cAMP-dependent protein kinase regulates desensitization of the capsaicin receptor (VR1) by direct phosphorylation

The capsaicin receptor, VR1 (also known as TRPV1), is a ligand-gated ion channel expressed on nociceptive sensory neurons that responds to noxious thermal and chemical stimuli. Capsaicin responses in sensory neurons exhibit robust potentiation by cAMP-dependent protein kinase (PKA). In this study, we demonstrate that PKA reduces VR1 desensitization and directly phosphorylates VR1. In vitro phosphorylation, phosphopeptide mapping, and protein sequencing of VR1 cytoplasmic domains delineate several candidate PKA phosphorylation sites. Electrophysiological analysis of phosphorylation site mutants clearly pinpoints Ser116 as the residue responsible for PKA-dependent modulation of VR1. Given the significant roles of VR1 and PKA in inflammatory pain hypersensitivity, VR1 phosphorylation at Ser116 by PKA may represent an important molecular mechanism involved in the regulation of VR1 function after tissue injury.     

Parathyroid hormone 2 receptor is a functional marker of nociceptive myelinated fibers responsible for neuropathic pain

We have previously demonstrated that parathyroid hormone 2 (PTH2) receptors are expressed in dorsal root ganglion (DRG) neurons and that its endogenous agonist tuberoinfundibular peptide of 39 residues (TIP39) causes nociceptive paw flexor responses after intraplantar administration. Here we found that the PTH2 receptor is selectively localized on myelinated A‐, but not unmyelinated C‐fibers using immunohistochemical labeling, based on PTH2 receptor expression on antibody N52‐positive medium/large‐sized DRG neurons, but not on TRPV1, substance P, P2X₃ receptor or isolectin B4‐binding protein‐positive small‐sized DRG neurons. Pharmacological studies showed that TIP39‐induced nociceptive responses were mediated by activation of G_s and cAMP‐dependent protein kinase. We also found that nociceptive responses induced by TIP39‐ or the cAMP analog 8‐bromo‐cAMP were significantly greater following partial sciatic nerve injury induced neuropathic pain, without changes in PTH2 receptor expression. Together these data suggest that activation of PTH2 receptors stimulates nociceptive A‐fiber through G_s‐cAMP‐dependent protein kinase signaling, and this pathway has elevated sensitization following nerve injury.     

Early painful diabetic neuropathy is associated with differential changes in the expression and function of vanilloid receptor 1

Diabetes mellitus is associated with one or more kinds of stimulus-evoked pain including hyperalgesia and allodynia. The mechanisms underlying painful diabetic neuropathy remain poorly understood. Previous studies demonstrate an important role of vanilloid receptor 1 (VR1) in inflammation and injury-induced pain. Here we investigated the function and expression of VR1 in dorsal root ganglion (DRG) neurons isolated from streptozotocin-induced diabetic rats between 4 and 8 weeks after onset of diabetes. DRG neurons from diabetic rats showed significant increases in capsaicin- and proton-activated inward currents. These evoked currents were completely blocked by the capsaicin antagonist capsazepine. Capsaicin-induced desensitization of VR1 was down-regulated, whereas VR1 re-sensitization was up-regulated in DRG neurons from diabetic rats. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate blunted VR1 desensitization, and this effect was reversible in the presence of the PKC inhibitor bisindolylmaleimide I. Compared with the controls, VR1 protein was decreased in DRG whole-cell homogenates from diabetic rats, but increased levels of VR1 protein were observed on plasma membranes. Of interest, the tetrameric form of VR1 increased significantly in DRGs from diabetic rats. Increased phosphorylation levels of VR1 were also observed in DRG neurons from diabetic rats. Colocalization studies demonstrated that VR1 expression was increased in large myelinated A-fiber DRG neurons, whereas it was decreased in small unmyelinated C-fiber neurons as a result of diabetes. These results suggest that painful diabetic neuropathy is associated with altered cell-specific expression of the VR1 receptor that is coupled to increased function through PKC-mediated phosphorylation, oligomerization, and targeted expression on the cell surface membrane.     

