Novel Mechanisms of Ion Channel Regulation Implicated in Neuropathic Pain

Neuropathic pain is thought to arise from injured nerves made hypersensitive in part by increased expression of sodium channels and decreased expression of potassium channels. Two different papers report new molecular players that regulate those ion channels in unexpected ways to foster hypersensitivity to touch and temperature in rodents. The findings may point to new mechanistic targets for understanding neuropathic pain in people.

One study finds that an endogenous long noncoding RNA (lncRNA) in dorsal root ganglion (DRG) neurons can promote allodynia in rat models of nerve injury by silencing the expression of Kcna2, which encodes a potassium channel, Kv1.2. The paper from Yuan-Xiang Tao, Johns Hopkins University School of Medicine, Baltimore, Maryland, US, and colleagues was published online June 23 in Nature Neuroscience.

A second study finds that a nerve injury-induced drop in a ubiquitin protein ligase contributes to allodynia in mouse nerve injury models by increasing the expression and altering the distribution of two sodium channels, Nav1.7 and Nav1.8. The paper from Hugues Abriel, University of Bern, Switzerland, and Isabelle Decosterd, University of Lausanne, Switzerland, appeared online June 17 in the Journal of Clinical Investigation.

The discoveries might someday lead to new treatment strategies for neuropathic pain, say the authors. Each study showed that both the pathological ion channel changes and allodynia can be reversed in animal models by targeting the newfound molecules controlling the action. Other experts contacted by the Pain Research Forum praised the papers for their rigorous methodologies and mechanistic insights, but pointed out critical questions about the translational prospects for the studies.

An endogenous antisense RNA

The study from Tao and co-investigators began five years ago when Tao asked what role, if any, lncRNAs might play in pain. Some lncRNAs, defined as RNAs more than 200 nucleotides long, are transcribed from the noncoding (complementary) DNA strand of genes and can function as negative regulators of gene expression (reviewed in Kung et al., 2013).

Tao's team began by selecting the top 10 genes implicated in neuropathic pain in people, as verified by multiple studies. Then they turned to genetic databases to see which genes might have lncRNA counterparts.

Of the 10 genes, the team found five with antisense lncRNA counterparts, including Kcna2. Co-first authors Xiuli Zhao, Zongxiang Tang, Hongkang Zhang, Fidelis Atianjoh, and Jian-Yuan Zhao and colleagues verified the presence of Kcna2 lncRNAs in rat, mouse, monkey, and human DRG. In rats, the lncRNA was also distributed in other tissues at varying levels. The team found that, in normal conditions, most DRG neurons expressed sense, protein-encoding Kcna2 messenger RNA (mRNA) at high levels. In pathological conditions, injured neurons downregulated the mRNA and upregulated the lncRNA. The findings were confirmed in two nerve injury models, L5 spinal nerve ligation (SNL) and chronic constriction injury (CCI).

In the SNL model, the researchers found a sustained increase in the Kcna2 lncRNA in injured neurons starting at day three and lasting at least two weeks after injury, with an inversely proportional decrease in Kcna2 mRNA. The drop was consistent with previous studies showing that Kcna2 mRNA and protein are downregulated on the same timetable after injury in several neuropathic pain animal models.

The researchers traced the lncRNA rise to an increase in myeloid zinc finger protein 1 (MZF1), a transcriptional activator that binds to the Kcna2 antisense RNA gene promoter. Ramping up MZF1-dependent Kcna2 expression in normal conditions mimicked the effects of nerve injury on Kcna2 lncRNA expression.

Next, the team explored the impact of Kcna2 lncRNAs on the potassium channels. In a human embryonic kidney cell line and rat DRG neurons, overexpression of the lncRNA specifically and selectively decreased the levels of mRNA and protein for the Kcna2 channel. As a result, the neurons showed reduced potassium channel currents and became more excitable.

"The action potential increases, which means DRG neurons are more prone to hyperexcitability," Tao said. In seminars, he uses a familiar Chinese yin yang symbol to illustrate how the relative expression levels of the Kcna2 transcripts change from the normal mRNA (yang) to the more pathological lncRNA (yin). The downregulated potassium channels in the primary sensory neurons prevent a stimulated nerve from calming down and may cause a surge in neurotransmitters and lead to spinal central sensitization, contributing to chronic neuropathic pain, Tao and his co-authors speculate.

Behavioral results support that idea. When normal rat DRG were injected with a viral vector that overexpressed lncRNA, the rats exhibited a lower threshold to mechanical and cold stimuli compared to controls. The phenomena mimic two major clinical symptoms of neuropathy—mechanical and cold allodynia—in people, Tao said.

Finally, through many months of trial and error, the researchers developed a way to block lncRNA transcription with a sense fragment complementary to the lncRNA at its 3' end. In both the SNL and CCI rat models, they were able to prevent the onset of hypersensitivity to touch, cold, and heat, as well as reverse established hypersensitivity.

