allodynia without compromising other pain sensations. Allodynia, a phenomenon in which normally innocuous sensations are perceived as painful, affects millions of people with chronic pain, including inflammatory pain from conditions like osteoarthritis, as well as neuropathic pain resulting from chemotherapy, diabetes, or some other insult.
The new research was published October 10 in two complementary papers in Science Translational Medicine.
Shining a light
The Patapoutian group first used optogenetics to demonstrate the effects of Piezo2 activation in vivo. Mice were genetically engineered to express channelrhodopsin-2 (ChR2) in cells expressing Piezo2. When the researchers activated the cells by shining blue light on the hindpaw, the animals responded with nocifensive behaviors like licking and flinching, suggesting that nociceptive sensory neurons contained Piezo2.
Conversely, small interfering RNA (siRNA) knockdown of Piezo2 in cultured dorsal root ganglia (DRG) neurons led to fewer responses to mechanical stimuli applied to the cell body, in calcitonin gene-related peptide (CGRP)-expressing peptidergic nociceptors and in Mas-related G protein-coupled receptor type D (MRGPRD)-expressing nonpeptidergic nociceptors.
The findings do not necessarily secure a role for Piezo2 in sensing noxious stimuli, Seal said. “So you drive expression of channelrhodopsin and activate those Piezo2 neurons optically, and you get pain behaviors. But if you activate those cells with a natural stimulus, then Piezo is dispensable—there is no phenotype,” she said, referring to previous experiments from Patapoutian’s group in mice with conditional knockout of Piezo2 (Ranade et al., 2014).
Swetha Murthy, lead author of the new study and a neuroscientist at The Scripps Research Institute, agreed. “There have been some reports that Piezo2 is expressed in Aδ and C-fiber nociceptors, but we’ve never seen a phenotype in the knockouts. The optogenetic strategy was another way to see if it’s expressed in pain-sensing neurons.”
Piezo2 is required for tactile allodynia
The two research teams independently set out to determine the effects of Piezo2 loss in mice. Knocking out Piezo2 entirely is lethal to mice at birth, so both groups got creative in designing mice lacking the channel in sensory neurons.
The Patapoutian lab used a genetic strategy in which Piezo2 was constitutively knocked out specifically in cells that also expressed the developmental transcription factor HoxB8 (Piezo2HoxB8). “HoxB8 is expressed mostly in the caudal region,” said Murthy. “The knockout mice only have proprioceptive and touch deficits in the bottom half of the body,” so the researchers tested responses to hindpaw stimulation. In a control experiment, mice reflexively blinked in response to a gentle poke to the eye, confirming that mechanical sensation was intact rostrally. Temperature sensation was unaffected by the loss of Piezo2. But the knockout mice displayed profound deficits in sensing innocuous mechanical stimuli like gentle brushing. Murthy and colleagues also saw subtle impairments in the animals’ responses to noxious pinprick or a pinch from an alligator clip.
In collaboration with Gary Lewin, Max Delbrück Center for Molecular Medicine, Berlin, Germany, the group also used an ex vivo saphenous skin-nerve preparation to make electrophysiological recordings of sensory neurons during stimulation of the attached skin. The researchers identified the neurons as Aβ, Aδ or C-fibers by their conduction velocity and other properties. In the Piezo2HoxB8 mice, significantly more Aβ fibers were mechanically insensitive compared to controls. The number of mechanically sensitive Aδ and C-fibers was similar to controls, but they required stronger stimuli to respond, and neurons lacking Piezo2 fired fewer spikes at the onset of stimuli, suggesting some role for Piezo2 in nociceptors as well as in touch-sensing neurons.
“That corroborates what we see with the behavior,” Murthy said, referring to the dampened responses to acute noxious stimuli. “But I want to emphasize: It’s not gone. The animals still do feel pain.”