Regulation of galanin and galanin receptor 2 expression by capsaicin in primary cultured dorsal root ganglion neurons

Galanin is a 29-amino-acid neuropeptide expressed in dorsal root ganglion (DRG) neurons which is thought to play a role in modulation of nociception in neuropathic states. Activation of galanin receptor 2 (GalR2) plays a pronociceptive role and enhances capsaicin-induced nociception in the periphery. GalR2 and vanilloid receptor 1 (VR1) are co-expressed in DRG neurons. Capsaicin evokes acute pain via activation of VR1 expressed in primary sensory neurons. It is not known to what extent galanin and its receptor GalR2 expression is regulated by capsaicin in DRG neurons. Effects of acute (4 h) or chronic (4 d) treatment with capsaicin at different concentrations (0.01, 0.1, 1 micromol/L) on galanin and GalR2 expression in primary cultured DRG neurons were investigated in the present study. Our results showed that acute exposure of high concentration capsaicin (1 micromol/L) increased galanin expression, whereas chronic exposure of low concentration capsaicin (0.01, 0.1 micromol/L) promoted galanin expression. Only chronic exposure of 0.1 micromol/L concentration capsaicin could elevate GalR2 expression, whereas capsaicin did not have this effect at any other conditions in this experiment. These results indicated that certain concentrations or exposure time of capsaicin stimulation may be relevant to upregulation of galanin and its receptor GalR2 expression in DRG cultures suggesting a response to peripheral neuronal stimulation. And also, capsaicin-induced GalR2 expression may be also modulated by capsaicin-induced galanin expression. The possible significance of the neurotransmission of nociceptive information involved in galanin or GalR2 expression caused by capsaicin is still to be clarified.     

Expression and function of proton-sensing G-protein-coupled receptors in inflammatory pain

Background

Chronic inflammatory pain, when not effectively treated, is a costly health problem and has a harmful effect on all aspects of health-related quality of life. Despite the availability of pharmacologic treatments, chronic inflammatory pain remains inadequately treated. Understanding the nociceptive signaling pathways of such pain is therefore important in developing long-acting treatments with limited side effects. High local proton concentrations (tissue acidosis) causing direct excitation or modulation of nociceptive sensory neurons by proton-sensing receptors are responsible for pain in some inflammatory pain conditions. We previously found that all four proton-sensing G-protein-coupled receptors (GPCRs) are expressed in pain-relevant loci (dorsal root ganglia, DRG), which suggests their possible involvement in nociception, but their functions in pain remain unclear.

Results

In this study, we first demonstrated differential change in expression of proton-sensing GPCRs in peripheral inflammation induced by the inflammatory agents capsaicin, carrageenan, and complete Freund's adjuvant (CFA). In particular, the expression of TDAG8, one proton-sensing GPCR, was increased 24 hours after CFA injection because of increased number of DRG neurons expressing TDAG8. The number of DRG neurons expressing both TDAG8 and transient receptor potential vanilloid 1 (TRPV1) was increased as well. Further studies revealed that TDAG8 activation sensitized the TRPV1 response to capsaicin, suggesting that TDAG8 could be involved in CFA-induced chronic inflammatory pain through regulation of TRPV1 function.

Conclusion

Each subtype of the OGR1 family was expressed differently, which may reflect differences between models in duration and magnitude of hyperalgesia. Given that TDAG8 and TRPV1 expression increased after CFA-induced inflammation and that TDAG8 activation can lead to TRPV1 sensitization, it suggests that high concentrations of protons after inflammation may not only directly activate proton-sensing ion channels (such as TRPV1) to cause pain but also act on proton-sensing GPCRs to regulate the development of hyperalgesia.     

