© 2013 Nature America, Inc. All rights reserved. sense RNA may act as a biologically active regulator and participate participate and regulator active biologically a as act may RNA sense RNA. We antisense it anti RNA, Kcna2 Kcna2 we that named found Kcna2 to complementary is sequence its of most Because neurons. we a report new native lncRNA that is in DRG expressed mammalian Correspondence should Correspondence be addressed to Y.-X.T. ( 7 Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. Baltimore, Maryland, USA. Nanjing University of Chinese Medicine, Nanjing, China. 1 elusive is development disease to linked how causally but are they processes, pathological and physiological some with ated and translation as transcription such identified in mammalian cells and implicated in gene-regulatory roles in of its study infancy lncRNAs is still lncRNAs RNAs, including noncoding widespread involves unknown. are still downregulation this underlie that mechanisms tribute to induction of neuropathic pain in DRG the channels for tein these expression downregulates of injury Peripheral nerve mRNA and pro DRG lation of receptors, enzymes and voltage-dependent ion channels in the may result from maladaptive changes in gene transcription and trans of DRG neurons the sensory first-order and neuromas in arises that activity spontaneous abnormal by gered neuropathic of pain induction the underlie that mechanisms the of ing disorder has had limited success owing to our incomplete understand Neuropathic pain is a major public health problem. Treatment for this the treatment of neuropathic pain. and maintenance of neuropathic pain. These findings suggest endogenous Kcna2 antisense RNA as a therapeutic target for symptoms. Blocking this increase reversed nerve of downregulation injury–induced DRG Kcna2 and attenuated development reduced total potassium voltage-gated current, increased excitability in DRG neurons and produced neuropathic pain a factor transcription that binds to the nerve injury increased Kcna2 antisense RNA expression in injured DRG through activation of myeloid zinc finger protein 1, potassium channel voltage-dependent mRNA, in the development of neuropathic pain is unclear. Here we identify a conserved lncRNA, named Kcna2 antisense RNA, for a sensory pathway. Long noncoding RNAs (lncRNAs) are emerging as new players in gene regulation, but how lncRNAs operate Neuropathic pain is a refractory disease by characterized maladaptive changes in gene and transcription translation in the Min Li Wei Wang Zhao Xiuli by silencing Kcna2 in primary afferent neurons A long noncoding RNA contributes to neuropathic pain nature nature Received 21 March; accepted 16 May; published online 23 June 2013; Howard Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. Department of and Anesthesiology Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. Recent studies suggest that the mechanism for gene regulation regulation gene for mechanism the that suggest studies Recent 1 4 . Nerve injury–induced neuropathic pain is thought to be trig be to thought is pain neuropathic injury–induced Nerve . . . Voltage-dependent potassium channels govern cell excitability. 3 NEUR , , Xinzhong Dong 1 1 , , Xiaowei Guan OSCI , 8 , , Zongxiang Tang EN C 4 E Institute Institute of Nautical Medicine, Jiangsu Key Laboratory of Nantong Neuroregeneration, University, Nantong, China. advance online publication online advance 3 , 7 1 & Yuan-Xiang Tao , , Sheng-Chin Kao 5– 1 18 0 18 2 , , a that may phenomenon con , , 1 , 11– 2 8 1 9 , [email protected] , , Hongkang Zhang 9 3 . Their expression is associ expression . Their . . A lncRNAs few have been . The abnormal excitability excitability . abnormal The Kcna2 1 4 . . However, the molecular 3 The The Solomon H. Snyder Department of Johns Neuroscience, Hopkins University School of Medicine, antisense RNA gene promoter. Mimicking this increase Kcna2, downregulated Kcna2 ). 1 , , Vinod Tiwari , , in first-order sensory neurons of rat dorsal root ganglion (DRG). Peripheral 1 18

15– , 1 9 doi:10.1038/nn.343 1 . Here, Here, . 7 . The . The 3 , 8 , , Fidelis E Atianjoh ------by blot northern analysis of RNA from adult rat DRG and spinal cord, RNA. noncoding ( at the 5 5 3 the sequence, coding the ing ( DRG Fig. 1b rat in RNA antisense Kcna2 2.52-kb a fied 5 of sequencing directional for ends cDNA organs of rats ( body other and regions brain various cord, spinal in detected transcript this also We species. among identical not were sequences the ( human and monkey mouse, rat, of DRGs transcript the in antisense Kcna2 identified we transcription, reverse for primers strand-specific Using direction. antisense the in were few a of portions reflected tags sequence expressed the published complete the using database a searched first we RNA, antisense Kcna2 Todetect Identification of natural Kcna2 antisense RNA in DRG neurons RESULTS DRG. the in specifically expression Kcna2 by silencing pain neuropathic of maintenance and induction in Supplementary Fig. 1b Fig. Supplementary ′ untranslated region, but Kcna2 antisense RNA but regions region, Kcna2 had unique antisense untranslated We further confirmed the Kcna2 antisense RNA at the expected size 1 6 , , Yong-Jing Gao Biomedical Biomedical Innovation Institute, Yangzhou University, Yangzhou, China. 8 ). ). Most of that overlapped of its sequence ′ and 3 ′ ends ( Supplementary Fig. 1a Fig. Supplementary 1 , 8 8 Fig. Fig. 1 , , Jian-Yuan Zhao These These authors contributed equally to this work. 4 Kcna2 , , Paul N Hoffman ), indicating that Kcna2 antisense RNA is is RNA antisense Kcna2 that indicating ), b ). ). It had no frame apparent reading open cDNA sequence. Although many of of many Although sequence. cDNA ′ untranslated region and part of the the of part and region untranslated ). Using rapid amplification ). of Using rapid amplification 2 College College of Basic Medicine, 1 , 8 ′ and 3 and , Lingli , Liang Lingli 5 , , Hengmi Cui Kcna2 5 Department Department of t r a Fig. 1 Fig. Kcna2 ′ ends, we identi we ends, Supplementary Supplementary RNA, includ

a transcript, transcript, ), although although ), C I

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© 2013 Nature America, Inc. All rights reserved. is is altered in rat after peripheral nerve injury. Consistent with previous Next, we examined whether expression of DRG Kcna2 antisense RNA injury nerve after expression RNA antisense Kcna2 DRG ( protein Kcna2 most Kcna2 RNA–positive antisense neurons express low amounts of overlappedpartially that of Kcna2 protein in DRG ( ( (CGRP) peptide for gene-related calcitonin 15.3% for 28.7% and P2X3, B4 for isolectin 18.1% protein, (NF-200) neurofilament-200 for positive were neurons RNA–positive antisense Kcna2 of 60.6% >35 (7%; large few a and <25 (24%; small were some although diameter), in (228 of 1,060) were labeled. Most were medium-sized (69%; 25–35 ( neurons DRG in weakly expressed was RNA antisense Kcna2 that showed chemistry ( weak were signals the although n DRG. rat in RNA AS Kcna2 of distribution the showing histochemistry rats. of 2) (lane cord spinal and 1) (lane DRG the in (arrowhead) RNA AS Kcna2 of ( RNA. AS of cDNAs the between ( control. no-template C, ladder; 100-bp M, primers. intron-spanning specific using tissues the in RNA AS Kcna2 native of existence the confirmed We further species. per experiments DNA. genomic of absence indicating samples, I–treated DNase in detected Gapdh Gapdh I. DNase excess with samples RNA extracted the pretreated we contamination, DNA genomic To primers. exclude strand-specific with PCR (RT)- transcription reverse using human and ( monkey (C57/BL6), mouse (Sprague-Dawley), rat of DRGs the in detected ( ganglion. root dorsal naive in RNA antisense 1 Figure t r a  b a Fig. Fig. = 5 rats. Scale bar, 40 Scale = 5 rats.

j g d a ) Native Kcna2 antisense (AS) transcripts transcripts (AS) antisense Kcna2 ) Native ) The extent of sequence overlap (blue boxes) boxes) (blue overlap sequence of extent ) The Kcna2 AS Kcna2 AS Kcna2 AS Kcna2 AS 2 nor Kcna2 AS PCR products were were products PCR AS Kcna2 nor neither RT primers, Without a control. is ). Although the distribution pattern of Kcna2 antisense RNA antisense Kcna2 of pattern distribution the Although ).

C I Identification and expression of Kcna2 Kcna2 of expression and Identification c ) Northern blot expression analysis analysis expression blot ) Northern e l Fig. 3 Fig. s Fig. 1 Fig. Kcna2 c ). µ k h e b n m. ( m. IB4 P2X3 CGRP NF200 d = 3 repeated = 3 repeated µ Macaca fascicularis Macaca ). Approximately 21.5% of DRG neurons neurons DRG of 21.5% Approximately ). RNA and Kcna2 Kcna2 and RNA m in diameter) ( diameter) in m e ) Histogram shows the distribution of Kcna2 AS RNA–positive somata in normal rat DRG. rat normal in somata RNA–positive AS Kcna2 of distribution the shows ) Histogram Fig. 1 Fig. c

). ). In situ

) Fig. 1 Fig. l i f c Merged Merged Merged Merged hybridization histo hybridization Kcna2 AS Kcna2 AS a c e Figs. Figs. 1e Gapdh Gapdh ). Approximately Approximately ). µ Mouse 2.5 Kb 3.0 Kb 9.0 Kb m in diameter) diameter) in m Ra t M M and 1 M + + + + + – – + – – 3a + – + – 2 GFP µ – , – b m C C ), ), -

