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• W2113123068 abstract "Article29 July 2011free access Bidirectional integrative regulation of Cav1.2 calcium channel by microRNA miR-103: role in pain Alexandre Favereaux Corresponding Author Alexandre Favereaux CNRS, IINS, UMR 5297 ‘Central Mechanisms of Pain Sensitization’, Bordeaux Cedex, France Université de Bordeaux, Bordeaux Cedex, France Search for more papers by this author Olivier Thoumine Olivier Thoumine Université de Bordeaux, Bordeaux Cedex, France CNRS, IINS, UMR 5297 ‘Biophysics of Adhesion and Cytoskeleton’, Bordeaux Cedex, France Search for more papers by this author Rabia Bouali-Benazzouz Rabia Bouali-Benazzouz CNRS, IINS, UMR 5297 ‘Central Mechanisms of Pain Sensitization’, Bordeaux Cedex, France Université de Bordeaux, Bordeaux Cedex, France Search for more papers by this author Virginie Roques Virginie Roques Université de Bordeaux, Bordeaux Cedex, France INSERM U862, Neurocentre François Magendie, Bordeaux Cedex, France Search for more papers by this author Marie-Amélie Papon Marie-Amélie Papon CNRS, IINS, UMR 5297 ‘Central Mechanisms of Pain Sensitization’, Bordeaux Cedex, France Université de Bordeaux, Bordeaux Cedex, France Search for more papers by this author Sherine Abdel Salam Sherine Abdel Salam CNRS, IINS, UMR 5297 ‘Central Mechanisms of Pain Sensitization’, Bordeaux Cedex, France Université de Bordeaux, Bordeaux Cedex, France Department of Zoology, University of Alexandria, Alexandria, Egypt Search for more papers by this author Guillaume Drutel Guillaume Drutel Université de Bordeaux, Bordeaux Cedex, France INSERM U862, Neurocentre François Magendie, Bordeaux Cedex, France Search for more papers by this author Claire Léger Claire Léger Université de Bordeaux, Bordeaux Cedex, France INSERM U862, Neurocentre François Magendie, Bordeaux Cedex, France Search for more papers by this author André Calas André Calas CNRS, IINS, UMR 5297 ‘Central Mechanisms of Pain Sensitization’, Bordeaux Cedex, France Université de Bordeaux, Bordeaux Cedex, France Search for more papers by this author Frédéric Nagy Frédéric Nagy CNRS, IINS, UMR 5297 ‘Central Mechanisms of Pain Sensitization’, Bordeaux Cedex, France Université de Bordeaux, Bordeaux Cedex, France Search for more papers by this author Marc Landry Marc Landry CNRS, IINS, UMR 5297 ‘Central Mechanisms of Pain Sensitization’, Bordeaux Cedex, France Université de Bordeaux, Bordeaux Cedex, France Search for more papers by this author Alexandre Favereaux Corresponding Author Alexandre Favereaux CNRS, IINS, UMR 5297 ‘Central Mechanisms of Pain Sensitization’, Bordeaux Cedex, France Université de Bordeaux, Bordeaux Cedex, France Search for more papers by this author Olivier Thoumine Olivier Thoumine Université de Bordeaux, Bordeaux Cedex, France CNRS, IINS, UMR 5297 ‘Biophysics of Adhesion and Cytoskeleton’, Bordeaux Cedex, France Search for more papers by this author Rabia Bouali-Benazzouz Rabia Bouali-Benazzouz CNRS, IINS, UMR 5297 ‘Central Mechanisms of Pain Sensitization’, Bordeaux Cedex, France Université de Bordeaux, Bordeaux Cedex, France Search for more papers by this author Virginie Roques Virginie Roques Université de Bordeaux, Bordeaux Cedex, France INSERM U862, Neurocentre François Magendie, Bordeaux Cedex, France Search for more papers by this author Marie-Amélie Papon Marie-Amélie Papon CNRS, IINS, UMR 5297 ‘Central Mechanisms of Pain Sensitization’, Bordeaux Cedex, France Université de Bordeaux, Bordeaux Cedex, France Search for more papers by this author Sherine Abdel Salam Sherine Abdel Salam CNRS, IINS, UMR 5297 ‘Central Mechanisms of Pain Sensitization’, Bordeaux Cedex, France Université de Bordeaux, Bordeaux