The team next examined the effects of Piezo2 loss during inflammation or nerve injury. “The phenotype was much more pronounced when we looked at sensitized pain,” Murthy said. After hindpaw injection with capsaicin, control mice responded to a poke with a von Frey hair at much lower thresholds than before, and displayed nocifensive behaviors after a gentle brush stroke. Piezo2HoxB8 mice, in contrast, did not display those changes after capsaicin, suggesting that Piezo2 is a crucial regulator of tactile allodynia.
Similarly, Piezo2HoxB8 mice showed little change in mechanical sensitivity after spared nerve injury (SNI), a model of chronic neuropathic pain. “Even on day 21 post-injury, knockout mice still don’t exhibit any kind of allodynia,” Murthy said. “So that means there is no compensation—Piezo2 is what mediates allodynia. That was a strong result.”
Clinically, patients describe allodynia in multiple ways, Murthy said. Dynamic allodynia occurs when a moving stimulus causes pain, like gently rubbing the skin. Punctate (static) allodynia can be measured by a gentle poke with a filament. “We tried to recapitulate those in mice. After injury, we run a cotton swab on the paw to recapitulate dynamic allodynia, and we tried to test static allodynia with filaments. From what we saw in mice, both these forms of allodynia are still Piezo2 dependent” after injury, she said.
Watching sensory neurons in action reveals a similar story
To generate mice lacking Piezo2, Chesler’s group took a mosaic approach. They injected newborn mice with a virus that could selectively remove Piezo2 from any neuron it infected (Piezo2cKO), creating individual knockout cells in a mosaic pattern throughout the sensory ganglia. In the same cells, the virus also drove production of the calcium-sensitive fluorescent marker GCaMP6f. “The neurons fluoresce green, but their brightness depends on intracellular calcium, which we can use as a proxy for neuronal activity,” Chesler said.
Labeled neurons acutely cultured from adult Piezo2cKO mice were unresponsive to mechanical stimuli, unlike neurons from control mice. The mice also displayed mechanical and proprioceptive behavioral deficits.
The group then used an in vivo imaging system recently developed by one of three co-lead authors, Nima Ghitani (Ghitani et al., 2017), also at NIH, and similar to other systems recently used in the DRG (Chisholm et al., 2018; Kim et al., 2016). The researchers removed the mouse forebrain to expose the trigeminal ganglia for direct optical imaging of the neurons expressing GCaMP6f. Aside from that, Chesler said, “We wanted to be as naturalistic as possible, to keep the animal as intact as possible. So it is breathing, its blood is circulating, and all the [spinal and brainstem] synaptic contacts are still there.”
Marcin Szczot, also a co-lead author at NIH, said, “The prep allows us to image in vivo, to see how neurons respond not to an artificial stimulus but to one that happens in a real-life setting. When we gently brush the cheek of the mouse, you lose the calcium transients in neurons when Piezo2 is knocked out, but the response to pinch is still there. It’s the same thing you’d predict from the phenotype in the human patients.” Neurons lacking Piezo2 did not respond to vibration but reacted normally to thermal stimuli.
And, Chesler added, “We are not imaging one cell but hundreds of neurons, which allows us to see the population code.” Globally, the activity of high threshold-activated neurons appeared similar to control animals in response to noxious mechanical stimuli, suggesting that Piezo2 is not critical for their function.
Szczot and colleagues then induced inflammation with either complete Freund’s adjuvant (CFA) or capsaicin, and also used the SNI model of neuropathic pain. In each case, injury or inflammation sensitized Piezo2cKO neurons to heat stimuli, but they still did not respond to gentle brushing. Piezo2cKO neurons responded normally to pinch. The findings confirmed that Piezo2 is critical for detecting innocuous mechanical stimuli under normal conditions, as well as during inflammatory and neuropathic pain states, in multiple mouse models.
Piezo2 knocked out—in humans
Collaborator Carsten Bönnemann, National Institute of Neurological Disorders and Stroke (NINDS), identified four patients with complete loss-of-function mutations in PIEZO2, leaving them devoid of any sense of light mechanical touch or vibration. They also displayed proprioceptive deficits. The individuals add to insights gleaned from two patients with PIEZO2 mutations previously identified by the group (Chesler et al., 2016; also see PRF related news).