Anandamide activates vanilloid receptor 1 (VR1) at acidic pH in dorsal root ganglia neurons and cells ectopically expressing VR1

The vanilloid receptor type 1 (VR1) is a heat-activated ionophore preferentially expressed in nociceptive neurons of trigeminal and dorsal root ganglia (DRG). VR1, which binds and is activated by capsaicin and other vanilloid compounds, was noted to interact with the endocannabinoid anandamide (ANA) and certain inflammatory metabolites of arachidonic acid in a pH-dependent manner. At pH < or = 6.5 ANA induced (45)Ca(2+) uptake either in primary cultures of DRG neurons or cells ectopically expressing C-terminally tagged recombinant forms of VR1 with an EC(50) = approximately 10 microm at pH 5.5. Capsazepine, a potent antagonist of vanilloids, inhibited ANA-induced Ca(2+) transport in both cell systems. Vanilloids displaced [(3)H]ANA in VR1-expressing cells, suggesting competition for binding to VR1. Ratiometric determination of intracellular free calcium and confocal imaging of the VR1-green fluorescent fusion protein revealed that, at low pH (< or =6.5), ANA could induce an elevation of intracellular free Ca(2+) and consequent intracellular membrane changes in DRG neurons or transfected cells expressing VR1. These actions of ANA were similar to the effects determined previously for vanilloids. The ligand-induced changes in Ca(2+) at pH < or = 6.5 are consistent with the idea that ANA and other eicosanoids act as endogenous ligands of VR1 in a conditional fashion in vivo. The pH dependence suggests that tissue acidification in inflammation, ischemia, or traumatic injury can sensitize VR1 to eicosanoids and transduce pain from the periphery.     

Role of sodium ferulate in the nociceptive sensory facilitation of neuropathic pain injury mediated by P2X(3) receptor

Neuropathic pain usually is persistent and no effective treatment. ATP plays an important role in the initiation of pain. P2X(3) receptors are localized in the dorsal root ganglion (DRG) neurons and activated by extracellular ATP. Sodium ferulate (SF) is an active principle from Chinese herbal medicine and has anti-inflammatory activities. This study observed the effects of SF on the nociceptive facilitation of the primary sensory afferent after chronic constriction injury (CCI) mediated by P2X(3) receptor. In this study, the content of ATP in DRG neurons was measured by high-performance liquid chromatography (HPLC). P2X(3) agonist-activated currents in DRG neurons was recorded by the whole-cell patch-clamp skill. The expression of P2X(3) mRNA in DRG neurons was analyzed by in situ hybridization. The ATP content of DRG was increased after CCI. In CCI rats treated with SF, the content of ATP in DRG neurons was reduced. SF decreased the increment of P2X(3) agonist-activated currents and P2X(3) mRNA expression in DRG neurons during CCI. SF may inhibit the initiation of pain and primary afferent sensitization mediated by P2X(3) receptor during CCI.     

Distribution and immunocytochemical characterization of dorsal root ganglion neurons innervating the lumbar intervertebral disc in rats: a review

Previously, it was believed that the lumbar intervertebral disc was innervated segmentally by dorsal root ganglion (DRG) neurons via the sinuvertebral nerves. Recently, it was demonstrated using retrograde tracing methods that the lower disc (L5-L6) is innervated predominantly by upper (L1 and L2) DRG neurons via the sympathetic trunks. Furthermore, we investigated the expression of various pain-related molecules such as calcitonin gene-related peptide (CGRP), isolectin B4 (IB4), P2X(3) receptor and vanniloid receptor 1 (VR1) in DRG neurons innervating the disc using a combination of immunostaining with the retrograde tracing method. This review outlines the distribution and immunocytochemical characterization of DRG neurons innervating the disc. Small nociceptive DRG neurons are classified into nerve growth factor (NGF)-dependent neurons and glial cell line-derived neurotrophic factor (GDNF)-dependent neurons and they can be distinguished by their reactivity for CGRP and IB4, respectively. We found that about half of the neurons innervating the disc were CGRP-immunoreactive (-ir), whilst, only 0.6% of the DRG neurons were IB4-positive, thereby indicating that NGF-dependent neurons are the main subpopulation which transmits and modulates nociceptive information from the disc. In addition, we also demonstrated P2X(3)- and VR1-immunoreactivity in DRG neurons innervating the disc and noted that they were mainly localized in NGF-dependent neurons. It is well known that NGF has sensitizing effects on DRG neurons, with a recent study demonstratng the presence of NGF in the painful intervertebral disc. Therefore, it is suggested that NGF is involved in the generation of discogenic low back pain.     