, which is not expressed in mammalian cells, was used as a negative control. control. a negative as used was cells, mammalian in expressed not is , which DNase RT DNase RT d 270 nt 452 nt 250 nt 452 nt but not sham surgery, time-dependently downregulated studies Arrows, double-labeled neurons. neurons. double-labeled Arrows, (CGRP; peptide gene-related (NF200; neurofilament-200 for and RNA (AS) antisense Kcna2 for double-labeled were Neurons rats. naive of DRG in neurons 2 Figure L5 the DRG contralateral from groups the ( sham-operated in that to compared 51.8% by reduced was DRG L5 ipsilateral the in for Kcna2 ratio corresponding the whereas groups, sham-operated the of antisense RNA was 2.2-fold greater in the injured L5 DRG than in that Kcna2 On day 7 axotomy,to after contralateral of ratio the ipsilateral rons ( SNL after 14 and 7 3, days ( on DRG L5 contralateral the in those than higher were DRG L5 ipsilateral the in neurons RNA–positive antisense Kcna2 of number and density staining the Furthermore, point, time per group per rats ( DRG L4 ipsilateral the in RNA of expression the ( SNL after DRG L5 ipsilateral the in time-dependently increased RNA antisense Kcna2 ( zinc finger protein 1 (MZF1) in the promoter region of the the of region promoter the in (MZF1) 1 protein finger zinc motif ( Using the online software TFSEARCH, we found a consensus injury? binding nerve after upregulated RNA antisense Kcna2 DRG is How SNL after activity gene RNA antisense Kcna2 promotes MZF1 injury. nerve peripheral after DRG injured the in induced RNA that can Kcna2 be antisense demonstrate results together, these ( SNL after d 7 neurons, DRG large and medium in ( from rats respectively, sham-operated neurons, DRG large and medium small, in 121 of RNA antisense and group, Kcna2 AS Fig. 4d Fig. 4 Fig. We the examined opposing further SNL-induced changes in Kcna2 I I Kcna2 Kcna2 AS Kcna2 AS n Figs. 1e Gapdh Gapdh = 3 repeated experiments. M, RNA marker. ( marker. RNA M, experiments. = 3 repeated mRNA was 75% lower ( lower 75% was mRNA Monkey −161 Human a P 5–

, ) and protein ( protein and ) e < 0.05; 0.05; < Subpopulation distribution of Kcna2 antisense RNA–containing RNA–containing antisense Kcna2 of distribution Subpopulation 1 to Kcna2 antisense RNA were approximately 82, 118 and and 118 82, approximately were RNA antisense Kcna2 to ). ). These changes occurred predominantly in large DRG neu 0 advance online publication online advance AGTGGGGA , unilateral fifth lumbar (L5) spinal nerve ligation (SNL), (SNL), ligation nerve spinal (L5) lumbar fifth unilateral , M M GFP and + + + + + Supplementary Fig. 2d Fig. Supplementary + – – 4 Kcna2 + – – f Kcna2 Fig. 4 Fig. ). ). Results were similar after sciatic nerve axotomy. – C Fig. 4 Fig. C Fig. 4 Fig. DNase RT DNase RT −154 mRNA, Kcna2 protein or Kcna2 antisense antisense Kcna2 or protein Kcna2 mRNA, 452 nt 555 nt 452 nt 555 nt c mRNA DRG in neurons. Ratios individual ). Neither SNL nor sham surgery changed changed surgery sham nor SNL Neither ). d h ) ) for the transcriptional activator myeloid – I I f ). These ratios decreased, particularly particularly decreased, ratios These ). b e ), P2X3 ( P2X3 ), n b ) in the ipsilateral L5 DRG. Notably, DRG. L5 ipsilateral the in ) P Fig. 4 Fig. Frequency = 5 rats. Scale bar, 40 Scale = 5 rats.

> 0.05; 0.05; > (%) Kcna2 AS Kcna2 RNA –2,690 to–2,441 Fig. 4a Fig. 10 12 14 3 5 –2,388 to–2,314 0 2 4 6 8 ′ ′ 0 –910 to–586 Unique regionsofKcna2AScDNA Unique regionof Overlapping region g Small g ). Additionally, Kcna2 protein protein Kcna2 Additionally, ). – ). i , Supplementary Fig. 2a Fig. Supplementary ) or isolectin B4 (IB4; (IB4; B4 isolectin ) or c Cross-sectional area

) or L5 spinal cord ( cord spinal L5 or ) 600 nature nature –163 to–1 Medium AT d

– Codingsequenc Kcna2 G ) ) In situ In 1 ,200 cDNA a NEUR Fig. 4 Fig. µ – c m. Kcna2 ( ), calcitonin calcitonin ), 1,501 to1,743 hybridization hybridization µ Large m 1,744 to1,783 T 2 OSCI e n ) h GA = 12 per ). Taken ). mRNA j Kcna2 – 2,000 3 n 5 l EN ′ ). ). ′ = 4 = – c C ). ). E -

© 2013 Nature America, Inc. All rights reserved. medium cells and 4.48 for large cells. * cells. large for 4.48 and cells medium surgery. or sham SNL 7 on day after neurons DRG in AS individual RNA Kcna2 ** mRNA. for 7.55 and AS RNA Kcna2 SNL. ( SNL. 14 on after L5 day DRG ipsilateral in the small 14.5% and medium, 39.1% large, are neurons ( 40 contralateralcorresponding side. Scale bars, F SNL. after DRGs ( Two-way ANOVA Tukeywith point. time corresponding at the group operated * point. time per group per rats surgery. sham or SNL after DRGs L4/5 ( in presented are blots Tukey with ANOVA Two-way point. time corresponding the at group sham-operated the of side contralateral * 14. day for F surgery. sham or SNL after DRG L5 in expression Tukey with Two-way ANOVA point. time corresponding the at group sham-operated the versus point. time per group contralateral. con, ipsilateral; Ipsi, surgery. sham or SNL after DRGs L4/5 injury. ( nerve peripheral after Kcna2 and RNA antisense 4 Figure ( 14 day on rats sham-operated density in the ipsilateral L5 DRG from SNL rats compared to that from gene antisense promoter, as demonstrated by a greater 4.12-fold band the to MZF1 of binding the increased SNL selective. and cific that the binding of MZF1 to the or after preabsorption of MZF1 antibody (data not rats shown), indicating sham-operated in DRGs ( from fractions nuclear in antibody could be amplified from the complex immunoprecipitated with MZF1 the of fragment a that revealed assay immunoprecipitation chromatin A ( DRG the in motif this to MZF1 of binding demonstrated gene RNA antisense nature nature f d c Fig. 5 P = 358.18. * = 358.18. = 6.90 for day 3, 74.11 for day 7 and 351.39 351.39 7 and day for 74.11 3, day for = 6.90 c a ) Histogram; AS RNA-positive of Kcna2 46.4% ) Kcna2 antisense (AS) RNA expression in expression RNA (AS) antisense ) Kcna2 , Kcna2 ASRNA < 0.05, ** < 0.05, e µ ) Kcna2 AS RNA–positive neurons in L5 neurons AS RNA–positive ) Kcna2 Kcna2 m. Two-way ANOVA with Tukey g b ) Expression of Kcna2 AS RNA and and AS RNA of Kcna2 ) Expression

). This amplification did not occur with normal serum ( n NEUR Changes in expression of DRG Kcna2 Kcna2 DRG of expression in Changes = 12 rats per group per time point. point. time per group per rats = 12 antisense RNA promoter that includes the binding motif motif binding the includes that promoter RNA antisense post-hoc P a P P < 0.05, ** < 0.05, ) ) < 0.01 versus the sham- the versus < 0.01 OSCI < 0.05, ** < 0.05, Kcna2 n = 5 rats per time point. point. = time per 5 rats post-hoc test. ( test. EN F Supplementary Figure 7 Figure Supplementary mRNA expression in expression mRNA = 60.05. ** = 60.05. 20 C , P E 2 b Kcna2 protein P

1 < 0.01 versus the the versus < 0.01 b ) Kcna2 protein protein ) Kcna2 test. Full-length Full-length test. post-hoc . An electrophoretic mobility shift assay assay shift mobility electrophoretic An . < 0.01 versus the versus < 0.01

advance online publication online advance Frequency (%) F n 0 1 2 3 4 5 6 7 8 = 35.51. = 35.51. = 12 rats per per rats = 12 0 post-hoc Small Kcna2 n P = 6 rats per group, group, per rats 6 = test. test. < 0.01 < 0.01 600 n P P = 12 < 0.01 versus the corresponding sham-operated group. Paired Student’s Paired group. sham-operated corresponding the versus < 0.01 antisense RNA promoter is spe Medium < 0.05, ** < 0.05,

test.

Kcna2

1,200

.

mRNA in L4/5 DRGs on day 7 after axotomy or sham surgery.or sham axotomy 7 on day after DRGs in L4/5 mRNA Merged P < 0.01 versus the corresponding sham-operated group. Paired Student’s Paired group. sham-operated corresponding the versus < 0.01 f c a Frequency (%) Relative amount (ipsi/con) Relative amount (ipsi/con) 0.5 1.0 1.5 0.5 1.0 1.5 10 12 Large 0 2 4 6 8 Days afterSNLorshamsurgery 4 0 1 2 3 4 0 0 0 1 2 3 P Kcna2 ASRNA 0 < 0.05). This This 0.05). < Days afterSNLorshamsurgery Kcna2 Small L5 DRG L4 DRG L5 DRG L4 DRG Cross sectionalarea

7 3 Sham ** 3 Fig. 5 Fig. mRNA 600 * Fig. 5 Kcna2 Medium 2,900 ** a b 1,200 7 ). ). ** SNL - )

SNL Sham 14 n ohr otg-ae ptsim hnes MF overexpres MZF1 channels. potassium voltage-gated other and 3b Fig. ( cells (HEK)-293T kidney embryonic human tured full-length overexpressed we expression, RNA sense promoter the to MZF1 of region more of binding the allowing expression, MZF1 DRG increases injury nerve peripheral that suggest findings ( neurons DRG the in RNA not Moreover,shown). (data DRG L4 ipsilateral and DRG L5 contralateral the in sion sham nor SNL or altered binding basal activity surgery MZF1 expres ( DRG L5 ipsilateral the in MZF1 of upregulation may increase result from time-dependent SNL-induced n staining. protein Kcna2 high-density display (arrows) a few although protein, Kcna2 of amounts low express DRG the in neurons RNA-labeled ( 8.6%. small, 18.6%; medium, 72.6%; large, somata: Kcna2-positive of 70% (855 of 1,220) of DRG neurons were positive for Kcna2. ( Approximately neurons. of the Kcna2-positive showing example distribution ( DRG. rat normal in protein Kcna2 with RNA antisense 3 Figure indicating that upregulation of Kcna2 antisense RNA was specific specific was RNA antisense Kcna2 of upregulation that indicating scrambled full-length with co-transfected Mzf1 cells in abolished were responses decreased ingly correspond and RNA antisense Kcna2 increased significantly sion ** Large n c = 5 rats. Scale bars, 50 50 bars, Scale = 5 rats. ( = 15 neurons per cell type per group. group. per type cell per = 15 neurons ) Representative examples showing that most Kcna2 antisense (AS) (AS) antisense Kcna2 most that showing examples ) Representative µ 14 To further examine whether MZF1 directly regulates Kcna2 anti Kcna2 regulates directly MZF1 whether examine Tofurther ** m 2 ) 2,100 vector and and vector b β Kcna2 -actin ,

