The results of this study are pretty amazing: injecting a “pain protein” from humans into normal mice caused the mice to feel pain.
This directly contradicts the popular belief that pain isn’t a physical entity in itself, but rather a bio-psycho-social disorder. Now we know that’s simply not true.
Unremitting pain may eventually create a bio-psycho-social disorder by leading to deconditioning, depression, and isolation, but those are consequences, not causes.
For decades, doctors have been puzzled why some patients with entirely different autoimmune disorders share pain from nerve injury (neuropathic pain) in common.
Recently, a clue emerged when researchers found that the immune system in many of these patients makes an autoantibody—an antibody that attacks the body’s own proteins—against CASPR2. CASPR2 is a protein found in the nervous system.
Now, in a new study, scientists show how these autoantibodies can cause pain.
a team of researchers led by David Bennett, from the University of Oxford in the UK, finds that human CASPR2 autoantibodies, when injected into mice, decrease the number of potassium channels in neurons (nerve cells), and prevent the channels from getting to the right place along nerve fibers, resulting in pain.
Potassium channels are proteins in the cell membrane that, when functioning properly, quiet the electrical activity of neurons.
When the channels function abnormally—when the “brakes” on the neurons are released—this electrical activity goes up, contributing to pain.
“This finding is helpful for patients with CASPR2 autoantibodies because these individuals can suffer very badly from pain and do not respond to normal treatments,” says Andreas Goebel from the University of Liverpool in the UK. Goebel was not involved in the study.
On the attack
One of the jobs of the immune system is to make antibodies that attack unwelcome invaders like bacteria and viruses. But in patients with autoimmune disorders, the immune system mistakenly produces antibodies against the body’s own proteins.
atients with autoantibodies to the CASPR2 protein can develop a range of disorders that each feature their own unique set of symptoms—but a common thread is neuropathic pain.
Fortunately, the researchers already knew that the CASPR2 protein is necessary so that potassium channels get to the right spot along nerve fibers.
So they wondered if the CASPR2 autoantibodies, by attacking the CASPR2 protein, were preventing the proper placement of the potassium channels, resulting in increased electrical excitability of neurons and pain.
From humans to mice
The team first wanted to confirm that the CASPR2 autoantibodies themselves could actually cause pain.
To do so, the researchers isolated CASPR2 autoantibodies from two patients with autoimmune disease who also had neuropathic pain.
The researchers then treated normal mice each day with these human autoantibodies for two to three weeks, keeping an eye on whether this led to pain in the animals.
And indeed that’s just what they saw: Within 11-15 days the mice became hypersensitive when the researchers poked their paws with a thin filament (a common experimental technique used to study pain in animals). This is called mechanical hypersensitivity.
After ruling out other potential causes of pain, the scientists saw fewer potassium channels in the animals’ nerve cells. So they concluded that the autoantibodies contributed to pain through effects on these channels.
Releasing the brakes makes neurons hyperexcitable
Since the CASPR2 autoantibodies are thought to inhibit the normal function of the CASPR2 protein, the team believed that animals without that protein would also show mechanical hypersensitivity.
To test this idea, the researchers genetically engineered mice so they would lack fully functioning CASPR2 protein. Similar to the normal mice treated with CASPR2 autoantibodies, these animals also had mechanical hypersensitivity to pokes of the paw.
Next, the team took a look at pain neurons from these mice and found that the cells had fewer potassium channels and were also hyperexcitable. They also saw that the channels were redistributed along nerve fibers away from their normal location.
Similarly, in other experiments, the team saw that the CASPR2 autoantibodies decreased the number of potassium channels, and increased cell excitability, when they were applied to neurons from normal mice.
With this evidence, the group concluded that the CASPR2 autoantibodies released the brakes that the channels normally apply to nerve cells, causing increased electrical excitability of neurons, with subsequent pain.
What about other types of pain?
For the wider population of people with pain who don’t have CASPR2 autoantibodies but may still have hyperexcitable neurons, the study casts the CASPR2 protein itself in an interesting light: Perhaps increasing the amount of CASPR2 protein could decrease the excitability of neurons and in doing so reduce pain.
To read about the research in more detail, see the related IASP Pain Research Forum news story below.
Author: Nathan Fried is a postdoctoral fellow at the University of Pennsylvania, Philadelphia, US.
I had already carefully annotated this more technical version of the research below before I looked at the article above, so here it is, with many more details:
A Pathogenic Autoantibody Causes Pain by Releasing the Brakes on Sensory Neurons – by Nathan Fried on 3 Apr 2018
Patients who develop autoimmunity to contactin-associated protein-like 2 (CASPR2), a neuronal adhesion protein, share a number of clinical features, with neuropathic pain being one of them.
Now, new research demonstrates that human autoantibodies against CASPR2 are pathogenic in mice, causing pain through an increase in sensory neuron excitability.
Led by David Bennett, University of Oxford, UK, a team of scientists across multiple universities finds that:
treating mice with CASPR2 autoantibodies from human patients, or genetically knocking out full-length CASPR2 protein in the animals, enhances mechanical pain sensitivity by decreasing proper localization of Kv1 potassium channels along peripheral nerve fibers.