Fascin-1 Contributes to Neuropathic Pain by Promoting Inflammation in Rat Spinal Cord

Neuropathic pain is a complicated clinical syndrome caused by heterogeneous etiology. Despite the fact that the underlying mechanisms remain elusive, it is well accepted that neuroinflammation plays a critical role in the development of neuropathic pain. Fascin-1, an actin-bundling protein, has been proved to be involved in the processing of diverse biological events including cellular development, immunity, and tumor invasion etc. Recent studies have shown that Fascin-1 participates in antigen presentation and the regulation of pro-inflammatory agents. However, whether Fascin-1 is involved in neuropathic pain has not been reported. In the present study we examined the potential role of Fascin-1 by using a rodent model of chronic constriction injury (CCI). Our results showed that Fascin-1 increased rapidly in dorsal root ganglions (DRG) and spinal cord (SC) after CCI. The increased Fascin-1 widely expressed in DRG, however, it localized predominantly in microglia, seldom in neuron, and hardly in astrocyte in the SC. Intrathecal injection of Fascin-1 siRNA not only suppressed the activation of microglia and the release of pro-inflammatory mediators, but also attenuated the mechanical allodynia and thermal hyperalgesia induced by CCI.     

Reduced pain behaviors and extracellular signal-related protein kinase activation in primary sensory neurons by peripheral tissue injury in mice lacking platelet-activating factor receptor

Peripheral tissue injury causes the release of various mediators from damaged and inflammatory cells, which in turn activates and sensitizes primary sensory neurons and thereby produces persistent pain. The present study investigated the role of platelet-activating factor (PAF), a phospholipid mediator, in pain signaling using mice lacking PAF receptor (pafr-/- mice). Here we show that pafr-/- mice displayed almost normal responses to thermal and mechanical stimuli but exhibit attenuated persistent pain behaviors resulting from tissue injury by locally injecting formalin at the periphery as well as capsaicin pain and visceral inflammatory pain without any alteration in cytoarchitectural or neurochemical properties in dorsal root ganglion (DRG) neurons and a defect in motor function. However, pafr-/- mice showed no alterations in spinal pain behaviors caused by intrathecally administering agonists for N-methyl-d-aspartate (NMDA) and neurokinin(1) receptors. A PAFR agonist evoked an intracellular Ca(2+) response predominantly in capsaicin-sensitive DRG neurons, an effect was not observed in pafr-/- mice. By contrast, the PAFR agonist did not affect C- or Adelta-evoked excitatory post-synaptic currents in substantia gelatinosa neurons in the dorsal horn. Interestingly, mice lacking PAFR showed reduced phosphorylation of extracellular signal-related protein kinase (ERK), an important kinase for the sensitization of primary sensory neurons, in their DRG neurons after formalin injection. Furthermore, U0126, a specific inhibitor of the ERK pathway suppressed the persistent pain by formalin. Thus, PAFR may play an important role in both persistent pain and the sensitization of primary sensory neurons after tissue injury.     

Spatial and temporal relationship between monocyte chemoattractant protein-1 expression and spinal glial activation following peripheral nerve injury

