Extracellular MicroRNA Emerges as Neuromodulator, Pain Signal
Tiny, non-coding bits of RNA, once mistaken for cellular debris, have emerged in the past decade as key regulators of gene expression and, more recently, as potential extracellular messengers. Now, work from Ru-Rong Ji and colleagues at Duke University Medical Center, Durham, North Carolina, US, shows a provocative new role for microRNA (miRNA) as a novel activator of pain-sensing neurons. When applied directly to neurons, the miRNA rapidly evoked action potentials, and in mice, let-7b acted as a pain mediator. The paper appeared April 2 in Neuron.
“I think that we are moving from the protein era to a new RNA era,” said Ji. “These findings could open a new field of RNA communication—not just in pain but in neurobiology altogether,” he told PRF.
Because RNA is vulnerable to rapid degradation outside of cells, researchers long assumed that its actions were confined to the inside of cells. But recently, research has shown that extracellular miRNAs travel far and wide in bodily fluids, protected in tiny vesicles called exosomes or bound to protein complexes. Efforts are underway to understand how miRNA-containing exosomes might be used as biomarkers or even therapies for disease.
“This very exciting work beautifully bridges the biology of nociceptors with clinical findings,” said Rohini Kuner, a pain researcher at Heidelberg University, Germany, who was not involved in the new work. Kuner was referring to a growing body of literature in which extracellular miRNAs have been detected in various disease states, including pain. For example, previous research has linked levels of several miRNAs in blood with symptoms in people with complex regional pain syndrome (Orlova et al., 2011).
In the new work, Ji and colleagues homed in on lethal-7b (let-7b), an abundant miRNA that is known to activate the immune receptor Toll-like receptor 7 (TLR7; Lehmann et al., 2012). Previous work from the Ji lab had shown that TLR7 is present in a subset of nociceptors that also contain the transient receptor potential A1 (TRPA1) and V1 (TRPV1) channels (Liu et al., 2010), and that synthetic TLR7 ligands evoked excitatory currents in these cells. “We wanted to find the endogenous ligand for TLR7 on nociceptors,” explained Ji. Based on the findings, one such ligand appears to be let-7b.
In the study, co-first authors Chul-Kyu Park, Zhen-Zhong Xu, and Temugin Berta investigated the effects of applying “naked” extracellular let-7b to isolated dorsal root ganglia (DRG) neurons. They found that in some neurons, let-7b application induced excitatory inward ion currents and, astonishingly, caused the cells to fire action potentials.
The response to let-7b required both TLR7 and TRPA1, the researchers found. DRG neurons from mice lacking either TLR7 or TRPA1 did not display let-7b-evoked currents, while the subset of neurons that fired action potentials in response to let-7b contained both TRPA1 and TLR7, as determined with single-cell polymerase chain reaction (PCR). The activation was specific to let-7b: Currents absolutely required a guanosine-and-uridine (GU)-rich motif found in let-7b and were not induced by a mutant miRNA with reduced GU content.
Unlike previously described actions of let-7b at TLR7, the excitatory currents did not require intracellular signaling. The team tested that by blocking activity of myeloid differentiation factor 88 (MyD88), an adapter protein associated with TLR7 and required for its canonical activation. Blocking of other intracellular signaling molecules also had no effect on let-7b-activated TRPA1 currents. “This direct modulation [of neuronal activity] by miRNA via TLR7 with such a rapid time course of activation—this is very striking,” said Kuner.
That rapid activation might be mediated by a novel interaction between TLR7 and TRPA1 that the authors detected. Co-immunoprecipitation of TLR7 and TRPA1 co-expressed in HEK293 cells suggested that the receptors form a physical connection. And immunocytochemistry in HEK293 cells expressing TLR7 and TRPA1 showed that the two receptors co-localized with let-7b, both on the cell surface and inside cells, indicating that the miRNA might bind the protein complex. The TRPA1/TLR7 channel complexes were directly activated by let-7b, as the researchers demonstrated with electrophysiological recordings from single TRPA1 channels in HEK293 cells. Single-channel recordings from DRG neurons also indicated that extracellular let-7b activated TRPA1 channels.
Could miRNA signal acute pain?