These findings illustrate that an autoimmune, peripheral neuropathic pain disorder can be passively transferred from one organism to another.
“Injection of patient-derived sera into mice, for the first time, recapitulated a neuropathic pain state experienced by the human patient donors,” writes Camilla Svensson, Karolinska Institute, Stockholm, Sweden, and colleagues in a “Preview” accompanying the paper.
“The fact that autoantibodies alone can directly cause a channelopathy is impressive, but showing that patient-derived autoantibodies could cause this in mice is quite a leap forward for our ability to understand and treat disease.”
Autoantibodies and pain
The immune system is intricately linked to chronic pain. When an injury or illness occurs, immune cells release substances that fight off infection and promote healing. They also sensitize pain neurons to promote tissue-protective behaviors (Pinho-Ribeiro et al., 2017).
The adaptive immune system produces antibodies that tag invading pathogens so they can be identified and destroyed. However, with autoimmune disorders, the immune system mistakes proteins of the host organism for those pathogens and produces antibodies against them. These “self-antibodies,” or autoantibodies, lead to a range of symptoms depending on the protein they target. But the antibody contribution to pain has often been overlooked (see PRF related news story).
“Antibodies and pain have been relatively ignored. There’s a lot of literature about how macrophages or microglia are involved in pain, but not so much on how antibody-mediated disorders can promote pain,” explains Bennett.
CASPR2 antibodies are pathogenic
The group first tested whether CASPR2 autoantibodies (CASPR2-Abs) could actually cause pain.
The researchers isolated antibodies from two patients who had high serum levels of CASPR2-Abs and were experiencing neuropathic pain.
First author John Dawes and colleagues treated wild-type mice daily with purified CASPR2-Abs from either patient 1 for 14 days or from patient 2 for 22 days, while assessing mechanical sensitivity with von Frey hairs. “We were looking for any indication these antibodies were pathogenic and causal of pain in the patients,” according to Dawes.
Mice treated with CASPR2-Abs from patient 1 developed mechanical hypersensitivity by day 11, and animals treated with CASPR2-Abs from patient 2 developed it by day 15. To control for nonspecific effects, the investigators treated other sets of mice with antibodies from healthy donors who lacked CASPR2-Abs and confirmed that no hypersensitivity was present. These experiments showed that an autoimmune, peripheral neuropathic pain disorder could be passively transferred.
“We show for the first time that these antibodies can be directly pathogenic and cause pain” in mice, said Dawes.
No inflammation or nerve injury, but still pain
It’s possible that introduction of a foreign antibody caused neuroinflammation that sensitized the mice. To rule this out, the team examined the peripheral and central nervous systems for signs of inflammation.
There was no increase in markers
Critically, the researchers found no trace of CASPR2-Abs in the spinal cord, suggesting they didn’t pass through the blood-brain barrier. Instead, CASPR2-Abs coated the cell bodies of DRG neurons.
Using intra-epidermal nerve fiber density in the paw as a measure of nerve damage and examining peripheral nerve structure with electron microscopy, they found no abnormalities in the nerves of antibody-treated mice.
However, what they did see in these animals, here with immunostaining, was a decrease in both CASPR2 protein and Kv1 along the sciatic nerve. “With no inflammation or nerve damage,” said Dawes, “it seemed likely that CASPR2-Abs were instead working through a novel mechanism involving Kv1 channels.”
Releasing the brakes
The team also looked at how the loss of full-length CASPR2 and a decrease in Kv1 expression affected sensory neuron excitability in a series of electrophysiological and imaging experiments.
The researchers explored physiological changes in the cell bodies of cultured DRG neurons from mice lacking full-length CASPR2
Here, too, small- and medium-diameter neurons showed increased excitability. Use of a potassium channel blocker revealed that this hyper-responsiveness was likely due to the loss of potassium channel function.
“We do see hyperexcitability in other cell types when recording from the cell body, so I suspect that potassium channels are having a different effect depending on the compartment of the neuron you look at,” said Bennett
Finally, the investigators tested whether treatment with CASPR2-Abs could cause changes in neuronal excitability similar to those seen in mice lacking full-length CASPR2 protein.
Given that CASPR2-Abs bound the cell bodies of DRG neurons, the team concluded that CASPR2-Abs likely enhanced pain by regulating Kv1 trafficking to the cell bodies and axons of sensory neurons.
rom a mechanistic perspective, the current study lays the foundation for using immune therapy to treat patients who have CASPR2-Abs and neuropathic pain.
For the wider population of people with chronic pain, the research also casts CASPR2 itself as an interesting therapeutic target. By increasing expression of this protein, it may be possible to decrease neuronal excitability.
Ultimately, Bennett hopes the findings encourage researchers to pay more attention to antibody-induced pain.
Author: Nathan Fried is a postdoctoral fellow at the University of Pennsylvania, Philadelphia, US.