Relative amount c 0.4 0.8 1.2 Distribution of Kcna2 protein and double labeling of Kcna2 Kcna2 of labeling double and protein Kcna2 of Distribution ), which express endogenous Kcna2 antisense RNA, Kcna2 Kcna2 RNA, antisense Kcna2 endogenous express which ), d 0 Day 14 Day 7 Day 3 Kcna2 Mzf1 Con g Contralateral Sham Relative amount Sham Con antisense gene in the injured DRG neurons. DRG injured the in gene antisense

(ipsi/con) Kcna2 iN) ( siRNA) Ipsi 0 1 2 3 4 Day Mzf1 Mzf1 mRN Kcna2 Con 3 Ipsi ** SNL SNL mRNA and Kcna2 protein ( protein Kcna2 and mRNA A µ -specific short interfering RNA (but not not (but RNA interfering short -specific mRNA was expressed with Kcna2 antisense Kcna2 antisense with mRNA was expressed * Ipsi m. Kcna2 AS Ipsilateral Axotomy Sham i. 5e Fig. RN 0.4 0.8 1.2 Supplementary Fig. 3a Fig. Supplementary ** 0 A n Con = 12 rats per group. group. = per 12 rats Sham t -test. ( -test. Sham , Ipsi f Day and and Fig. 5c Fig. t = 1.01 for small cells, 3.35 for 3.35 cells, small for = 1.01 h e Con h Ratios (Kcna2/Kcna2 AS) 7 ) The ratios of ) ratios The SNL 100 120 140 160 180 Percentage Kcna2 AS– 20 40 60 80 SNL upeetr Fg 3d Fig. Supplementary 0 positive neurons per section Ipsi ** 10 20 30 40 50 60 70 , d 0 t -test. Error bars, s.e.m. bars, Error -test. ). As expected, neither neither expected, As ). Small cell SNL Sham 0.4 0.8 1.2 0 Con Con Ipsi t r a Sham a Days afterSNL t 3 Fig. 5e Fig. = −14.19 for = −14.19 ) A representative ) A representative * Supplementary Supplementary ). These These ). Medium Sham Ipsi Kcna2 Day 14 cell Mzf1 b * ) Distribution Con 7 ** C I SNL , mRNA to mRNA f ). These These ). SNL in cul in Large Ipsi in vivo in cell 14 ** e l ** **

s ), ),  - - - - -

© 2013 Nature America, Inc. All rights reserved. 0.78 for Kcna4 and 0.48 for Scn10a. ** Scn10a. for 0.48 and Kcna4 for 0.78 ( AS. AAV5-Kcna2 or AAV5-EGFP Tukey Two-way with ANOVA group. for 20.45 RNA, AS AS. AAV5-Kcna2 or AAV5-EGFP with injection after 8 weeks 4 and DRGs L4/5 (con) contralateral and ANOVA Tukeywith One-way AS alone. AAV5-Kcna2 for AS. + AAV5-Kcna2 SE AAV5-Kcna2 or AS AAV5-Kcna2 ( ## ** Scn10a. for 0.45 and Kcna4 for 0.61 analysis. densitometric the of summary statistical Right, AS. + Kcna2 SE Kcna2 or vector (AS) antisense Kcna2 vector, (SE) fragment sense Kcna2 vector, EGFP control with transfected cells HEK-293T in protein Scn10a and Kcna4 Kcna1, Kcna2, of amounts the showing blots 6 Figure length full- expressed that (AAV5) 5 virus adeno-associated recombinant of RNA and downregulation of antisense Kcna2 upregulation triggered of sion expres basal increased and expression RNA antisense Kcna2 basal MZF1. to response in Tukey with ANOVA One-way alone. (pGL3-Basic). vector Vec, control shown. as transfected cells 293T Tukey Two-way with ANOVA alone. AAV5-MZF1 corresponding for 17.79 and RNA AS Kcna2 for 30.84 mRNA, neuron. AAV5-EGFP-labeled an Inset, shown. as transduced neurons cultured DRG rat in mRNA control. * protein. for 7.93 and mRNA for 12.92 RNA, AS n scrambled Scram, siRNA; Ctl, shown. as ( protein ( RNA post-hoc Tukey with ANOVA One-way 0). (day group * n control. a loading as serves (H3) H3 Histone surgery. sham or SNL after DRGs L5 ipsilateral ( marker. ladder M, fragments. purified total Input, surgery. sham or SNL after 14 day on DRGs L5 ipsilateral the in MZF1 to antibody rabbit by immunoprecipitated ( 5). (lane MZF1 to antibody and extract nuclear or 4), (lane probe mutant unlabeled 50-fold and extract nuclear 3), (lane probe unlabeled 50-fold and extract nuclear 2), (lane extract nuclear plus or 1) (lane alone probe Labeled promoter. antisense the for MZF1 of specificity binding ( RNA. antisense Kcna2 DRG of upregulation 5 Figure t r a  post-hoc a b a b Scn10a P = 5 repeats per treatment. treatment. per = 5 repeats group. per point time per = 9 rats

β Kcna Kcna4 Kcna1 ) Electrophoretic mobility shift assay showing showing assay shift mobility ) Electrophoretic ) Amounts of Kcna2 AS RNA and of mRNAs for various ion channels in rat DRG cultured neurons transduced with AAV5-EGFP, SE, with AAV5-Kcna2 transduced neurons cultured DRG rat in channels ion various for mRNAs of and RNA AS Kcna2 of ) Amounts ) P -actin < 0.05 versus the corresponding naive naive corresponding the versus < 0.05 Kcna2 Kcna1 Kcna2 < 0.01 versus the corresponding Kcna2 AS vector alone. One-way ANOVA with Tukey with ANOVA One-way alone. vector AS Kcna2 corresponding the versus < 0.01 2 e EGFP ), ), Mzf1 Kcna2 #

C I f test. Error bars, s.e.m. Full-length blots are presented in in presented are blots Full-length s.e.m. bars, Error test. ( test. ) in HEK-293T cells transfected transfected cells HEK-293T ) in Kcna2 P MZF1 mediates nerve injury–induced injury–induced nerve mediates MZF1 Kcna2 antisense RNA specifically targets Kcna2. ( Kcna2. targets specifically RNA antisense Kcna2 mRNA in DRG cultured neurons that with were transduced P mRNA, 0.65 for for 0.65 mRNA, antisense (AS) promoter fragments fragments promoter (AS) antisense < 0.05, < 0.05, < 0.05, ** < 0.05, SE ( e l EGFP AS Fig. 5 Fig. mRNA and protein ( protein and mRNA e mRNA ( mRNA , f ) Amounts of Kcna2 AS Kcna2 of ) Amounts SE s AS c ## Kcna2 control; siRNA, siRNA, control; , + d g P P ) MZF1expression in the the in ) MZF1expression ). < 0.01 versus versus < 0.01 < 0.01 versus EGFP EGFP versus < 0.01 n

Relative amount e Mzf1 = 4 repeats per treatment, treatment, per = 4 repeats 0. 1. 1. ) and Kcna2 Kcna2 ) and mRNA, 0.39 for for 0.39 mRNA, Mzf1 5 0 5 0 Kcna4 n = 3 repeats. = 3 repeats. F EGFP = 8.53 for for = 8.53 siRNA transfection alone also reduced reduced also alone transfection siRNA Scn10a Kcna2 siRNA. siRNA. e mRNA and 0.87 for for 0.87 and mRNA ) Statistical summary of the densitometric analysis. analysis. densitometric the of summary ) Statistical post-hoc post-hoc Kcna2 F SE Mzf1 = 14.13. = 14.13. Mzf1

Fig. 5e Fig. Kcna4 Kcna1

* *

P P

AS

< 0.01 versus corresponding contralateral sides of the AAV5-EGFP-treated group. One-way ANOVA with Tukey with ANOVA One-way group. AAV5-EGFP-treated the of sides contralateral corresponding versus < 0.01 groups. EGFP corresponding the versus < 0.01

test. Error bars, s.e.m. Full-length blots are presented in in presented are blots Full-length s.e.m. bars, Error test. alone. One-way ANOVA with Tukey with ANOVA One-way alone. test. ( test. Kcna1

SE +AS ## , f ). We confirmed MZF1- We ). confirmed Kcna2 d mRNA and 0.56 for for 0.56 and mRNA post-hoc ) Representative western blots of ipsilateral and contralateral L4/5 DRGs 8 weeks after injection with with injection after 8 weeks DRGs L4/5 contralateral and ipsilateral of blots western ) Representative F g d a = 21.37 for Kcna2, 0.65 for Kcna1, Kcna1, for 0.65 Kcna2, for = 21.37 Scn10a b Relative amount mRNA. * mRNA.

n Relative amount 0 1 2 3 4 5 6 7 Relative amount 0 2 4 6 = 3 repeats per treatment. treatment. per = 3 repeats 5 4 3 2 1 0 2 1 3 or shamsurgery Days afterSNL Naive Sham test. ( test. 0 a Kcna2 Kcna2 ASRNA Mzf1 ) Left, representative western western representative ) Left, EGFP post-hoc Kcna1 Kcna2 Kcna mRNA. ** mRNA. 7 3 * mRNA P mRNA * EGFP c < 0.05, ** < 0.05, Supplementary Figure 7 Figure Supplementary 2 SNL ASRNA mRNA mRNA ) Levels of Kcna2 AS RNA and mRNAs for various ion channels in the ipsilateral (ipsi) (ipsi) ipsilateral in the channels ion various for mRNAs and AS RNA of Kcna2 ) Levels n 14 * * = 3 repeats per treatment. treatment. per = 3 repeats * SE test. ( test. Scn10a e probe MZF1 MZF1 Free ** P Mzf1 - Relative amount * *