Peripheral nerve injury can induce spinal microglial/astrocyte activation. Substances released by activated glial cells excite spinal nociceptive neurons. Pharmacological disruption of glial activation or antagonism of substances released by activated glia prevent or reverse pain hypersensitivity. It is not known, however, what causes spinal cord glia to shift from a resting to an activated state. In an attempt to understand the potential role of monocyte chemoattractant protein-1 (MCP-1) in triggering spinal glial activation and its contribution to the development of neuropathic pain, we investigated the effect of peripheral nerve injury on MCP-1 expression in dorsal root ganglia (DRG) and the spinal cord, and established its temporal relationship with activation of spinal microglia and astrocytes. We observed that MCP-1 was induced by chronic constriction of the sciatic nerve in DRG sensory neurons, spinal cord motor neurons and in the superficial dorsal horn, ipsilateral to the injury. Neuronal MCP-1 induction was followed by surrounding microglial activation. After peaking at day 7 after injury, MCP-1 levels began to decline rapidly and had returned to baseline by day 150. In contrast, microglial activation peaked by day 14 and declined afterwards to reach a lower, yet significantly raised level beyond day 22 and remained increased until the end of the test period. Astrocyte activation became detectable later, progressed more slowly and also remained increased until the end of the test period, in parallel with a decreased nociceptive threshold. Our results suggest that neuronal MCP-1 may serve as a trigger for spinal microglial activation, which participates in the initiation of neuropathic pain. Delayed, sustained astrocyte activation may participate with microglia in the persistent phase of pain hypersensitivity.     

cAMP反应元件结合蛋白介导磷酸化细胞外信号调节激酶在神经病理性痛形成中的作用

采用行为学、免疫组织化学和Western blot方法,观察鞘内注射细胞外信号调节激酶(extracellular signal-regulate kinase,ERK)信号转导通路阻滞剂对慢性压迫性损伤(chronic constriction injury,CCI)大鼠痛行为及脊髓背角内磷酸化cAMP反应元件结合蛋白(phosphorylated cAMP response-element binding protein,pCREB)和Fos表达变化的影响,探讨ERK／CREB转导通路在神经病理性疼痛中的作用。结果表明,CCI可明显增加双侧脊髓背角pCREB、损伤侧脊髓背角浅层Fos阳性神经元表达,以CCI后3与5d时尤为显著。鞘内沣射促分裂原活化蛋白激酶激酶(mitogen-activated protein kinase kinase,MEK)阻滞剂U0126及ERK反义寡核苷酸在减轻大鼠痛行为的同时,能明显抑制双侧脊髓背角内pCREB的表达,同时,Fos阳性神经元的表达也明显减少。大鼠痛行为及脊髓背角pCREB和Fos的表达在时相上一致。上述结果提示pCREB参与pERK介导的神经病理性疼痛。     

Transduction and transmission properties of primary nociceptive afferents.

The prototypical primary nociceptive afferent is the polymodal C-fiber nociceptor, which responds to noxious thermal, mechanical, and chemical stimuli. C-fiber nociceptors are peripheral terminals of small neurons in the dorsal root ganglia (DRG). DRG neurons must therefore supply their peripheral terminals with the molecular machinery for the encoding of noxious stimuli into trains of action potentials. The following phenomena are known for this encoding process in vivo: 1) adaptation: for a constant stimulus intensity the action potential discharge decreases slowly within 2-3 seconds, 2) fatigue: recovery from adaptation may take ten minutes or more, 3) sensitization: preceding tissue damage enhances the response, particularly to heat stimuli. Recent studies in vitro have provided important clues about the molecular mechanisms underlying these phenomena. Several membrane receptors and channels are specifically expressed in small nociceptive neurons, such as vanilloid receptors (VR1), purinergic receptors (P2X3), acid sensing ion channels (ASIC), and TTX-resistant Na-channels. In the near future, we may therefore expect major advances in our understanding of the transduction of noxious stimuli into generator potentials and transformation into trains of action potentials. Along the axon that leads from the innervated tissue to the spinal cord, primary nociceptive afferents have a limited capacity to transmit high impulse rates, suggesting a different composition of voltage-gated channels than in other primary afferents (low-threshold mechanoreceptors and thermoreceptors). Finally, the DRG neuron also supplies its central terminals with the molecular machinery for synaptic transmission and its presynaptic modulation. Progress in understanding the cellular mechanisms at both ends of the primary nociceptive neuron promises to lead to new analgesic treatment modalities for both acute and chronic pain.     