Cedex, France Department of Zoology, University of Alexandria, Alexandria, Egypt Search for more papers by this author Guillaume Drutel Guillaume Drutel Université de Bordeaux, Bordeaux Cedex, France INSERM U862, Neurocentre François Magendie, Bordeaux Cedex, France Search for more papers by this author Claire Léger Claire Léger Université de Bordeaux, Bordeaux Cedex, France INSERM U862, Neurocentre François Magendie, Bordeaux Cedex, France Search for more papers by this author André Calas André Calas CNRS, IINS, UMR 5297 ‘Central Mechanisms of Pain Sensitization’, Bordeaux Cedex, France Université de Bordeaux, Bordeaux Cedex, France Search for more papers by this author Frédéric Nagy Frédéric Nagy CNRS, IINS, UMR 5297 ‘Central Mechanisms of Pain Sensitization’, Bordeaux Cedex, France Université de Bordeaux, Bordeaux Cedex, France Search for more papers by this author Marc Landry Marc Landry CNRS, IINS, UMR 5297 ‘Central Mechanisms of Pain Sensitization’, Bordeaux Cedex, France Université de Bordeaux, Bordeaux Cedex, France Search for more papers by this author Author Information Alexandre Favereaux 1,2, Olivier Thoumine2,3, Rabia Bouali-Benazzouz1,2, Virginie Roques2,4, Marie-Amélie Papon1,2, Sherine Abdel Salam1,2,5, Guillaume Drutel2,4, Claire Léger2,4, André Calas1,2, Frédéric Nagy1,2,‡ and Marc Landry1,2,‡ 1CNRS, IINS, UMR 5297 ‘Central Mechanisms of Pain Sensitization’, Bordeaux Cedex, France 2Université de Bordeaux, Bordeaux Cedex, France 3CNRS, IINS, UMR 5297 ‘Biophysics of Adhesion and Cytoskeleton’, Bordeaux Cedex, France 4INSERM U862, Neurocentre François Magendie, Bordeaux Cedex, France 5Department of Zoology, University of Alexandria, Alexandria, Egypt ‡These authors contributed equally to this work *Corresponding author. CNRS, IINS, UMR 5297 ‘Central Mechanisms of Pain Sensitization’, Université de Bordeaux 2, 146 rue Léo Saignat, Bordeaux Cedex 33077, France. Tel.: +33 55 757 4077; Fax: +33 55 757 5051; E-mail: [email protected] The EMBO Journal (2011)30:3830-3841https://doi.org/10.1038/emboj.2011.249 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Chronic pain states are characterized by long-term sensitization of spinal cord neurons that relay nociceptive information to the brain. Among the mechanisms involved, up-regulation of Cav1.2-comprising L-type calcium channel (Cav1.2-LTC) in spinal dorsal horn have a crucial role in chronic neuropathic pain. Here, we address a mechanism of translational regulation of this calcium channel. Translational regulation by microRNAs is a key factor in the expression and function of eukaryotic genomes. Because perfect matching to target sequence is not required for inhibition, theoretically, microRNAs could regulate simultaneously multiple mRNAs. We show here that a single microRNA, miR-103, simultaneously regulates the expression of the three subunits forming Cav1.2-LTC in a novel integrative regulation. This regulation is bidirectional since knocking-down or over-expressing miR-103, respectively, up- or down-regulate the level of Cav1.2-LTC translation. Functionally, we show that miR-103 knockdown in naive rats results in hypersensitivity to pain. Moreover, we demonstrate that miR-103 is down-regulated in neuropathic animals and that miR-103 intrathecal applications successfully relieve pain, identifying miR-103 as a novel possible therapeutic target in neuropathic chronic pain. Cacna1c 3′UTR Fw: 5′-GCGCGCGGTACCTCGTTTCAATGTTCCTAATGGG-3′ Cacna1c 3′UTR Bw: 5′-GCGCGCGGATCCCGACACCTTTGTTCACTTTGC-3′ Cacna2d1 3′UTR Fw: 5′-GCGCGCGGTACCCCCTCTAAACCCATGATCTG-3′ Cacna2d1 3′UTR Bw: 5′-GCGCGCGGATCCAAACCTCCCACAGAGAATGAG-3′ Cacnb1 3′UTR Fw: 5′-GCGCGCGGTACCCACCATATCCATTCCATAACC-3′ Cacnb1 3′UTR Bw: 