< 0.01 versus AAV5-EGFP alone. alone. AAV5-EGFP versus < 0.01 ** 0 1 2 0 4 8 * AS * h P shown). MZF1-triggered downregulation of Kcna2 is thus not likely likely not thus is Kcna2 of downregulation MZF1-triggered shown). not (data antibody MZF1 by immunoprecipitated complex nuclear Kcna2 the activates of the activity the not enhance does MZF1 motif. MZF1-binding a not contain consensus does gene ) ) Kcna2 SE Kcna4 Scn10a < 0.01 versus the corresponding naive condition. condition. naive corresponding the versus < 0.01 mRNA. * mRNA. post-hoc Ct Mzf1 Kcna2 ** SE +AS A software prediction showed that the promoter region of the l ## b

# mRNA Mzf1 # Kcna2 AS

+ ** F mRNA ** gene promoter fragments were not amplified from the DRG DRG the from amplified not were fragments promoter gene

= 10.06 for for = 10.06 gene promoter and and promoter gene n M h

test. ( test. = 10 rats per group. group. per rats = 10 siRNA P Relative activity advance online publication online advance post-hoc 0 1 2 3 4 5 6 Sham < 0.05, ** < 0.05,

. * *

c Kcna2 Naiv Input Kcna2 ASRNA + Relative amount Kcna2 siRN g e Mzf1 ) Amounts of of ) Amounts SNL ##

(ipsi/con) # n F 0 1 2 3 Scn10a A = 3 repeats per treatment. treatment. per = 3 repeats = 82.09. ** = 82.09. β Mzf1 test. test. d Kcna2 Kcna4 Kcna1 Kcna2 Kcna2 -actin antisense gene promoter ( promoter gene antisense Scram mRNA Sham Supplementary Figure 7 Figure Supplementary Normal P AS EGFP serum 8 4 n * < 0.01 versus the corresponding EGFP-treated EGFP-treated corresponding the versus < 0.01 ASRNA = 12 rats per treatment. treatment. per rats = 12

Con Vec + Scram EGFP SNL mRNA, 11.90 for Kcna2 AS RNA, 0.24 0.24 RNA, AS Kcna2 for 11.90 mRNA, Mzf1 ** ** Kcna2 *

lpsi Vec Mzf1 Sham F ## EGFP = 15.51 for Kcna2, 0.35 for Kcna1, Kcna1, for 0.35 Kcna2, for = 15.51

0. 1. 1. + P f β Kcna2 MZF1 P Kcna2 Anti- -actin 5 0 5 0 < 0.01 versus pGL3-Kcna2 AS vector vector AS pGL3-Kcna2 versus < 0.01 Con Relative < 0.01 versus the corresponding corresponding the versus < 0.01 AS gene promoter activities in HEK- in activities promoter gene AS Vec Kcna2 AS mRNA, Kcna2 AS RNA and and RNA AS Kcna2 mRNA,

amount SNL Mzf1 + 0 1 2 3 lpsi 8 4 ** gene promoter, but it markedly

mRNA Ct Ct Weeks afterinjection Kcna2 l l e ** DNA Relative amount No Kcna2 Mzf1 Mzf1

0. 0. 1. 0. 1. 1. 4 8 2 0 * . F 5 0 5 0 nature nature EGFP

= 168.61 for for = 168.61 + Kcna1 Fig. 5 Fig. siRN siRNA Con

F Kcna2 Kcna4 # Kcna c ** = 15.91 for Kcna2 Kcna2 for = 15.91 8 4 P MZF1 MZF1

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mRNA + H3 H3 2 siRNA h Mzf1 NEUR lpsi Mzf1 #

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© 2013 Nature America, Inc. All rights reserved. cells cells were positive for NF-200, 4.21% for substance P, 6.32% for CGRP ( minals ter and fibers their and neurons DRG L4/5 ipsilateral the to limited L5 DRGs. (L4/5) Four AAV5EGFP-labeled weeks after injection, was and L4 the into unilaterally AAV5-EGFP or AAV5–Kcna2antisense not but expression, protein and mRNA Kcna2 decreased RNA markedly or EGFP (AAV5-EGFP) into cultured DRG AAV5 that Kcna2 RNAexpressed antisense (AAV5-Kcna2 antisense) transduced and cells HEK-293T cultured into vector EGFP control or vector proviral RNA antisense Kcna2 transfected we end, Tothis and excitability DRG neuronal and function, expression Kcna2 DRG alters RNA antisense Kcna2 DRG of ulation We next investigated whether mimicking nerve injury–induced upreg DRG Kcna2 antisense RNA leads to neuropathic pain symptoms expression. gene antisense Kcna2 may downregulation be attributable to MZF1-triggered in reduction MZF1-induced the reversed and RNA antisense Kcna2 in increase MZF1-induced the blocked fragment sense Kcna2 the that ( neurons DRG or cells HEK-293T cultured in Kcna2 ( expression RNA antisense Kcna2 blocked significantly fragment This +40). to (−311 fragment RNA sense Kcna2 a expresses that AAV5 an vector cloned we effect, this mediates RNA antisense Kcna2 whether the To examine to MZF1 of binding direct by occur to Tukey Two-way with ANOVA points. time corresponding ( cold TukeyTwo-way with ANOVA in as same ( neurons. DRG in (AP) ** rats). (14 group small). for 0.73 ( pulses for threshold t Tukey * treatment. MTX nM 100 after ( neuron. recording (green)-labeled EGFP an Inset, (MTX). maurotoxin nM 100 of perfusion bath after or before rats (AS)-injected antisense Kcna2 and control- from neurons DRG large in current potassium voltage-gated total of traces ( symptoms. pain neuropathic produces and in large and medium DRG neurons excitability increases current, potassium voltage-gated total reduces DRG in overexpression 7 Figure nature nature ( for P2X3 and 10.0% a = −4.21 for large, −7.04 for medium and −0.32 for small) and current current and small) for −0.32 and medium for −7.04 large, for = −4.21 b P

) ) Control < 0.05, ** < 0.05, AS Kcna2 I Before MTX Kcna1 - V F curve for control- and AS-treated large DRG neurons before or before neurons DRG large AS-treated and control- for curve post-hoc mRNA or Kcna2 protein or produce truncated Kcna2 protein protein Kcna2 truncated or produce protein mRNA or Kcna2 = 65.77) stimuli from control and AS-injected rats; w, weeks. w, rats; weeks. AS-injected and control from stimuli = 65.77)

Supplementary Fig. 4a−g Fig. Supplementary NEUR Kcna2 antisense RNA RNA antisense Kcna2 mRNA in DRG neurons ( neurons DRG in mRNA , , c a Kcna4 . . ) Representative ) Representative F P test. ( test. = 18.45 for large, 20.65 for medium and 0.67 for small cells. * cells. small for 0.67 and medium for 20.65 large, for = 18.45 < 0.01 versus the AS group. Two-way ANOVA with Two-way with ANOVA group. AS the versus < 0.01 OSCI n After MTX = 33 large, 42 medium and 30 small cells from the control group (12 rats). rats). (12 group control the from cells small 30 and medium 42 large, = 33 P or < 0.01 versus the corresponding control group. Unpaired Student’s Student’s Unpaired group. control corresponding the versus < 0.01 i. 6a Fig. EN I c threshold , Scn10a f Supplementary Fig. 4h Fig. Supplementary d − C ) Resting membrane potential (RMP, potential membrane ) Resting h E ) Numbers of evoked APs from control- and AS-injected rats after application of different currents. Numbers of cells recorded recorded cells of Numbers currents. different of application after rats AS-injected and control- from APs evoked of ) Numbers

+50 mV –80 mV post-hoc

, advance online publication online advance , , b

n

d expression ( expression ) but did not alter basal expression of of expression basal alter not did but ) = 14 cells per group. group. per cells = 14

; ;

t

= 3.22 for large, 6.28 for medium and and medium for 6.28 large, for = 3.22 5 nA 100 ms 40 ms test. ( test. f ). Approximately 87.1% of labeled of labeled ). Approximately 87.1% Number of APs 0.5 1.0 1.5 2.0 2.5 3.0 Fig. 5 Fig. 0 i b 1 ) Ipsilateral (ipsi) and contralateral (contra) paw withdrawal responses to mechanical ( mechanical to responses withdrawal paw (contra) contralateral and (ipsi) ) Ipsilateral Fig. Fig. 6a Current density 4 3 2 Stimulation intensity

g

neurons. neurons. Kcna2 antisense (pA/pF) * 100 150 200 250 300 ). Thus, MZF1-induced MZF1-induced Thus, ). Control AS

50 ), a distribution similar similar a ), distribution nociceptive thresholds. thresholds. nociceptive Kcna2 * 0 –80 Fig. 6a Fig. , 8 5 * b F ). Then we injected ). we Then injected 6 * –60 = 139.21, = 139.21, post-hoc Large 1 7 * gene promoter. promoter. gene Control +MTX Control AS +MTX AS –40 * , Voltage (mV) b 9 * –20 ). We found ). c ( * × ; 1 0 100 pA)

test. Error bars, s.e.m. bars, Error test.