Nerve growth factor activates brain-derived neurotrophic factor promoter IV via extracellular signal-regulated protein kinase 1/2 in PC12 cells

Brain-derived neurotrophic factor (BDNF) is a neuromodulator of nociceptive responses in the dorsal root ganglia (DRG) and spinal cord. BDNF synthesis increases in response to nerve growth factor (NGF) in trkA-expressing small and medium-sized DRG neurons after inflammation. Previously we demonstrated differential activation of multiple BDNF promoters in the DRG following peripheral nerve injury and inflammation. Using reporter constructs containing individual promoter regions, we investigated the effect of NGF on the multiple BDNF promoters, and the signaling pathway by which NGF activates these promoters in PC12 cells. Although all the promoters were activated 2.4-7.1-fold by NGF treatment, promoter IV gave the greatest induction. The p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, phosphatidylinositol 3-kinase (PI-3K) inhibitor, LY294003, protein kinase A (PKA) inhibitor, H89, and protein kinase C (PKC) inhibitor, chelerythrine, had no effect on activation of promoter IV by NGF. However, activation was completely abolished by the MAPK kinase (MEK) inhibitors, U0126 and PD98059. In addition, these inhibitors blocked NGF-induced phosphorylation of extracellular signal-regulated protein kinase (ERK) 1/2. Taken together, these results suggest that the ERK1/2 pathway activates BDNF promoter IV in response to NGF independently of NGF-activated signaling pathways involving PKA and PKC.     

Altered gene expression of NIDD in dorsal root ganglia and spinal cord of rats with neuropathic or inflammatory pain

Nitric oxide and nitric oxide synthases are key players in synaptic plasticity events in spinal cord (SC), which underlies the chronic pain states. To date, little is known about the molecular mechanisms regulating the activity of nitric oxide synthases in nociceptive systems. The present study was aimed at the determination of the gene expression of nNOS-interacting DHHC domain-containing protein with dendritic mRNA (NIDD), a recently identified protein regulating nNOS enzyme activity, in rat SC and dorsal root ganglia (DRG) and studying its regulation in states of nociceptive hypersensitivity in a rat model of neuropathic or inflammatory pain. It was found that NIDD mRNA was predominantly expressed in nociceptive primary neurons and in neurons of the spinal dorsal horn (DH) and the number of NIDD-positive neurons in the corresponding DRG or SC increased significantly following induction of chronic hyperalgesia. Meanwhile, remarkable changes of nNOS were detected under such pain conditions. Our data suggest a potential role for NIDD in the maintenance of thermal pain hypersensitivity possibly via regulating the nNOS activity. Meng-Ling Chen and Chun Cheng are contributed equally to this work.     

Activation of extracellular signal-regulated protein kinases 5 in primary afferent neurons contributes to heat and cold hyperalgesia after inflammation

Heat and cold hyperalgesia is a common feature of inflammatory pain. To investigate whether activation of extracellular signal-regulated protein kinase 5 (ERK5), also known as big mitogen-activated protein kinase 1, in primary sensory neurons participates in inflammatory pain, we examined the phosphorylation of ERK5 in the dorsal root ganglion (DRG) after peripheral inflammation. Inflammation induced by complete Freund's adjuvant produced heat and cold hyperalgesia on the ipsilateral hind paw and induced an increase in the phosphorylation of ERK5, mainly in tyrosine kinase A-expressing small- and medium-size neurons. In contrast, there was no change in ERK5 phosphorylation in the spinal dorsal horn. ERK5 antisense, but not mismatch, oligodeoxynucleotide decreased the activation of ERK5 and suppressed inflammation-induced heat and cold hyperalgesia. Furthermore, the inhibition of ERK5 blocked the induction of transient receptor potential channel TRPV1 and TRPA1 expression in DRG neurons after peripheral inflammation. Our results show that ERK5 activated in DRG neurons contribute to the development of inflammatory pain. Thus, blocking ERK5 signaling in sensory neurons that has the potential for preventing pain after inflammation.     