5′-GCGCGCGGATCCCAGTGCTATCTTCTAACCTG-3′ Cacna1d 3′UTR Fw: 5′-GCGCGCGGTACCGTACGAGTGATTTCACGCCT-3′ Cacna1d 3′UTR Fw: 5′-GCGCGCGGATCCTGCAGTTCAAATACGGTACGA-3′ miR-103 Fw: 5′-GCGCGCAAGCTTTTGAATGCACAGTCTAGATCTAAATGGG-3′ miR-103 Bw: 5′-GCGCGCGGATCCAAAGGGTCCTCCTGTCTCCTC-3′ miR-103-mut Fw: 5′-GCCTTGTTGCATATGGATCAAATAGCATTGTACAGGGCTATG-3′ miR-103-mut Bw: 5′-CATCGCCCTGTACAATGCTATTTGATCCATATGCAACAAGGC-3′ Cacna1c 3′UTR-mut Fw: 5′-GGATTCACTTCTTGTATATTTTGTAAAGTAAATCATTTTG-3′ Cacna1c 3′UTR-mut Bw: 5′-CAAAATGATTTACTTTACAAAATATACAAGAAGTGAATCC-3′ Cacna2d1 3′UTR-mut Fw: 5′-GGAAACTGAAACTCGTGTGAACTCATCTGTGTCCAACATC-3′ Cacna2d1 3′UTR-mut Bw: 5′-GATGTTGGACACAGATGAGTTCACACGAGTTTCAGTTTCC-3′ Cacnb1 3′UTR-mut1 Fw: 5′-GAGGAGTGAGCAACTCAAGCCCCCGCAACCCCTTCCAG-3′ Cacnb1 3′UTR-mut1 Bw: 5′-CTGGAAGGGGTTGCGGGGGCTTGAGTTGCTCACTCCTC-3′ Cacnb1 3′UTR-mut2 Fw: 5′-GTCTCGCCACCTCCTGTGCCCCCACCCCTTCCGGGGG-3′ Cacnb1 3′UTR-mut2 Bw: 5′-CCCCCGGAAGGGGTGGGGGCACAGGAGGTGGCGAGAC-3′ MiR-103: 5′-TCATAGCCCTGTACAATGCTGCT-3′ SDHA Fw: 5′-TGCGGAAGCACGGAAGGAGT-3′ SDHA Bw: 5′-CTTCTGCTGGCCCTCGATGG-3′ Cacna1c Fw: 5′-AGACAGCCTTGCCCTTGCAT-3′ Cacna1c Bw: 5′-GGCCTGGGGAACGTGGA-3′ Cacna2d1 Fw: 5′-ATGTGCTGGAGGATTATACCG-3′ Cacna2d1 Bw: 5′-CCAAAGATAGACCATAAGGAAGG-3′ Cacnb1 Fw: 5′-AGGGCTATGAGGTAACTGAC-3′ Cacnb1 Bw: 5′-GGAAATCCTGCCATCAAACC-3′ miR-103 Fw: 5′-AGCAGCATTGTACAGGGCTATGA-3′ Introduction In chronic pain states, modifications of intrinsic amplification properties of dorsal horn spinal cord neurons lead to long-term sensitization (Derjean et al, 2003). Moreover, sensitization to pain involves the activation of voltage-gated calcium channels (VGCC) (Matthews and Dickenson, 2001; Bourinet et al, 2005; Altier et al, 2007), and in particular Cav1.2-LTC that regulates gene expression underlying long-term plastic changes (Dolmetsch et al, 2001; Fossat et al, 2010). MiRNAs are short non-coding transcripts targeting mRNA 3′ untranslated region (3′UTR) for cleavage or translation inhibition (Lee et al, 1993; Kertesz et al, 2007). Recent findings have demonstrated a key role for miRNAs in synaptic plasticity through the regulation of NMDA receptor expression (Edbauer et al, 2010; Wibrand et al, 2010) or dendritic spine morphology (Schratt et al, 2006). Previous screening studies addressed the involvement of miRNA in the nociceptive system. Indeed, miRNA repertoires in trigeminal root ganglia, spinal cord and prefrontal cortex are modulated after inflammatory pain (Bai et al, 2007; Kusuda et al, 2011; Poh et al, in press). Similarly, peripheral nerve injury modulates miRNA expression in spinal cord and dorsal root ganglion (Kusuda et al, 2011; von Schack et al, 2011). Taken together, these data suggest a role in pain pathophysiology. In addition, two studies provided evidence for a causal role of miRNAs. First Zhao et al (2010) demonstrated that miRNAs regulate Nav1.8 sodium channel expression in dorsal root ganglion and therefore have a role in inflammatory pain. Second, Li et al (2011) suggested that miR-146a controls knee joint homeostasis and osteoarthritis-associated algesia. The regulatory role of miRNAs is likely to involve multiple mRNAs since perfect matching to target sequence is not required for inhibition (Lim et al, 2005). We show here for the first time that such multi-target (or integrative) silencing regulates the different subunits of a macromolecular complex, namely Cav1.2-LTC. In an animal model for neuropathic pain, we demonstrate that miR-103 is down-regulated leading to an up-regulation of Cav1.2-LTC subunits. We further demonstrate a novel causal role of miRNA in the maintenance of pain sensitization, showing that miR-103 intrathecal applications repress Cav1.2-LTC up-regulation and consequently relieve