* 1 1 0

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*

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n * = 14 rats per group. ** group. per rats = 14 * 4 ** 40

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g i Paw withdrawal Number of APs 60 treated group retained 92.3 92.3 retained antisense– group Kcna2 the treated from neurons medium and large but ment, 81.7 retained group control the in neurons medium and mV,large +50 0.01; or ( voltages depolarized at group antisense–treated Kcna2 the antisense, ( large in current potassium voltage-gated total in reductions greater produced MTX of 100 nM maurotoxin (MTX), a selective Kcna2 current reduction was due to Kcna2 downregulation, we used bath application ( neurons medium-diameter and large- in lower significantly was sity den current potassium voltage-gated total group, antisense-treated group) and recorded only green DRG neurons ( of AAV5-Kcna2 antisense plus AAV5-EGFP (Kcna2 antisense-treated AAV5-EGFP (control group) injected alone or solution a viral mixed we efficiency, recording the Toincrease injection. after weeks 8–12 DRGs L4/5 injected the from dissociated freshly neurons in current Kcna2. targets selectively and RNA specifically antisense Scn10a ( was and significantly temporally reduced in the ipsilateral L4/5 DRGs injection ( viral after weeks 12 least at for high remained and weeks 8 at peak a reached weeks, 4 at DRGs L4/5 the in increased ficantly after SNL ( to that of RNA–positive Kcna2 antisense neurons in the injured DRG 0.5 1.0 1.5 2.0 2.5 3.0 Fig. 6c Fig. Fig. 6 Fig. Fig. 7a Fig. threshold (g) 0 P Using a voltage-clamp technique, we recorded Kcna2-related Kcna2-related recorded we technique, voltage-clamp a Using 10 15 20 25 c < 0.05 versus the same stimulation intensity in the control group. group. control the in intensity stimulation same the versus < 0.05 0 5 RMP (mV) 4 3 2 1 ± –65 –60 –55 –50 –45 1.7% and 85.1 Stimulation intensity –1 c were ( unaffected Time pointsafterinjection – ). In contrast, the expression of of expression the contrast, In ). , * AS Control 3 d Fig. 7a Fig. b Large e t * and and ). The amounts of mRNA and protein of of protein and mRNA of amounts The ). n -test. ( -test. Fig. Fig. 4 ** = 15) neurons from the control group than from in those Control AS * 8 5

8 w

Mechanical Medium * 6 Medium n P Supplementary Fig. 5a−c Fig. Supplementary * , 1 7 f = 43 large, 70 medium and 32 small cells from the AS the from cells small 32 and medium 70 large, = 43 e < 0.01 versus control on the ipsilateral side at the the at side ipsilateral the on control versus < 0.01 b ). ). Expression of the Kcna2 antisense RNA was signi ** 4 ) Representative traces of the evoked action potentials potentials action evoked the of traces ) Representative n ** and and * w = 14 per group) and medium and group) per 14 = * 9 Small ± ( * × 2.2% of treat respectively, after MTX current, 1 0 100 pA) ** Supplementary Fig. Supplementary w * 1 1 Fig. Fig. 6c * 1 2 12 * 1 3 ** d * w ± l (pA) 4 threshold – 0.9% and 94.9 94.9 and 0.9% 1,000 e 200 400 600 800 ). ). These results indicate that Kcna2 Paw withdrawal h Number of APs 0

latency (s) 0.5 1.0 1.5 2.0 2.5 3.0 Large 10 20 30 40 50 60 0 0 ** Control AS 1 –1 Kcna2 Medium 5 4 3 2 Stimulation intensity ). To verify whether this this whether verify To ). Time pointsafterinjection 3 d

AS Control 5a−c ** Control oncontra Control onipsi AS oncontra AS onipsi

w Fig. Fig. 7 F mRNA and protein protein and mRNA Small = 38.31) and and = 38.31) ± 4 t r a .% of 1.6% ). When tested at tested When ). 6

6 w (control: (control: Cold Kcna1 Small 1 7 a ). ). In the Kcna2 **

9 8 inhibitor w e 8 C I ( **

, , AS Control × w 1 0

Kcna4 100 pA) current, current, P 1 1 n 12 ** < 0.05 0.05 < e l = 17; 17; = 40 ms 1 2 40 mV w 22– 1 3 or 2 s 4 4  ­ - - .

© 2013 Nature America, Inc. All rights reserved. tions of Kcna2 antisense RNA showed behavioral changes in noci in changes behavioral showed RNA antisense Kcna2 of tions excitability. neuronal DRG increases inhibition 1 Table ( effects similar produced ( threshold, duration, overshoot and after-hyperpolarization amplitude amplitude, as such parameters, input potential action membrane other or in resistances groups two the between differences apparent ( neurons DRG small in observed were ( neurons number control medium and the large in in average group intensity the stimulation than corresponding the by greater evoked was group antisense–treated of stimulation by evoked potentials action of ( respectively pA, 344 and ( respectively neurons, medium and large in mV 10.52 and mV 6.74 by potentials, membrane resting increased significantly treatment RNA antisense Kcna2 group, control the to Compared injection. after weeks 8–12 recording current-clamp whole-cell out carried we excitability, nal neurons. DRG all in current related Kcna2- decreases and neurons DRG medium and large in densities current potassium voltage-gated total RNA reduces antisense Kcna2 group, ( significant still was groups antisense–treated Kcna2 and control between difference the but prominent, less was MTX by reduction current the neurons, DRG small In ­respectively. Tukey Two-way with ANOVA point. time corresponding the at group EGFP-treated the of ( SNL after and post-hoc * group. per rats thermal). for 12.45 and cold for 267.42 ( CCI or thermal) for 15.31 and cold for 545.13 mechanical, ( SNL after and before shown times the at responses withdrawal Paw hypersensitivities. pain CCI-induced or SNL- of ( in presented are Tukey with Two-way ANOVA group. EGFP-treated the of side # group. EGFP-treated the of side contralateral * sham-operated. group. per rats groups. SE–treated Kcna2 and treated EGFP- the in SNL or surgery sham after 14 day on DRGs L5 contralateral and ipsilateral the post-hoc Tukey Two-way with ANOVA CCI. or SNL after group EGFP-treated corresponding the versus surgery. sham after group ** group. groups. SE)-treated (Kcna2 fragment sense Kcna2 and EGFP-treated the in surgery sham or mRNA) for 8.73 and RNA AS ( CCI mRNA), for 10.26 and RNA ( SNL after 14 day on DRGs L5 (Con) contralateral and (Ipsi) ipsilateral the in expression RNA (AS) antisense Kcna2 and ( pain. neuropathic attenuates RNA antisense Kcna2 DRG of upregulation 8 Figure t r a  of not but antisense, AAV5–Kcna2 of Injection thresholds. ceptive c Supplementary Table 1 Table Supplementary P

, < 0.05 versus the corresponding ipsilateral ipsilateral corresponding the versus < 0.05 Lastly, we examined whether rats that received L4/5 DRG injec DRG L4/5 received that rats whether examined we Lastly, neuro DRG modulates RNA antisense Kcna2 whether Toassess d ) Effect of Kcna2 SE on the development development the on SE Kcna2 of ) Effect P

C I ). Our findings indicate that Kcna2 knockdown or current current or knockdown Kcna2 that indicate findings Our ). test. ( test. ( test. < 0.05; < 0.05; P Blocking nerve injury–induced injury–induced nerve Blocking < 0.01 versus the EGFP-treated EGFP-treated the versus < 0.01 post-hoc e l e b Fig. 7 Fig. F F ) Effect of Kcna2 SE on the maintenance of SNL-induced pain hypersensitivities. Paw withdrawal responses at the times shown before before shown times the at responses withdrawal Paw hypersensitivities. pain SNL-induced of maintenance the on SE Kcna2 of ) Effect ) Kcna2 protein expression in expression protein ) Kcna2 P = 9.26 in SNL and 0.53 in 0.53 and SNL in = 9.26 = 22.66 for mechanical, 104.16 for cold and 7.64 for thermal). thermal). for 7.64 and cold for 104.16 mechanical, for = 22.66 F Supplementary Figure 7 Figure Supplementary P Supplementary Fig. 5d−f Fig. Supplementary < 0.05, ** < 0.05, = 22.51 for mechanical, mechanical, for = 22.51 s < 0.05 versus corresponding corresponding versus < 0.05 test. Full-length blots blots Full-length test. c ), and reduced current thresholds by 217 pA pA 217 by thresholds current reduced and ), # P Supplementary Fig. 6 Supplementary ). Application of MTX into DRG neurons neurons DRG into MTX of Application ). F n < 0.05, < 0.05, P = 41.03 for AS for = 41.03 F = 12 rats per per rats = 12 P a < 0.01 versus the ipsilateral side of the EGFP-treated group at the corresponding time point. Two-way ANOVA with Tukey Two-way with ANOVA point. time corresponding the at group EGFP-treated the of side ipsilateral the versus < 0.01 = 23.25 for for = 23.25 < 0.01; 0.01; < ) ) Kcna2 F = 35.91 for for = 35.91 ## n mRNA mRNA P = 8 Fig. 7 Fig. < 0.01 < 0.01 . n

= 8 Fig. 7e Fig.

Fig. 7c Fig.

). These data indicate that that indicate data These ). d ). The average number number average The ). ≥ – , 300 pA in the Kcna2 Kcna2 the in pA 300 g and d n ). No such changes changes such No ). , = 11 neurons per per neurons 11 = a e d c h

). There were no no were There ). Paw withdrawal Paw withdrawal Paw withdrawal Relative amount (ipsi/con) 0. 1. 1. Supplementary Supplementary threshold (g) threshold (g) threshold (g) 0 1 2 3 4 5 0 5 0 10 15 20 25 30 10 15 20 25 30 10 15 20 25 30 0 5 0 5 0 5 –14 Sham –28 –28 injectio Vira injectio Vira Kcna2 SE EGFP Kcna2 ASRNA injectio Vira Kcna2 –1 l l l –1 –1 Days afterSNL n n Days afterCCI Days afterSNL n mRNA 7 5 3 Mechanical ** ** SNL 5 3 5 3 * * ## # - - -

** * 14 * SNL produced mechanical, cold and thermal hypersensitivities on on hypersensitivities thermal and cold mechanical, produced SNL block SNL-induced Kcna2 antisense RNA expression before that time. to fragment sense Kcna2 little too was there that showed work pilot tion, we subjected rats to SNL 4 weeks after DRG viral injection, as our the role examine of RNAKcna2 antisense pain in induc neuropathic ( rats sham-operated of DRG L5 ipsilateral the RNA in of expression basal alter not did alone fragment sense AAV5–Kcna2 of Injection 4 weeks and were ( maintained for at least 12 weeks after viral (CCI) injection. injury constriction Kcna2 blocked upregulation of Kcna2 antisense RNA and downregulation of AAV5-EGFP, not significantly but fragment, sense AAV5–Kcna2 of injury. nerve after our with Consistent thresholds nociceptive and expression Kcna2 in of lation DRG RNA DRG reductions Kcna2 antisense affect would upregu injury–induced Finally,nerve blocking we whether inquired Blocking DRG Kcna2 antisense RNA attenuates neuropathic pain pain. neuropathic of symptoms clinical main two hypersensitivities, cold and mechanical Kcna2 antisense RNA–triggered DRG Kcna2 that downregulation induces suggest findings These shown). not (data functions locomotor ( weeks 12 least at for maintained were and weeks 8 at peak a reached P ( respectively latency, withdrawal paw and threshold withdrawal paw in decreases ipsilateral by as demonstrated hypersensitivities cold and mechanical produced AAV5-EGFP, Fig. 7 Fig. ** 1 7 1 7 **