"We think there is clinical potential in targeting this novel mechanism of neuropathic pain involving antisense RNA," said Tao, perhaps through viral vectors being developed to deliver the therapeutic gene products to neurons (Simonato et al., 2013).

"The new paper is significant because it shows yet another example of an antisense transcript that is playing a functional role in regulating gene expression," said Jeannie Lee, a geneticist at Massachusetts General Hospital and Harvard Medical School, Boston, US, and an expert in lncRNA. "We can expect to see more," said Lee, who was not involved in the study and predicted that the Kcna2 lncRNA was one of thousands of transcripts formerly known as "junk" that may influence pain-sensing fibers. In most cells, about 80 to 90 percent of the genome is transcribed, Lee said, and only about 2 percent of that comes from protein-coding regions.

"This study proposes a novel mechanism of neuropathic pain," said Ru-Rong Ji, a neurobiologist at Duke University Medical Center in Raleigh, North Carolina, US. "It will be of general interest to the field to know whether lncRNAs can also regulate the expression of other genes to control pain sensitivity. For example, many types of potassium channels are known to be downregulated after nerve injury. It will be important to figure out how many of these downregulations are mediated by long noncoding RNAs and whether targeting one type of potassium channel is sufficient to alleviate neuropathic pain in human beings." (See full comment from Ji below).

A post-translational regulator of sodium channels

The paper from Abriel, Decosterd, and colleagues also posits a new player in neuropathic pain, using a spared-nerve injury (SNI) model in mice. The researchers show that reduced levels of the ubiquitin ligase NEDD4-2, as a result of nerve injury or genetic knockout, leads to increased expression and activity of the Nav1.7 and Nav1.8 sodium channels, two key regulators of pain signaling (for review, see Dib-Hajj et al., 2012). Restoring NEDD4-2 function in the animals decreased the expression of the sodium channels and alleviated the mechanical allodynia usually seen after SNI.

NEDD4-2 works by attaching ubiquitin moieties to lysine residues in the proteins, triggering a common way for cells to recycle or degrade proteins that are dysfunctional or no longer needed. A previous study (Moss et al., 2002) had implicated ubiquitination-dependent mechanisms in neuropathic pain, and Abriel’s group and others had shown that NEDD4-2 ubiquitinates Nav1.7 and other sodium channels (Fotia et al., 2004; van Bemmelen et al., 2004). And Laedermann and colleagues had previously shown that NEDD4-2 was expressed in all cells, but especially in small and medium DRG neurons (Cachemaille et al., 2012).

In the latest paper, the team explored the function of NEDD4-2 in DRG. In one key experiment, the group showed that a decrease in NEDD4-2 in mouse DRG after nerve injury was sufficient to increase Nav1.7 and Nav1.8 expression. NEDD4-2 knockout animals demonstrated similar heat hypersensitivity as the SNI mice, compared to controls. In another test, the knockout animals showed increased persistent pain behaviors in the formalin injection model, which suggests that removing NEDD4-2 from the periphery will also impact processes of central sensitization, Laedermann said.

To ask whether the loss of NEDD4-2 caused the increase in Nav1.7 and Nav1.8, the researchers overexpressed NEDD4-2 in DRG after nerve injury by using a viral vector that preferentially infects small neurons (Towne et al., 2009). That led to a decrease of Nav1.7 and Nav1.8 current amplitudes, as measured by electrophysiological recording of the neurons, and alleviated the mechanical allodynia induced by SNI. Interestingly, in uninjured animals, the extra NEDD4-2 delivered in the viral vector did not appear to modify the basal sensitivity of the animals.

Increasing NEDD4-2 expression "is an alternative way to look at how to reduce the activity of those ion channels," said Abriel, who noted that therapeutics such as lidocaine for pain and anti-arrhythmia drugs now target overabundant sodium channels directly. "Here you can target the ion channels by regulating their number." It will also be necessary to target the neuron directly because of the other proteins known to be regulated by NEDD4-2, Laedermann added. The team is now investigating what happens to the sodium channels after they are ubiquitinated and internalized from the cell membrane.

The finding is an important mechanistic insight, commented John Wood, a neuroscientist at University College London, UK, by e-mail. "This paper further emphasizes the importance of sodium channels as analgesic targets," wrote Wood, who nonetheless expressed skepticism about NEDD4-2 as a clinical approach to treating pain because of the widespread presence of NEDD4-2 in the body and potentially widespread side effects. “The idea that a ubiquitin ligase could be a target for intervention in pain is not very appealing, as this enzyme has many other functions.” (See full comment from Wood below.)

Top image: Kcna2 expression in DRG neurons. Reprinted by permission from Macmillan Publishers Ltd: Nature Neuroscience, advance online publication, 23 June 2013 (doi: 10.1038/nn.3438).

Carol Cruzan Morton covers science, health, and the environment, and is based near Boston, Massachusetts, US.