Once the team had identified let-7b as a rapid activator of TLR7-TRPA1 complexes in nociceptors, they asked whether the miRNA could act as an endogenous pain signal. Activation of DRG neurons with capsaicin or depolarization with potassium chloride caused the cells to release let-7b, suggesting a possibility that the miRNA could act in an autocrine fashion on nociceptors, or perhaps constitute a novel signaling pathway in the DRG itself (for more, see comment below from Marshall Devor). Researchers already knew that miRNAs could be up- or downregulated inside DRG neurons in some pain states, said Kuner. “But to know that it is possible for neurons to release them—that opens up so many other possibilities to study,” she said, such as whether neurons release miRNA from their central terminals in the spinal cord.
To further elucidate the role of let-7b, the team tested the effects of the miRNA in the setting of pain signaling. Formalin, an inflammatory pain mediator, triggers pain-like behaviors when injected into the mouse hindpaw, primarily by activating TRPA1 currents. In DRG neurons, formalin-activated TRPA1 currents were blocked by a let-7b inhibitor. Similar to formalin, intraplantar injection of let-7b in mice elicited pain behaviors that were dose dependent and were prevented by pretreatment with the let-7b blocker.
In their experiments, the researchers applied let-7b at micromolar concentration, much higher than the picomolar level they measured in DRG culture medium. But the authors point out that let-7b likely reaches even higher concentrations locally at its site of release and action. For example, the extracellular concentration of glutamate at nerve terminals has been estimated to reach into the millimolar range.
Although microRNAs have been previously shown to regulate ion channel gene expression, this report provides the first evidence of a direct effect on ion channel activity. “Here, we focused on the non-genomic actions of let-7,” Ji said. It is also possible that let-7 could activate canonical signaling through TLR7 to trigger the production of chemokines and cytokines thought to contribute to inflammatory pain, he added. “I am sure that there will be genetic effects important to the chronic phase,” Ji said, “whereas this coupling to TRPA1 seems to be especially important in the acute phase.”
The researchers found that let-7b was released from DRG neurons themselves upon excitation, but it could also potentially come from other sources during inflammation or injury, postulated Ji. Although let-7b is highly enriched in DRG neurons, it is also found in skin cells, which could spill the miRNA upon tissue injury.
The work from the Ji group not only raises the exciting possibility that miRNA works to modulate pain signals, but it also debuts miRNA as a neurotransmitter of sorts. “We need to think about how miRNA can work not just to regulate gene expression but also how it acts as an extracellular signaling molecule, which will have broad implications throughout biology and medicine,” he predicted.
The big picture
Investigations of the role of miRNAs in pain pathways have proliferated in the past four years (for a recent review, see Kress et al., 2013). In some studies, researchers are tracking genomewide miRNA changes in cells throughout the pain pathway, from sensory neurons to emotional circuits. Early indications suggest that each pain state comes with its own specialized changes in miRNA expression, fostering the hope that miRNA profiles can serve as biomarkers of disease states or as therapeutic targets. Last year, for example, Kuner and colleagues profiled miRNA expression in a cancer pain model in mice, discovered a number of novel miRNAs, and validated a new potential therapeutic target (Bali et al., 2013).
In a recent study published March 19 in Molecular Pain, Kuner and Marshall Devor at the Hebrew University of Jerusalem, Israel, used the genomewide sequencing strategy to identify two particular miRNAs that could contribute to the risk for developing pain after nerve injury (Bali et al., 2014). They screened DRG from two genetically related lines of rats that differ in pain response following spinal nerve injury. In order to home in on miRNAs specifically related to neuropathic pain, the authors looked for miRNAs that were differentially regulated between the high-pain and low-pain lines. Of 284 mature miRNAs detected, two abundant miRNAs in particular differed between the lines, making them “prime candidates as novel PNS [peripheral nervous system] determinants of neuropathic pain,” the authors wrote. Supporting that idea, among the messenger RNA targets for the two miRNAs are three pro-nociceptive molecules with major roles in pain physiology—namely, tumor necrosis factor (TNF), brain-derived neurotrophic factor (BDNF), and the pain-related signal transducer and activator of transcription 3 (STAT3). Other genes regulated by the differentially expressed miRNAs may also contribute to the differences in pain between the two lines. The study illustrates yet another way that miRNAs are emerging as powerful determinants of the pain experience, and potential targets for novel treatments.
Stephani Sutherland, PhD, is a neuroscientist, yogi, and freelance writer in Southern California, US.
Image credit: Chul-Kyu Park, Duke University
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