< 0.01; < 0.01; Relative amount (ipsi/con) 21 0. 1. 1. 2. 2. 0. 1. 1. ** 5 0 5 0 5 5 0 5 0 0 n ** 28 ** ** = 8 rats per group. * group. per = 8 rats 4 4 mRNA and protein in the injured DRGs after SNL or chronic chronic or SNL after DRGs injured the in protein and mRNA Sham i Kcna2 ASRNA ). Neither AAV5-Kcna2 antisense nor AAV5-EGFP affected affected AAV5-EGFP nor antisense AAV5-Kcna2 Neither ). Kcna2 post-hoc Fig. 7 Fig. Paw withdrawal Paw withdrawal Paw withdrawal advance online publication online advance latency (s) latency (s) latency (s) mRNA 30 40 50 60 10 20 30 40 50 60 10 20 30 40 50 60 10 20 0 0 0 Kcna2 ** i CCI * –14 ). These hypersensitivities developed by 4 to developed 6 weeks, hypersensitivities ). These –28 –28 Vira injectio injectio Vira injectio Vira test. Error bars, s.e.m. bars, Error test. # # –1 l l l –1 –1 mRNA and Kcna2 protein or Kcna2 antisense antisense Kcna2 or protein Kcna2 and mRNA in vitro in Days afterSN n EGFP contralateral Days afterCCI Days afterSN n n 7 5 3 EGFP ipsilateral * b β 7 5 3 5 3 Cold Kcna2 P * -acti

< 0.05, ** < 0.05, Relative amount 0. 0. 1. ** n work ( work ** 4 8 2 0 i. 8a Fig. 14 Con * L L ** EGFP ** 1 7 EGFP 21 ** Con Ipsi Sham ** ** 14 28 Fig. 6a Fig. ** 4 P , Con Kcna2 SE Kcna2 SE b < 0.01 versus the ipsilateral side side ipsilateral the versus < 0.01 Ipsi . hs efcs curd at occurred effects These ).

Paw withdrawal Paw withdrawal Paw withdrawal Ipsi

, latency (s) latency (s) latency (s) 10 12 10 12 10 12 b 6 8 0 2 4 6 8 0 2 4 6 8 0 2 4 nature nature ), ), n β Kcna2 –14 –28 –28 -acti = 14 rats per group, group, per rats 14 = in vivo in Viral injectio Viral injectio Viral injectio Relative amount 0. 0. 1. –1 n 4 8 2 0 Kcna2 SEcontralateral –1 –1 Days afterSN Con Days afterCCI Days afterSN n n n 5 3 EGFP Kcna2 SEipsilateral Therma NEUR EGFP DRG injection injection DRG 5 3 5 3 * Ipsi Fig. 8a Fig. * SN 1 7 * * Kcna2 SE Con l Kcna2 SE L OSCI 2 4 L 1 7 1 7 * * L Ipsi * # , 1 b EN * ). To). * 28 4 4 * C E - -

© 2013 Nature America, Inc. All rights reserved. subunits was unaffected, likely because they lack complementary complementary lack they because likely unaffected, was subunits heteromultimers functional are DRG sites. Kcna subunits initiation and translation transcription the including regions, complementary their of overlap extensive the to ited inhib specifically and selectively neurons DRG or cells HEK-293T antisense RNA, as overexpression of Kcna2 antisense RNA in cultured rons and Kcna2 protein were correspondingly downregulated in these neu medium-sized neurons in the injured DRG. Conversely,and large- to pattern distribution subpopulation its altered also but not nerve only Kcna2 peripheral RNAincreased antisense expression neurons DRG large-sized or medium- most in medium- expressed Kcna2 was protein highly DRG whereas neurons, sized) (mostly few a in level low a at expressed was RNA sense of studies. future our in possibilities addressed be will and excluded These be cannot modification. epigenetic other and/or stability RNA in increases by caused be might RNA antisense in increase the trigger activation of also Kcna2 antisense transcription is unknown. Additionally, factors transcription other Whether activation. MZF1 DRG through triggered was RNA antisense Kcna2 of upregulation injury–induced Nerve activation. by can be regulated transcriptional investigation. further merit and unusual are gene antisense Kcna2 the of mechanisms splicing rule), GT-AG the the of junctions the the as patterns exhibits splicing RNA same antisense Kcna2 the note, a Of is lncRNA. RNA encoded antisense Kcna2 that suggesting strongly sequence, the of most to complementary and kb 2.5 than more is genes RNA the of that from different in or locus genomic same the at genes RNA the in transcribed be can They mammals in naturally occur to shown recently were lncRNAs DISCUSSION treatment. pain neuropathic in applications clinical have may expression its blocking that and maintenance and development pain neuropathic to contributes RNA antisense Kcna2 that indicate rats not (data Our findings or shown). in sham-operated cold stimuli mechanical to responses basal altered or shown) not (data function ( side contralateral the on latency or threshold withdrawal paw affected AAV5-EGFP nor fragment sense ( rats fragment–injected sense Kcna2 the in SNL after 28 and 21 14, on days attenuated markedly were ties EGFP-injected rats on day 7 after SNL ( completely in developed both the Kcna2 and sense fragment–injected were hypersensitivities and cold thermal Mechanical, injection. viral pathic pain maintenance, we rats subjected to SNL 2 weeks after DRG ( well as model CCI the in ment of AAV5-Kcna2 effects on pain develop fragment neuropathic sense ( SNL after 14 to 3 days from group rats than in the EGFP-injected in fragment–injected the Kcna2 sense higher were stimuli thermal and to cold latency withdrawal paw and ( rats hypersensitivity was attenuated in the Kcna2 sense fragment–injected group ( in EGFP-injected the side ipsilateral the nature nature occurs RNA antisense Kcna2 of effect inhibitory the and sequences Kcna2 antisense RNA functions as a biologically active regulator regulator active biologically a as functions RNA antisense Kcna2 RNA, of that like of native Kcna2 antisense mRNA, the Expression neuro in RNA antisense Kcna2 of role the investigate Tofurther Kcna2 Kcna2 5– Fig. 8 Fig. 1 0 . This downregulation is likely caused by the increase in Kcna2 NEUR mRNA in primary afferent neurons. Normally, anti Kcna2 neurons. afferent mRNA primary in mRNA and protein expression. This effect may related be effect This mRNA expression. and protein c ). Paw withdrawal threshold to mechanical stimulation stimulation mechanical to threshold withdrawal Paw ). OSCI Kcna2 EN C sense gene are canonical (that is, they follow follow they is, (that canonical are gene sense E advance online publication online advance cis Kcna2 from the opposing DNA strands of of strands DNA opposing the from 9 Fig. 8 Fig. , 26– 2 sense RNA. Because the splice splice the Because RNA. sense 8 Fig. 8 Fig. Fig. 8c Fig. . The expression of other Kcna Kcna other of expression The . Fig. 8 Fig. d 2 Fig. Fig. 8 ). 5 . Rat Kcna2 antisense RNA antisense Kcna2 Rat . e c ). Neither AAV5–Kcna2 Neither ). e – 9 ). We observed similar similar observed We ). ). ). These hypersensitivi . Of note, injury to the the to injury note, Of . e ), affected locomotor locomotor affected ), Fig. 8 Fig. trans c ). By contrast, ). By contrast, Kcna2 from a locus locus a from Kcna2

mRNA RNA 18 cis , 1 9 ------.

membrane. In addition, the depolarized resting membrane potential potential membrane resting depolarized the addition, In membrane. gated channels example, Kcna1, Kcna4), hyperpolarization-activated cyclic nucleotide- resistance also depends on other voltage-gatedinput membrane potassium that channelsfact the (for with consistent is that observation an in DRG neuronal increase membrane inputinsignificant, resistances, but modest, a RNAproduced antisense Kcna2 by knockdown Kcna2 Naby predominantly determined be may parameters two affect action potential threshold or amplitude in DRG neurons, as these lation was also reported previously DRG neuronal resting membrane potential by DRG Kcna2 downregu action potentialsof number in large the and increased medium and DRG neurons.potentials Depolarizationaction of of activation for old depolarized the resting membrane potential, decreased current thresh Kcna2 antisense RNA decreased total voltage-gated potassium current, neurons those in expressed poorly is Kcna2 because possibly neurons, DRG small in observed was reduction Minimal neurons. in these function channel potassium voltage-gated determining in subunit key a is Kcna2 that indicates neurons DRG medium and large in density current sium that potas fact voltage-gated total the reduced markedly However, downregulation Kcna2 heteromultimers. of formation the before could create positive feedback to further reduce DRG reduce further to feedback positive create could activity This expression. gene govern that factors transcription ing includ DRG genes, of other expression the in changes SNL-induced formation. sensitization Persistent central reduction spinal in DRG of neuronal attenuation excitability in may also resulting block further that may enhance the decrease in primary afferent transmitter release, Kcna2 downregulation causes persistent reduction in DRG excitability DRG blocking We that points. think time late at the hypersensitivity pain SNL-induced abolishes almost Kcna2 DRG of downregulation cold attenuated and and mechanical Kcna2 and induction maintenance of injury–induced nerve DRG in reduction the reversed RNA anti Kcna2 sense of upregulation SNL-evoked blocking that fact the by supported is conclusion This pain. neuropathic of maintenance and to development the contributes which sensitization, central to spinal leads and terminals afferent these primary of their from release the neurotransmitters drives neurons DRG medium and large of ity d injury 2 nerve as after early as increased markedly are neurons DRG medium and large in and fibers myelinated injured the in CGRP and P Substance stimuli. cold sig noxious and exhibited mechanical to RNA hypersensitivities nificant antisense Kcna2 overexpressed that animals Indeed, hyperexcitability. to prone more neurons those render may neurons DRG of potential membrane resting the of RNA–induced depolarization antisense Kcna2 neurons. DRG large and in medium mainly RNA antisense Kcna2 increased injury nerve Peripheral considered to play a role leading in the genesis of neuropathic pain bodies neuronal DRG medium and large corresponding the in have which found injured and myelinated been primarily afferents injury nerve after peripheral reported has been resting membrane potentials are markedly depolarized in DRG neurons larization only in the of absence a resting membranesignificant depo potential observed was neurons DRG in current potassium voltage-gated ing resistance. input The membrane increase in in membrane input increase resistance caused by deficiency-induced block Kcna2 counteract by may itself conductance resting potassium increase We found that selective reduction of Kcna2 expression in DRG by DRG in expression Kcna2 of reduction Weselective that found Nerve injury-induced increases in spontaneous ectopic activity, activity, ectopic spontaneous in increases injury-induced Nerve

hypersensitivities. It is still a puzzle how blocking SNL-induced SNL-induced how blocking a puzzle It still is hypersensitivities. 3 4 . The fact membrane input resistance is unchanged but that 31 , 3 9 2 . and chloride channels 3 , 3 8 . It is very likely that the increase in excitabil in increase the that likely very is It . 29 , 3 0 . . Kcna2 antisense RNA did not 3 3 expressed on DRG neuronal 3 5 . t r a 31 , 3 2

, which may, which excitability. excitability. + C I channels. channels. 36 , e l 3 7 , are , 1 , s 3  ------.