Co-lead author Jaquette Liljencrantz, NCCIH, evaluated the patients for the new study. To test whether the patients lacking PIEZO2 developed tactile allodynia, she applied capsaicin to one area of the arm, hidden from view, and a placebo cream to another. When the investigators gently brushed the skin, control subjects rated brushing of the capsaicin area as more painful than the placebo area every single time. But people with the mutation could not distinguish between capsaicin and placebo; when forced to choose which was more painful, their answers were no better than chance.
“If you block this channel, you will block this type of pain. It’s a clear-cut, single-result finding: In mice and humans, if you don't have Piezo2, you don’t have allodynia,” said Chesler.
The individuals with PIEZO2 mutations did respond normally to pinch and pinprick. “When we pinch humans, they still say ‘ouch.’ They still feel it,” Chesler said.
As for a potential therapeutic targeting PIEZO2, Chesler said, “The gulf is large, but you could imagine a PIEZO2 antagonist applied topically in a cream to attenuate touch responses but keep all other responses intact.”
A slight discrepancy
Chesler’s findings in mice and people contrast slightly with Patapoutian’s finding that Piezo2 plays a functional role in sensing acute noxious mechanical stimuli, something Chesler’s team did not observe. The authors offered several possible explanations.
While Chesler’s group did not see a significant decrease in calcium signaling during noxious stimuli in neurons lacking Piezo2, he said, “Calcium is not neuronal activity—it is a proxy. The [fluorescent] signal was incredibly sensitive—it’s fantastic. But it’s much slower than action potentials.” In the Patapoutian work, in which the group observed a decrease in early spiking in Piezo2-deficient nociceptive neurons, Chesler said, “They’re looking at single spikes. We couldn’t have gotten to that subtle level of information, so maybe that’s where they see a role in acute pain.”
Previous work that did not find a phenotype (Ranade et al., 2014) used PiezocKO mice, as did the Chesler study. The Patapoutian team suggests that the depletion of Piezo2 in Piezo2HoxB8 mice is more extensive, and the total loss of Piezo2 might have revealed a minor role for the channel in sensing acute noxious mechanical stimuli. Precisely what that role might be remains unknown.
In any case, Patapoutian explained, “The noxious pain phenotype is rather subtle in mice, and I am not sure you would expect to see it in humans, particularly with such a small number of patients” used in the Chesler study.
Settling a debate?
There are two main theories of how tactile allodynia arises. The first—supported by the new studies—posits that low-threshold mechanoreceptors (LTMRs) transduce signals to the spinal cord in response to gentle touch, but those signals somehow get interpreted at higher brain centers as painful. “There are specialized neurons that sense touch and pain,” Seal said. “But with injury, you get the recruitment of LTMRs into the nociceptive network. Usually they’re not able to elicit pain—they just mediate touch.”
What changes in the spinal dorsal horn after injury to turn gentle touch painful? “Lots of mechanisms have been proposed,” Seal said, “but the basic idea is that there are mechanisms of disinhibition that decrease inhibitory tone, that allows LTMRs to access nociceptive pathways.”
“Under physiological conditions, there are interneurons that inhibit the signal from Aβ neurons,” Murthy said. “Under conditions after injury, that inhibition is lost. So information from touch neurons intersects with the pain pathway.”
The second theory, still debated, proposes that an unknown mechanosensitive channel—on either nociceptors or mechanosensitive neurons—becomes sensitized with inflammation or injury and responds to light mechanical stimuli to produce allodynia.
“Both these studies together come down firmly against that idea,” Chesler said. Piezo2 is required for allodynia, “but this does not settle what specific cell types are responsible.”
Piezo’s role in nociceptors also remains unclear. “When we look at expression at the messenger RNA level, we find it in over 70 percent of neurons, and it’s clearly in nociceptors that we know do not respond to gentle touch. We still don’t know what it’s involved in, in those cells—it may play a modulatory role,” Chesler said.
Stephani Sutherland, PhD, is a neuroscientist and freelance journalist in Southern California. Follow her on Twitter @SutherlandPhD