GPR35 is a functional receptor in rat dorsal root ganglion neurons

GPR35, previously an orphan G-protein coupled receptor, is a receptor for kynurenic acid. Here we examine the distribution of GPR35 in the rat dorsal root ganglion (DRG) and the effects of its selective activation. GPR35 was expressed predominantly by small- to medium-diameter neurons of the DRG. Many of these same neurons also expressed the transient receptor potential vanilloid 1 channel, a nociceptive neuronal marker. The GPR35 agonists kynurenic acid and zaprinast inhibited forskolin-stimulated cAMP production by cultured rat DRG neurons. Inhibition required G_i/o proteins as the effect was completely abolished by pretreatment with pertussis toxin. This is the first study to report the expression and function of GPR35 in rat nociceptive DRG neurons. We propose that GPR35 modulates nociception and that continued study of this receptor will provide additional insight into the role of kynurenic acid in pain perception.     

MicroRNA-143 expression in dorsal root ganglion neurons

The unpleasant sensory and emotional experience of pain is initiated by excitation of primary afferent nociceptive neurons. Nerve damage or inflammation induces changes in nociceptive DRG neurons which contribute to both peripheral and central sensitization of pain-sensitive pathways. Recently, blockade of microRNA synthesis has been found to modulate the response of nociceptive neurons to inflammatory stimuli. However, little is known about the contributions of individual miRNAs to painful conditions. We compared miRNA expression in mouse sensory neurons and focussed on the localisation and control of miR-143. Using miRNA-arrays we compared the microRNA expression profile of intact lumbar DRG with one-day-old DRG cultures and found that nine miRNAs including miR-143 showed lower expression levels in cultures. Subsequent RT-qPCR confirmed array data and in-situ hybridisation localised miR-143 in the cytosol of sensory DRG neurons in situ and in vitro. Analysis of microbead-enriched neuron cultures showed significantly higher expression levels of miR-143 in isolectin B4 (I-B4) binding sensory neurons compared with neurons in the I-B4 negative flow-through fraction. In animal models of peripheral inflammation (injection of Complete Freund's Adjuvant, CFA) and nerve damage (transection of the sciatic nerve), we found that expression levels of miR-143 were significantly lower in DRGs ipsilateral to CFA injection or after nerve damage. Taken together, our data demonstrate for the first time miR-143 expression in nociceptive neurons. Since expression levels of miR-143 were higher in I-B4 positive neurons and declined in response to inflammation but not axotomy, miR-143 could selectively contribute to mRNA regulation in specific populations of nociceptors.     

microRNA-222 Targeting PTEN Promotes Neurite Outgrowth from Adult Dorsal Root Ganglion Neurons following Sciatic Nerve Transection

Dorsal root ganglia (DRG) neurons spontaneously undergo neurite growth after nerve injury. MicroRNAs (miRNAs), as small, non-coding RNAs, negatively regulate gene expression in a variety of biological processes. The roles of miRNAs in the regulation of responses of DRG neurons to injury stimuli, however, are not fully understood. Here, microarray analysis was performed to profile the miRNAs in L4-L6 DRGs following rat sciatic nerve transection. The 26 known miRNAs were differentially expressed at 0, 1, 4, 7, 14 d post injury, and the potential targets of the miRNAs were involved in nerve regeneration, as analyzed by bioinformatics. Among the 26 miRNAs, microRNA-222 (miR-222) was our research focus because its increased expression promoted neurite outgrowth while it silencing by miR-222 inhibitor reduced neurite outgrowth. Knockdown experiments confirmed that phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a major inhibitor of nerve regeneration, was a direct target of miR-222 in DRG neurons. In addition, we found that miR-222 might regulate the phosphorylation of cAMP response element binding protein (CREB) through PTEN, and c-Jun activation might enhance the miR-222 expression. Collectively, our data suggest that miR-222 could regulate neurite outgrowth from DRG neurons by targeting PTEN.