© 2013 Nature America, Inc. All rights reserved. 7. 6. 5. 4. 3. 2. 1. reprints/index.htm at online available is information permissions and Reprints The authors declare no competing interests.financial Y.-X.T. wrote the manuscript. All authors read and the manuscript.discussed X.Z., Z.T., H.Z., F.E.A., J.-Y.Z. L.L., W.W., X.G., V.T. and Y.-X.T. H.Z. and analyzed the M.L. data. performed voltage-clamp experiments. P.N.H. did microinjection. and behavioral experiments. Z.T. and X.D. performed current-clamp experiments. F.E.A., J.-Y.Z., L.L., W.W., X.G., S.-C.K. and V.T. performed molecular, biochemical F.E.A., J.-Y.Z., Y.-J.G., H.C., M.L., X.D. and Y.-X.T. the designed project. X.Z., Y.-X.T. conceived the project most and experiments. X.Z., Z.T.,supervised H.Z., NS072206) and the grants from the Rita Allen Foundation to Y.-X.T. grants (MH084691 and GM078579) to M.L. and by NIH grants (DA033390, Institutes of Health (NIH) grants (NS054791 and GM087369) to X.D., by NIH work was supported by the Howard Hughes Medical Institute and US National University) for MZF1 antibody. We thank C.F. forLevine editorial assistance. This for EGFP-MZF1the full-length plasmid and D.Y.H. Tuan (Georgia Health Sciences J. Zhou (Institute ofof Biochemistry Biology,Cell AcademyChinese of Sciences) We thank R.J. Samulski (University of North Carolina) for the rAAV5 plasmid, Note: Supplementary information is available in the the in version available are references associated any and Methods M fields. other in research of conduct the affect the will also but pain regarding neuropathic of advances development conceptual provide only not findings our Therefore, RNAs. antisense and sense both of transcription reverse in RNAs antisense endogenous detect they as controls, negative as designed For example, we ular methodologies. cannot probes use sense usually demonstration of Kcna2 antisense RNA may challenge current molec addition, In processes. pathological other in implicated be may they RNA, sense anti Kcna2 Because disorder. this of for therapies of targets development potential the and pain, neuropathic in mechanism novel excitability, a neuronal and expression channel Kcna2 of regulation pain. neuropathic treating for target a be may expression channel Kcna2 of maintenance. Regulation and development pain neuropathic in trigger enous together, our suggest that findings Kcna2 RNAantisense is an endog potential These t r a  COM AUTH Acknowledgmen

ethods In summary, identification of Kcna2 antisense RNA may point to to point may RNA antisense Kcna2 of identification summary, In i, .. Co, .. Rm HD & h, .. oneuain f voltage-gated of Downregulation H.J. Cho, & H.D. Rim, J.O., Choi, D.S., Kim, of expression in Changes S.G. Waxman, & J.A. Black, M., Tanaka, K., Ishikawa, conductance potassium maintains factor growth Nerve J.D. Kocsis, & B. Everill, response maladaptive a pain: Neuropathic C.J. Woolf, & J. Scholz, M., Costigan, A in in discharge neurons Ectopic DRG M. of Devor, hyperexcitability of Importance K. Chung, & J.M. Chung, pain. neuropathic of Meyer,Mechanisms & R.A. J.N. Campbell, (2002). chronic constriction injury of following the rat sciatic ganglia nerve. root dorsal in expression gene alpha channel potassium Nerve Muscle neurons. ganglion voltage-gated potassium channels in dorsal root ganglion neurons following axotomy. root dorsal afferent cutaneous adult in Neuroscience injury nerve after damage. to system nervous the of Res. Brain Exp. pain. neuropathic (2006). P O E TI R R C I of the pape the of CON NG in situ in Kcna2 e l FI

TRIBUTI

100 22 N l .

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© 2013 Nature America, Inc. All rights reserved. tions. The 3 The tions. 5 the amplificationof (2nd kit RACE 5 The a Indianapolis,IN). Diagnostics, Roche Generation, using by then and primers strand-specific with RT-PCR 56 °C, and 20 s at 72 °C for 35 cycles. 0.5 20 in Inc.) Laboratories, (Clontech ase Table 2 ( database genome UCSC the from determined were primers primers, RNA(1 cDNAfromsingle-stranded transcribed reverse we strand-specific with CA) Valencia, (QIAGEN, kit RT Omniscript (MountainUsingView, the Inc.CA). Laboratories, Clontech from purchased were DRG human from samples RNA DNase-treated purified, Highly MA). Ipswich,Biolabs, England (New I DNaseexcess with treated(Invitrogen) and RT-P quantitative and Reverse transcription (RT)-P to each well. Cells were collected 4 d later. 5% CO St. Louis, MO) and laminin (10 glass coverslips or in a six-well plate coated with poly- were resuspended in DH10 and plated at a density of 1.5 × 10 out Ca enzyme solution (3.5 mg/ml dispase, 1.6 mg/ml collagenase type I in HBSS with lin-streptomycin (Quality Biological, Gaithersburg, MD)) and then treated with (Gibco, Grand Island, NY), 10% FBS (JR Scientific, Woodland, CA), DMEM/F-121% (90% penicil DH10 cold in collected were DRGsisoflurane. with death D (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. 2000 Lipofectamine with cells HEK-293T the into transfected were plasmids modified Eagle’s medium at 37 °C in a humidified incubator with 5% CO C described protocols reflexes, were performed before and after viral injection according to previously ously previ described as USA) Instruments,WoodlandCA, Science Hills,Inc./Life latencies to noxious heat were measured with Model 336 Analgesia Meter (IITC avoidto used wascutoff s ofAtissue 60time damage. Finally, pawwithdrawal was repeated three times at 10-min intervals for the paw on the ipsilateral side. paw licking and flinching, was defined as the paw withdrawal latency. Each trial placement of the hind paw on the plate and the animal jumping, with or without chamber on the cold plate, which was set at 0 °C. Plexiglas The a length in of placed time betweenwas thecontinuously.animal monitored Each was which of temperaturetheplate, cold a with measured then were °C) (0 coldnoxious to up–down testing paradigm as described previously drawal thresholds in response to mechanical stimuli were first measured withwere the carried out. Each behavioral test was carried out at 1-h intervals. Paw with Behavioral analysis. procedures but without transection of the respective nerve. carried out as described previously. Sham-operated groups underwent identical (CCI) injury N to treatment blind condition. were experimenters the of All used. animals of number the reduce to and suffering animal minimize to made were efforts All Pain. of Study the for Association International the and Health of Institutes guidelines National US the ethical of the with consistent and University Hopkins Johns the at Committee Use and Care Animal the by approved were used procedures All standard 12-h light/dark cycle, with water and food pellets available Animals. ONLINE METHODS doi: hydroxyl group of the RNA, followed by gene- and adaptor-specific amplification. ell line culture and transfection. erve erve injury models. R RNA fragments amplified from the rat DRG were extended first by using by first extended were DRG rat the from amplified fragments RNA G 10.1038/nn.3438 µ M of PCR primer. PCR amplification consisted of 30 s at 94 °C, 20 s at s 20 °C, 94 at s 30 of consisted amplification PCR primer. PCR of M 4 neuronal culture and AAV5and cultureneuronal transduction. 1 2+ 2 . Tests of locomotor function, including placing, grasping and righting andgrasping placing,Testsincluding locomotorfunction, . of ). Template (1 at 37 °C. One day later, 1 and Mg ae pau-aly as egig 0–5 g ee et n a in kept were g 200–250 weighing rats Sprague-Dawley Male ′ 4 RACE analysis was performed by ligating an adaptor to the 3- the to adaptor an ligating by performed was analysis RACE 2 and sciatic nerve axotomy nerve sciatic and 2+ (Gibco)) at 37 °C. After the centrifugation, dissociated cells 39 Mechanical, cold, thermal and locomotor behavioral tests µ , 40 ′ L5 spinal nerve ligation (SNL) -end of cDNA according to the manufacturer’sthe tocDNAaccording instrucof -end l) was amplified by PCR with TaKaRa Taq DNA polymer , 4 C 3 R. . Total RNA was extracted by the Trizol method method Trizol the by extracted was RNA Total C µ R, R, rapid amplification of c µ g/ml, Invitrogen). The cells were incubated in l of AAV5 virus (titer HEK-293T cells were cultured in Dulbecco’s µ 3 ttl ecin oue containing volume reaction total l 9 models of neuropathic pain were pain neuropathic of models Adult male rats were put to putwererats Adult male 3 9 l . Paw withdrawal latencies 39– -lysine (0.5 mg/ml, Sigma, ≥ 4 1 × 10 1 , chronic constriction ′ RACE was used for used was RACE DN 5 to 4 × 10 µ 12 g). RT and PCR andRT g). Supplementary A ends (RA /ml) was added ad libitum 5 cells on 2 . The CE ------) .