spinal sensitization to pain, therefore identifying miR-103 as a possible therapeutic target. Results Neuronal Cav1.2-LTC comprises three subunits: Cav1.2-α1, α2δ1 and β1 encoded by Cacna1c, Cacna2d1 and Cacnb1 mRNAs, respectively. Bioinformatics analyses using miRanda and Targetscan algorithms (John et al, 2004; Lewis et al, 2005) revealed that all these mRNAs are putative targets of various miRNAs (Supplementary Table S1). They also suggested that a single miRNA (miR-103) possibly binds and inhibits all three subunits forming Cav1.2-LTC (Figure 1A). Indeed, Cacna1c and Cacna2d1 each contain one miR-103 target site while Cacnb1 has two miR-103 target sites. Moreover, the miR-103 target sequences in the Cacna1c, Cacna2d1 and Cacnb1 3′UTRs are well conserved across vertebrates and miR-103 does not target any other VGCC subunits (Supplementary Figure S1; Supplementary Table S2). In mammals, miRNAs are thought to regulate the expression of target mRNAs predominantly through the inhibition of productive translation (Bartel, 2004). Thus, to confirm these theoretical interactions, we generated reporter plasmids by fusing the 3′UTR of each Cav1.2-LTC subunit mRNA (containing miR-103 target sites) to the 3′ terminus of a Renilla luciferase coding sequence (Figure 1B). In COS-7 cells, co-expression of these Renilla luciferase constructs with a miR-103-encoding plasmid prevented luciferase expression in a dose-dependent manner (Figure 1C), whereby demonstrating miR-103 potency to inhibit translation of the three Cav1.2-LTC subunits. Interestingly, the amount of inhibition was different according to the Cav1.2-LTC subunit 3′UTR considered. Cacna2d1 3′UTR mediated significant inhibition at the lowest miR-103 dose tested whereas Cacna1c and Cacnb1 3′UTRs reporter activities remained unaffected. At higher miR-103 doses, the activity of all three Cav1.2-LTC subunit reporters was significantly repressed. Differences in the number and the location of miR-103 sites in 3′UTRs could explain these observations (Grimson et al, 2007; Kertesz et al, 2007). To demonstrate that inhibition was sequence specific, we cloned into the reporter plasmid, the 3′UTR of Cav1.3, another LTC-α1 subunit that contains no miR-103 target site. As expected, miR-103 had no effect on Cav1.3 reporter gene translation, even at the highest amounts tested (Figure 1C, Cacna1d). To illustrate the specificity of miR-103 targeting, we designed a miR-103 mutant (seed-mut-miR-103) differing from the original sequence in the ‘seed region’ of miR-103 (residues at positions 2–8), that controls hybridization with mRNAs (Lewis et al, 2003; Stark et al, 2003; Doench and Sharp, 2004). Nucleotide sequence of the ‘seed’ region was mutated to antisense sequence (Figure 1D) and using miRNA hybridization algorithm (Lewis et al, 2005), we confirmed that it theoretically results in a complete loss of targeting Cav1.2-LTC components. As expected, seed-mut-miR-103 was unable to inhibit any of the Cav1.2-LTC subunit reporters, even at the highest amount tested (Figure 1C). To further demonstrate the role of the seed regions of Cacna1c, Cacna2d1 and Cacnb1 3′UTRs in miR-103 specificity, we deleted the seed regions of all Cav1.2-LTC luciferase reporters. As a result, miR-103 was then unable to mediate translation inhibition of these modified reporters (Figure 1C, seed-mut-3′UTR). As miR-103 sequence is conserved between rat and COS-7 cells, we tested whether endogenous miR-103 expressed by COS-7 cells could regulate Cav1.2-LTC. Therefore, we knocked-down endogenous miR-103 by transfecting LNA-modified miR-103 inhibitor (miR-103-KD). As a result, miR-103-KD enhanced luciferase activity of all Cav1.2-LTC reporters (Figure 1C), demonstrating an endogenous and bidirectional regulation of Cav1.2-LTC by miR-103 in COS-7 cells. It is now well admitted that miRNAs can inhibit translation of target mRNAs either with or without mRNA degradation (Huntzinger and Izaurralde, 2011). To determine whether miR-103 induces target mRNA decay, we measured luciferase mRNA levels by qRT–polymerase chain reaction (PCR). Compared with seed-mut-miR-103 over-expression, miR-103 transfection induced a significant down-regulation of luciferase mRNA for all Cav1.2-LTC reporters (Figure 1E). This result indicates that the inhibition of Cav1.2-LTC subunit translation by miR-103 implies, at least, mRNA decay. Thus, we demonstrated, at the molecular level, the ability of miR-103 to silence simultaneously the three subunits of Cav1.2-LTC through binding their 3′UTR. Figure 1.Integrative regulation of Cav1.2-LTC by miR-103. (A) Bioinformatics analyses revealed that all Cav1.2-LTC subunit mRNA contain a target site for miR-103. Cacna1c and Cacna2d1 each contain one miR-103 site while Cacnb1 has two miR-103 sites. Hybridization to Cacna1c, Cacna2d1 and Cacnb1 3′UTR, yellow indicates perfect base pairing, green indicates wobble base pairing and grey indicates no match. (B) Luciferase reporters consisted of a Renilla luciferase coding sequence under CMV promoter control that was fused on its 3′ end to the 3′UTR of the different Cav1.2-LTC subunits. (C) Compared with control conditions, Cacna2d1 3′UTR mediated significant inhibition of Renilla luciferase activity at the lowest miR-103 dose (150 ng, −24%, P<0.001) whereas Cacna1c and Cacnb1 3′UTRs reporter activities were still unaffected. At higher miR-103 dose (500 ng), all Cav1.2-LTC subunit reporter activities were significantly repressed: −40.51%, P<0.001, −56.45%, P<0.001 and −31.51%, P<0.001 for Cacna1c, Cacna2d1 and Cacnb1, respectively. At the highest miR-103 dose (750 ng), inhibition reached a plateau for Cacna1c and Cacna2d1 (−31.59%, P<0.001 and −53.94%, P<0.001, respectively) whereas Cacnb1 reporter was more efficiently inhibited (−53.07%, P<0.001). Mutation of miR-103 seed region (seed-mut-miR-103) or reporter 3′UTR seed region (seed-mut-3′UTR) resulted in a complete abolition of miR-103 inhibition. Endogenous miR-103 knockdown resulted in a moderate but significant increase of all Cav1.2-LTC subunit reporter activities (miR-103-KD). #P<0.001, ANOVA, Holm–Sidak post hoc test. Data are expressed as mean values±s.e.m. n=6. (D) Mutation of miR-103 (seed-mut-miR-103) at nucleotide positions 2–8 (antisense of original sequence, highlighted in red). (E) qRT–PCR analysis of luciferase mRNA levels showed that miR-103 over-expression induced reporter mRNA decay for all Cav1.2-LTC subunits (Cacna1c −36.88%, P=0.021; Cacna2d1 −28.94 %, P=0.013; Cacnb1 −36.74%, P=0.004). *P<0.05, **P<0.01. Data are expressed as mean values±s.e.m. n=3. Download figure Download PowerPoint Next, we assessed the effect of miR-103 regulation on Cav1.2-LTC expression in neurons. We performed immunolabelling for Cav1.2-α1, α2δ1 and β1 subunit (Figure 2A, C and E, respectively) on primary spinal cord neuron cultures and we quantified immunostaining intensity on confocal images. Compared with control non-transfected conditions, over-expression of miR-103 induced a significant decrease in the intensity of all Cav1.2-LTC subunit staining, namely Cav1.2-α1, α2δ1 and β1 subunits (Figure 2B, D and F, respectively). On the contrary, miR-103 knockdown with miR-103-KD caused an increase in all Cav1.2-LTC subunit labelling intensity (Figure 2B, D and F), demonstrating a role for endogenous miR-103 to limit Cav1.2-LTC expression in neurons. As a control, we used either