Atsushi Sakai

Long non–coding RNAs (lncRNAs), which are generally defined as non–coding RNAs that are longer than 200 nucleotides, have recently emerged as an important regulator of pain. Most lncRNAs exhibit tissue– or even cell type–specific expression, and a large number of lncRNAs are preferentially expressed in the nervous system, suggesting their importance in neurological functions. Accordingly, expression changes of lncRNAs in the somatosensory nervous system have been reported in various pain disorders. lncRNAs affect various gene expression processes, including epigenetic regulation and transcription, through interaction with DNA, RNA and protein at several subcellular compartments. Dysregulation of lncRNAs significantly impacts pain perception by affecting mechanisms relevant to pain, including neuronal excitability and neuroinflammation. Intriguingly, lncRNAs are released into the extracellular space with encapsulated in extracellular vesicles, such as exosome. The extracellular lncRNAs may have a role in regulating nociceptive signaling through intercellular communication, as with other non–coding RNAs such as microRNAs. In addition, extracellular lncRNAs that entered the bloodstream have great potential as diagnostic biomarkers for pain disorders. In this review, we summarize current insights into potential applications of lncRNAs as an analgesic target and a biomarker in pain disorders.

Corticotropin‐releasing factor (CRF) plays roles in stress‐related responses through its type 1 (CRF ₁ ) and type 2 receptors. Both CRF and CRF ₁ are expressed in the rat colon. Peripheral CRF administration and various stressors increase colonic motility and defecation. Stress induces CRF release in the colon, suggesting CRF may mediate stress‐related responses of the colon. The vagal nodose ganglion (NG) transduces visceral information, including colonic sensation, to the brain. However, it remains unclear whether the CRF/CRF ₁ system is involved in vagal afferent functions. This study, therefore, aimed to clarify the involvement of the CRF/CRF ₁ system in relaying visceral sensory information to the brain and the effect of stress exposure on vagal nerve function. The experiments were conducted in male rats. First, CRF ₁ ‐like immunoreactivity (CRF ₁ ‐LI) was characterized in the NG. Second, the effects of vagotomy on CRF ₁ ‐LI in the NG, intraperitoneally administered CRF‐induced fecal output, and c‐Fos expression in the nucleus tractus solitarius (NTS) were evaluated. Subsequently, a fast blue retrograde tracer was microinjected into the proximal colon. Finally, we analyzed CRF‐ or stress‐induced phosphorylation of cyclic AMP‐response element‐binding protein (pCREB) in the NG. CRF ₁ mRNA and CRF ₁ ‐LI were detected, and CRF ₁ ‐LI accumulated on the proximal side of the ligated region of the nerve trunk, and CRF ₁ ‐LI was detected in most cholinergic neurons. CRF ₁ siRNA suppressed the expression of CRF ₁ ‐LI in the NG. Subdiaphragmatic vagotomy decreased the number of CRF ₁ ‐positive cells in the NG while it did not affect CRF‐induced fecal output. CRF‐induced c‐Fos expression in the NTS was suppressed by vagotomy. A neuronal tracing study showed that approximately half of CRF ₁ ‐positive cells expressed fast blue in the NG. Intraperitoneal CRF, a selective CRF ₁ agonist, or immobilization stress induced pCREB expression and increases in CRF ₁ ‐positive cells in the NG. In contrast, a CRF ₁ antagonist reduced the immobilization‐induced increase in the expression of pCREB in the NG. These results suggest that the CRF/CRF ₁ system is involved in the signal transduction of colonic sensory information to the central nervous system via the NG.

Oxaliplatin, a platinum-based chemotherapeutic agent, frequently causes acute and chronic peripheral sensory neuropathy, for which no effective treatment has been established. In particular, chronic neuropathy can persist for years even after treatment completion, thus worsening patients’ quality of life. To avoid the development of intractable adverse effects, a predictive biomarker early in treatment is awaited. In this study, we explored extracellular long non-coding RNAs (lncRNAs) released from primary sensory neurons as biomarker candidates for oxaliplatin-induced peripheral neuropathy. Because many human-specific lncRNA genes exist, we induced peripheral sensory neurons from human induced pluripotent stem cells. Oxaliplatin treatment changed the levels of many lncRNAs in extracellular vesicles (EVs) released from cultured primary sensory neurons. Among them, the levels of release of lncRNAs that were considered to be selectively expressed in dorsal root ganglia were correlated with those of lncRNAs in plasma EV obtained from healthy individuals. Several lncRNAs in plasma EVs early after the initiation of treatment showed greater changes in patients who did not develop chronic neuropathy that persisted for more than 1 year than in those who did. Therefore, these extracellular lncRNAs in plasma EVs may represent predictive biomarkers for the development of chronic peripheral neuropathy induced by oxaliplatin.

