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. But the underlying mechanisms behind this phenomenon have remained a mystery. 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.
“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, University of Liverpool, UK, who was not involved in the study. “We knew some things about how autoantibodies can cause pain, but not to this level of detail.”
“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.”
The new research and accompanying Preview were published in the February 21 issue of Neuron.
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.
For some autoimmune disorders such as Morvan’s syndrome or neuromyotonia, both of which feature hyperexcitability of peripheral nerves and thus spontaneous muscular activity, the body produces autoantibodies against CASPR2, a protein that localizes in specialized domains of myelinated neurons. Although these disorders present with their own unique set of symptoms, neuropathic pain is common among them (Klein et al., 2012).
CASPR2 localizes a voltage-gated potassium channel called Kv1 to the juxtaparanode of myelinated axons, where the channel remains electrically inactive. Following injury, however, the channels are relocated to the paranode, where they can suppress hyperexcitability (see figure below). In the current paper, the authors found that both CASPR2 and Kv1 are also expressed in the cell bodies of sensory neurons.
“These potassium channels essentially put the brake on neuronal excitability, and in their absence neurons become hyperexcitable,” explains Bennett.
When Bennett first moved to the University of Oxford, he had a conversation with coauthor Angela Vincent about a unique observation she made in her patients. “She told me that, of her patients with CASPR2 autoantibodies, many seemed to experience pain,” he said.
This led the two to wonder if these patients experienced neuropathic pain because the CASPR2 autoantibodies caused mislocalization of Kv1 channels, resulting in hyperexcitability of sensory neurons.
CASPR2 antibodies are pathogenic
The group first tested whether CASPR2 autoantibodies (CASPR2-Abs) could actually cause pain. “We used a classic passive immunization paradigm where you take an antibody you think is pathogenic and put it into an animal,” according to Bennett.
The researchers isolated antibodies from two patients who had high serum levels of CASPR2-Abs and were experiencing neuropathic pain. “We chose these patients because neuropathic pain was a big component of their clinical presentation,” said Bennett.
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 for neutrophils, macrophages, lymphocytes, or inflammatory cytokines within the dorsal root ganglion (DRG), and no elevation in markers for neutrophils, lymphocytes, or activated astrocytes in the spinal cord. 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.
“We then wanted to see whether the antibodies caused damage to the peripheral nervous system, which could also cause a general neuropathy,” said Dawes.
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.”
Next, the team obtained mice genetically engineered to lack full-length CASPR2. Instead, these mice only expressed a version of the protein missing most of its extracellular portion, a modification that would interfere with its ability to interact with Kv1 channels.
Similar to the mice treated with patient-derived CASPR2-Abs, these animals were hypersensitive to mechanical stimulation. They also featured enhanced pain behaviors in response to hind paw injection of either capsaicin or formalin, chemicals that result in inflammation and activate pain neurons.
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.
Using in vivo calcium imaging to directly observe cell body activity of sensory neurons in anesthetized animals while mechanically stimulating the hind paw, they found hyperexcitability in small- and medium-diameter neurons, which transmit nociceptive signals.
The researchers explored physiological changes in the cell bodies of cultured DRG neurons from mice lacking full-length CASPR2 using patch-clamp electrophysiology. 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.
The group then turned to in vivo extracellular recordings of spinal cord neurons in anesthetized mice to examine changes of sensory neuron integration into the spinal cord. This, too, revealed hyperexcitability in response to mechanical stimulation of the paw with von Frey hairs.
Next, use of a tibial nerve-skin ex vivo preparation allowed for recording from the axons of tibial nerve fibers during mechanical stimulation of the paw. Considering known electrophysiological properties and using various mechanical stimulation parameters to categorize sensory neuron subtypes, the authors found that only D-hair afferents were hyperexcitable when recording at the axon; these afferents are a type of low-threshold, A-delta mechanoreceptor that forms lanceolate endings around hair follicles in the skin and play a role in pain under pathological conditions.
“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. Patch-clamp electrophysiology at the level of the cell body showed that treating cultured sensory neurons from normal mice with patient-derived, CASPR2-Ab-containing plasma for 24 hours also increased neuronal excitability and decreased Kv1 expression.
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.
Wider implications?
From a mechanistic perspective, the current study lays the foundation for using immune therapy to treat patients who have CASPR2-Abs and neuropathic pain. However, the patients in the new work had very high levels of CASPR2-Abs and were already going to be treated for their other neurological signs with immune therapy in order to decrease systemic levels of the antibodies.
“Clinically, treatment for these patients may not yet change much,” said Goebel. “More studies are needed to determine whether a strategy of immune modulation will work for patients with lower levels of 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.
“We should be considering antibodies in the generation of neuropathic pain much more,” he said. “As we show here, modulation of CASPR2 levels can have quite a big impact on sensory neuron hyperexcitability.”
Nathan Fried is a postdoctoral fellow at the University of Pennsylvania, Philadelphia, US.