seed-mut-miR-103 or scrambled miRNA inhibitor that both had no effect on Cav1.2-LTC expression (Figure 2B, D and F). These results confirmed in neurons the luciferase assay results, indicating an integrative regulation of the three Cav1.2-LTC subunits by miR-103. Moreover, these experiments indicate that the regulation of Cav1.2-LTC by miR-103 is bidirectional since knocking-down or over-expressing miR-103, respectively, up- or down-regulate the level of Cav1.2-LTC translation. Among the three subunits forming Cav1.2-LTC, Cav1.2-α1 is specific of this calcium channel subtype whereas α2δ1 and β1 subunits could be associated with other VGCC. Combinatorial subunit associations are still poorly understood (Davies et al, 2007); however, α1 subunits of other calcium channels implicated in pain such as N-type Cav2.2 (Chaplan et al, 1994; Altier et al, 2007) or T-type Cav3.2 (Matthews and Dickenson, 2001; Bourinet et al, 2005) could be associated with α2δ1 and β1 subunits. To evaluate a possible impact of α2δ1 and β1 regulation by miR-103 on other pain-related calcium channel expression, we quantified immunolabelling of Cav2.2 and Cav3.2. Neither miR-103 over-expression, nor miR-103 knockdown had an effect on Cav2.2 and Cav3.2 expression (Supplementary Figures S2 and S3). We further tested for changes in Cav1.2, Cav2.2 and Cav3.2 trafficking by measuring the ratio between membrane and cytoplasm immunostaining. We first validated this method to quantify membrane trafficking by monitoring the GABAB receptor, which is known to be trafficked to the membrane only when associated as a heterodimer (Supplementary Figure S4). Then, we quantified the membrane to cytoplasm ratio for Cav1.2, Cav2.2 and Cav3.2 with and without miR-103 application. This ratio remained constant in both conditions for Cav1.2, Cav2.2 and Cav3.2 proteins, thus strongly suggesting that miR-103 does not induce changes in their trafficking (Figure 3). Figure 2.MiR-103 bidirectionally regulates Cav1.2-LTC expression in spinal cord neurons. To assess all Cav1.2-LTC subunit expressions, we performed a specific labelling for Cav1.2-α1, α2δ1 and β1 subunits (A, C, E, respectively). In non-transfected neurons (a, b), labelling appears more intense, particularly at the plasma membrane (arrows), than in miR-103 over-expressing cells (star in c, d). Quantification of immunostaining intensity on confocal images demonstrated that miR-103 over-expression induced a significant decrease in Cav1.2-α1, α2δ1 and β1 labelling intensity compared with control conditions (−23.47% (B), −32.75% (D) and −25.66% (F), respectively, ***P<0.001). In miR-103 knockdown experiment, Cav1.2-α1, α2δ1 and β1 expressions were significantly increased (+59.64% (B), +57.62% (D) and +64.20% (F), respectively, P<0.001). Transfection with seed-mut-miR-103 or scrambled miRNA inhibitor had no effect on Cav1.2-LTC subunit labelling intensities. Data are expressed as mean values±s.e.m. of four experiments with a number of quantified neurons ranging from 11 to 19 for each condition, bar=10 μm. Download figure Download PowerPoint Figure 3.MiR-103 does not affect Cav1.2, Cav2.2 and Cav3.2 trafficking. We assessed Cav1.2, Cav2.2 and Cav3.2 trafficking by measuring the ratio between membrane and cytoplasm immunostaining with and without miR-103 application. Membrane is identified with a lipophilic dye, DiD (blue in A, C, E). (A, B) miR-103 over-expressing neurons (red, A, star) showed an overall decrease in Cav1.2 labelling (green, B, arrow and arrowhead) but trafficking was not affected. In contrast, miR-103 over-expression (red, star in C, E) did not alter Cav2.2 nor Cav3.2 labelling (green; D, F). (G) Statistical analysis showed no