Background: Peripheral nerve injury to dorsal root ganglion (DRG) neurons develops intractable neuropathic pain via induction of neuroinflammation. However, neuropathic pain is rare in the early life of rodents. Here, we aimed to identify a novel therapeutic target for neuropathic pain in adults by comprehensively analyzing the difference of gene expression changes between infant and adult rats after nerve injury. Methods: A neuropathic pain model was produced in neonatal and young adult rats by spared nerve injury. Nerve injury-induced gene expression changes in the dorsal root ganglion (DRG) were examined using RNA sequencing. Thymic stromal lymphopoietin (TSLP) and its siRNA were intrathecally injected. T cells were examined using immunofluorescence and were reduced by systemic administration of FTY720. Results: Differences in changes in the transcriptome in injured DRG between infant and adult rats were most associated with immunological functions. Notably, TSLP was markedly upregulated in DRG neurons in adult rats, but not in infant rats. TSLP caused mechanical allodynia in adult rats, whereas TSLP knockdown suppressed the development of neuropathic pain. TSLP promoted the infiltration of T cells into the injured DRG and organized the expressions of multiple factors that regulate T cells. Accordingly, TSLP caused mechanical allodynia through T cells in the DRG. Conclusion: This study demonstrated that TSLP is causally involved in the development of neuropathic pain through T cell recruitment.

Primary sensory neurons regulate inflammatory processes in innervated regions through neuro-immune communication. However, how their immune-modulating functions are regulated in concert remains largely unknown. Here, we show that Neat1 long non-coding RNA (lncRNA) organizes the proinflammatory gene expressions in the dorsal root ganglion (DRG) in chronic intractable neuropathic pain in rats. Neat1 was abundantly expressed in the DRG and was upregulated after peripheral nerve injury. Neat1 overexpression in primary sensory neurons caused mechanical and thermal hypersensitivity, whereas its knockdown alleviated neuropathic pain. Bioinformatics analysis of comprehensive transcriptome changes indicated the inflammatory response was the most relevant function of genes upregulated through Neat1. Consistent with this, upregulation of proinflammatory genes in the DRG following nerve injury was suppressed by Neat1 knockdown. Expression changes of these proinflammatory genes were regulated through Neat1-mRNA interaction-dependent and -independent mechanisms. Notably, Neat1 increased proinflammatory genes by stabilizing its interacting mRNAs in neuropathic pain. Finally, Neat1 in primary sensory neurons contributed to spinal inflammatory processes that mediated peripheral neuropathic pain. These findings demonstrate that Neat1 lncRNA is a key regulator of neuro-immune communication in neuropathic pain.

MicroRNAs (miRNAs) are extracellularly released by cells for intercellular communication, while intracellularly, they inhibit the expression of specific genes. An increasing number of studies suggest that extracellular miRNAs have great potential as both therapeutic targets and disease-specific biomarkers in a variety of diseases, including pain disorders. However, little is known about miRNA release from dorsal root ganglion (DRG) neurons in neuropathic pain caused by peripheral nerve injury. In this study, we investigated the changes in the extracellular release of miRNAs from DRG neurons in a rat model of neuropathic pain induced by chronic constriction injury of the sciatic nerve. We found increased release of six miRNAs (let-7d, miR-21, miR-142-3p, miR-146b, miR-203-3p and miR-221) from primary cultured DRG neurons prepared from rats 7 days after nerve injury. Among these, miR-221 was also increased in serum from days 7 to 28 after nerve injury. In contrast, serum miR-221 levels and its release from DRG neurons were unchanged in an inflammatory pain model produced by intraplantar injection of complete Freund’s adjuvant. These results suggest that the increased release of specific miRNAs by DRG neurons may be involved in the pathophysiology of neuropathic pain through extracellular as well as intracellular mechanisms. Furthermore, serum miR-221 may be useful as a biomarker of neuropathic pain caused by peripheral nerve injury.

Neuropathic pain is chronic pain caused by damage of somatosensory system. However, existing analgesics are insufficient in effectiveness and have serious adverse effects. Although non-coding RNAs, especially microRNA, have been increasingly recognized as a critical modulator for neuropathic pain, involvement of long non-coding RNA (lncRNA) that is longer than 200 nucleotides is poorly understood. Here, we report a role of Neat1 lncRNA in the neuropathic pain. A rat neuropathic pain model was produced by ligation of the lumbar fifth spinal nerve. RNA sequencing of the dorsal root ganglion (DRG) revealed Neat1 was the most highly expressed among putative lncRNAs and was significantly upregulated in the injured DRG. Neat1 knockdown using AAV vector expressing shRNA alleviated mechanical allodynia and thermal hyperalgesia, and blocked expression changes in multiple genes related to inflammatory process or neuronal function. Many of these mRNAs were predicted to interact directly with Neat1. Neat1 knockdown in the DRG suppressed spinal microglial activation and elevated expressions of several pro-inflammatory cytokines after nerve injury. Neat1 may contribute to neuroinflammation in neuropathic pain through direct and indirect interaction with inflammatory genes.