difference in Cav1.2, Cav2.2 and Cav3.2 trafficking, data are expressed as mean values±s.e.m. n=10 for each condition, bar=20 μm. Download figure Download PowerPoint To assess the physiological relevance of such an integrative regulation of Cav1.2-LTC by miR-103, we imaged calcium transients elicited by KCl-induced depolarization in spinal cord neurons. Upon stimulation, neurons exhibited a large increase in intracellular calcium concentration due in part to ion influx through several types of voltage-dependent calcium channels including Cav1.2-LTC (Figure 4A). To demonstrate that miR-103 levels can dynamically regulate calcium influx through Cav1.2-LTC, we used either a miR-103 over-expression plasmid or miR-103-KD. MiR-103 plasmid transfection significantly decreased evoked calcium signals (−58.44%, P<0.001; Figure 4A and B), whereas miR-103-KD significantly increased calcium influx (+22.23%, P<0.01; Figure 4B). To assess the specificity of the miR-103 regulation, we used seed-mut-miR-103 and scrambled miRNA inhibitor, which had no effect on calcium transients. To evaluate the calcium signal mediated by Cav1.2-LTC in the global calcium response, we compared these data with the results obtained with anti-Cav1.2-α1 siRNAs. As shown in Figure 4B, in the presence of anti-Cav1.2-α1 siRNA, the decrease in calcium transients was equivalent to that induced by miR-103 (−55.69%, P<0.001, compared with control). This last result confirms that Cav1.2-LTC has an important role in calcium transients induced by KCl depolarization. Taken together, these calcium imaging experiments confirm that the integrative regulation of Cav1.2-LTC by miR-103 is bidirectional. Expression of Cav1.2-LTC is determinant for the dorsal horn neuron's firing properties (Morisset and Nagy, 1999; Fossat et al, 2010). Hence, our results strongly suggest that Cav1.2-LTC regulation by miR-103 controls neuron excitability in vitro. Figure 4.MiR-103 regulation modulates neuronal calcium transients in vitro. (A) Spinal cord neurons were loaded with Fluo4-AM calcium indicator and depolarization was induced by KCl bath application (25 mM). (B) MiR-103 over-expression strongly reduced calcium responses compared with control (−58.44%, P<0.001) while miR-103 knockdown increased calcium transients (+22.23%, P<0.01). Like miR-103, anti-Cav1.2 siRNA application significantly reduced calcium signals compared with control, (−55.69%, P<0.001). Seed-mut-miR-103 and scrambled miRNA inhibitor did not change calcium transients. **P<0.01, ***P<0.001, ANOVA, Holm–Sidak post hoc test. Data are expressed as mean values±s.e.m. n=16/7, 21/7, 30/8, 46/8, 44/9 and 18/7 (first and second numbers indicate the number of tested cells and cultures, respectively) for control, miR-103, miR-103-KD, seed-mut-miR-103, siRNAs and scrambled miRNA inhibitor experiments, respectively. Download figure Download PowerPoint We previously demonstrated that up-regulation of Cav1.2-LTC in spinal dorsal horn is a key mechanism involved in chronic neuropathic pain. Indeed, specific knockdown of Cav1.2-α1 in the spinal dorsal horn reversed the neuropathy-associated mechanical hypersensitivity and hyperexcitability, and increased responsiveness of dorsal horn neurons (Fossat et al, 2010). Therefore, we investigated the role of miR-103 in neuronal sensitization in vivo. In the present study, we demonstrated that miR-103 regulates Cav1.2-LTC expression in vitro. Thus, modulating miR-103 in vivo should also modify Cav1.2-LTC expression and should therefore modify neuron excitability and pain sensitivity. To test this hypothesis, we performed four daily intrathecal injections of miR-103-KD in naive animals and measured their sensitivity to mechanical stimulations. Compared with control animals that received scrambled miRNA inhibitor, miR-103-KD-injected rats demonstrated a moderate but significant hypersensitivity to pain (−14.57% in mechanical threshold after treatment, P<0.01; Figure 5), suggesting a basal anti-nociceptive control by endogenous miR-103. Figure 5.MiR-103 knockdown in naive animals induces hypersensitivity to pain. Naive animals were subjected to four daily intrathecal injections of miR-103-KD and their sensitivity to pain was measured with mechanical stimulations. Compared with control animals who received scrambled miRNA inhibitor, miR-103-KD-injected rats demonstrated a slight but significant hypersensitivity to pain (−14.57% in mechanical threshold after treatment, P<0.01). Data are expressed as mean values±s.e.m. n=6 for miR-103-KD and scrambled miRNA inhibitor injected rats, **P<0.01, Wilcoxon signed rank test. Download figure Download PowerPoint To uncover further the role of Cav1.2-LTC regulation by miR-103 in pain mechanisms, we used a rat model of chronic neuropathic pain. The neuropathy was induced by spinal nerve ligation (SNL) (Kim and Chung, 1992) and rats were tested for pain-related sensitization of mechanical sensitivity. We first investigated miR-103 expression in the spinal cord with in situ hybridization. Endogenous miR-103 was widely expressed throughout the grey matter, with the most intense signal in motoneurons and superficial dorsal horn (Figure 6A). In neuropathic animals, labelling intensity in spinal cord dorsal horn was clearly decreased ipsilaterally to the lesion (−15.92% compared with contralateral side, P=0.006; Figure 6B). Labelling was seen as clusters in cell bodies and processes (Figure 6C) and often co-localized with the somato-dendritic marker MAP2, indicating that miR-103 was expressed in neurons (Figure 6D and E). Electron microscopy confirmed this neuronal localization, in both soma and processes (Figure 6F and G). MiR-103 was found in the cytosol, but also in the vicinity of the plasma membrane, and in the nucleus, near the nuclear envelope. The localization of miR-103 in processes and its possible role in local translation remain to be studied. Figure 6.MiR-103 expression in the spinal cord is modulated in neuropathic pain conditions. (A) MiR-103 was detected in SNL rats by in situ hybridization with a double-digoxigenin-labelled probe. MiR-103 expression is enriched in superficial laminae of the dorsal horn and in ventral motoneurons. The signal intensity is weaker in the dorsal horn ipsilateral to the peripheral nerve injury (red mask). Bar: 500 μm. (B) Quantification of miR-103 labelling intensity showed a significant decrease in miR-103 expression in the ipsilateral dorsal horn of SNL animals (−15.92% compared with controlateral side, P=0.006). Data are expressed as mean values±s.e.m. n=5. (C–E) Double detection of miR-103 (green) and MAP2 (red) shows neuronal expression of the miRNA. MiR-103 is expressed as discrete clusters throughout cell bodies and dendritic processes (arrows). n=nucleus; bar=10 μm. (F, G) Ultrastructural localization of miR-103 in dorsal horn neurons by in situ hybridization, double-digoxigenin-labelled probe was visualized with silver-enhanced ultra-small gold particles. The expression of miR-103 is restricted to subdomains of the soma (arrowheads in F). It is also present in the nucleus, near the nuclear envelope (arrows in F). In processes, miR-103" @default.
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• W2113123068 title "Bidirectional integrative regulation of Cav1.2 calcium channel by microRNA miR-103: role in pain" @default.
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