turer’s instructions with the single-cell real-time RT-PCR assay kit (Signosis). RT-PCR procedure was carried out as described Aftercentrifugation, supernatantsthe were collected. Theremaining real-time a PCR tube with 6 with a glass micropipette, without contamination by other neurons, with anda micromanipulatorplaced in and microinjector. A single living neuron was selected largeDRGneurons wererandomly invertedanunder collected microscope fit firstprepared below. described as Four hours after plating, small,medium and was demonstrated to be stable after SNL ( efficiency ( amplification reactions of the target genes and reference genes have similar PCR ∆ Ratios of ipsilateral mRNA to contralateral mRNA were calculated by using the 96-well plates in an ABI 7500 Fast real-time PCR system (Applied Biosystems). kitmix(Applied Biosystems, Grand Island, ReactionsNY).were performedin run in quadruplicate in a 20- and0.5 Template(1 providetowere pooled enoughRNA. cDNA preparedwas above. described as RNA sequence determined. antisense Kcna2 full-length the and analyzed were sequences All sequencing. 5 All primers are listed in were treated as described above except that they were hybridized only with only hybridized were they that except above described as treated were situ in of labeling double the Forsubstrates. chloride tetrazoliumnitro-blue and salt production. virus and constructs rAAV5plasmid nals were developed with 5-bromo-4-chloro-3with developed were nals were incubated with alkaline phosphatase–conjugated anti-digoxigenin. The sig antisense RNA and tions were hybridized with digoxigenin-dUTP-labeled cRNA probes for Kcna2 IN). After treatment with proteinase K and prehybridization, the two sets of sec according to the manufacturer’s instructions (Roche Diagnostics, Indianapolis, ment) were prepared by Kcna2 20- modification minor with previously described as out carried In situ membrane was washed and dried, autoradiography was carried out. at 68 °C with ultraviolet light. After prehybridization, the membrane was hybridized overnighta BrightStar-plus positively charged nylon membrane and cross-linked by using RNA (10 generated from ing. The plasmid construct was linearized by Acc65I and XhoI. A riboprobe was DNA template, and identified the sequence using double-strand DNA sequenc antisense RNA, we constructed the pSC-A plasmid, which contained a 0.946-kb N produced at the University of North Carolina Vector Core. cytomegalovirus promoter. rAAV5 viral particles carrying the four cDNAs were Kcna2 antisense RNA, Kcna2 sense fragment and MZF1 under the control of the GFP (EGFP) and the S-D sequence. The resulting four vectors expressed EGFP, enhanced replace to Hill) Chapel Carolina, North(University of plasmids ral clone sequencing. positive clones were identified by restriction enzyme analysis (BspEI/NotI) and productswerecloned cloningeasybypGEM-T usingthe(Invitrogen). kit The 5 the at introduced were sites nested by amplified were RT-PCR (primers +40) in to (−311 fragment cDNA sense Kcna2 and full-length cDNA, antisense Kcna2 full-length DRG, the from ′ Ct(2 method orthern blotting. RACE and 3 For single-cell quantitative RT-PCR, freshly dissociated rat DRG neurons were rats individual three from DRGs RT-PCR,three quantitativereal-time For Northern blot analysis was performed as described previously Theidentified fragments were ligated into the BspEI/NotI sites of the provi µ m sections were collected from each DRG by grouping every third section. cRNA probe (0.268-kb fragment) and hybridization histochemistry and immunohistochemistry, the sections immunohistochemistry,andthe histochemistry hybridization hybridization histochemistry. µ M of each primerofeachlisted inM µ g) was separated on a 1.5% agarose/formaldehyde gel, transferred to Supplementary Fig. 8a µ 32 l) was amplified by real-time PCR by using 1 using by PCR real-time byamplified was l) P-dUTP-labeled cRNA probes for Kcna2 antisense RNA. After the ′ − RACE were extracted, purified and verified by automated DNA in vitro ∆ ∆ µ Ct To prepare complementary RNA (cRNA) probes of rat Kcna2 GFP l of cell lysis buffer (Signosis, Sunnyvale, CA) as described ) atthreshold)a ourofaspilot0.02, dataindicated that the Supplementary Table 2 transcription and labeled with in vitro RNA for 18 h at 68 °C. After being washed, the sections Supplementary Table 2 µ l reaction with the TaqMan Universal PCR master transcription and labeled with digoxigenin-dUTP ′ and 3 and ). All data were normalized to SupplementaryTable 2 ′

ends of the three fragments. The PCR PCR The fragments. three the of ends In situ Supplementary Fig. 8b hybridization histochemistry was ). Restriction enzyme recognition GFP ′ 4 -indolyl phosphate-indolyl . PCR products from RT-PCR, 4 or according to the manufac cRNA probe (0.187-kb frag nature nature After RNA was extracted extracted was RNA After 32 P-dUTP. NEUR µ . Each sampleEach . was 46 M of each probe each of M 4 5 , 4 . The extracted Gapdh − 7 . Two sets of Two sets . Mzf1 d OSCI p ). -toluidine cDNA , which EN C 4 4 E ------.

© 2013 Nature America, Inc. All rights reserved. n. Frigae N) o ha crmtn n poue .- o -b DNA 1-kb to 0.2- produce and chromatin shear to NY) Farmingdale, Inc., disruptor(Misonix cell ultrasonic an withsonicated was suspension The tors. SDS, 1% sodium deoxycholate and 1% NP-40 in the presence of protease inhibi collected,waswashedand lysispelletsuspended buffer inthe containing 0.1% centrifugation, After glycine. M 0.25 of addition the by terminated reaction the and min 5 for °C 37 atformaldehyde 1% with cross-linked was DRG the C 25 °C for 30 min before the assay. assay, the nuclear extracts were incubated with rabbit MZF1 antibody (2 carriedout after the gel was dried. For supershift electrophoretic mobility shift ation, the DNA-protein complexes were subjected to probePAGE. at 25 Autoradiography°C for 20 min. wasLabeled probe alone was used as alabeled control.probe (9 ng)After alone incub or with 50× unlabeled probe or 50× unlabeled mutant Healthcare, Silver Spring, MD). DRG nuclear extract (5 shown in competitors.similarlypreparedas foruse oligonucleotideTheir aresequences were probemutant unlabeled and probeUnlabeled min. 30 for °C 72 at then containing solution polymerase PCR H-Star in oligonucleotide synthetic annealing by prepared was E quantified with densitometry. was blots of intensity The film. to exposure and NJ) Piscataway, and visualized by ImmunoResearch) chemiluminescence regents Jackson (ECL; Amersham (1:3,000 Pharmacia Biotech, antibody secondary anti-rabbit or proteinsThe horseradishbyweredetected peroxidase–conjugated anti-mouse Biotechnology) and rabbit anti–histone H3 (1:1,000, Cell Signaling Technology). Tuan,Medical ofCollegeGeorgia) NeuroMab) 1:1,000; (Nav1.8, PKC Technology,Signaling anti-mTORMA) Danvers,Cell (1:1,000, (Kv1.2, 1:200, NeuroMab) CA) NeuroMab,Davis, (Kv1.1,1:200, anti-Kcna1 mouse used: were antibodies primary following The h. 1 Tween-20for 0.1% taining stripssmall and thenblocked with nonfat3% Tris-bufferedin milk salinecon several into cut were membranes the weights, molecular protein targeted the to According MA). Billerica, (Immobilon-P,membraneMillipore, difluoride polyvinylidene a onto CA). transferred electrophoretically then were Hercules, proteins The Laboratories, (Bio-Rad gel SDS-polyacrylamide separating the samples were heated at 99 °C for 5 min and loaded onto a 4% stacking/7.5% fornuclearpellet proteins. theand proteinAfter concentration measured,was 1,000 at fluoride, 1 mM EDTA, 1 ultrasonicatedin chilled lysis buffer (50 mM Tris, 1mM phenylmethylsulfonyl to provide enough protein. The tissues were homogenized and the cultured cells W cal methods as described camera.Single-and double-labeled neurons were counted byusing stereologi spot CCD a with captured and Japan) Co., (Nikon microscope fluorescence TE2000ENikon a under examined immunofluorescence-labeledwereimages All antiserum. primary the ofomission and antiserum primary the for serum temperature (25 °C). Control experiments included substitution ImmunoResearch)of normalor with mouseFITC-labeled avidin D (1:200, Sigma) for 2 h at room Cy2 (1:400, Jackson ImmunoResearch, West Grove, PA) or Cy3 (1:400, Jackson 4 °C. The sections were then incubated with goat anti–rabbit IgG conjugated to anti-GFAP (1:500, Sigma) CGRP (1:500, EMD, Billerica, MA) (1:500, Neuromics, Edina, MN) MO) Louis, St. Sigma-Aldrich, (1:500, anti-NF200 rabbit with incubatedwere Tritonsections 0.3%the andX-100, serum goat 10% ing Immunohistochemistry. the fluorescent signals were and developed with Fast RNA Red. antisense Kcna2 for probe cRNA digoxigenin-dUTP–labeled nature nature fragments. After the samples were precleared with protein G–agarose, they were lectrophoretic mobility shift assay. hromatin immunoprecipitation assay.immunoprecipitationhromatin estern blotting. α (1:500, Santa Cruz Biotechnology, Santa Cruz, CA) g , the supernatant was collected for cytosolic and membrane proteinsmembraneand cytosolic for collected supernatantwas the , Supplementary Table 2 NEUR OSCI For DRG, three DRGs from three individual rats were pooled EN µ 4 C 9 After being blocked for 1 h at 37 °C in PBS containPBS in °C at37 h forblocked1 being After 48 9 32 , mouse anti-Kcna4 (Kv1.4, 1:300, NeuroMab) M leupeptin). After centrifugation at 4 °C for 15 min . E or mouse anti-OX-42 (1:400, Sigma) P-dCTP, dATP and dTTP at 56 °C for 1 min and min 1 for °C 56 at P-dCTP,dTTP and dATP 5 0 48 , rabbit anti-MZF1 (1:200, provided by D.Y.H.by provided (1:200, anti-MZF1 rabbit , . All probes were purified in a G50 column (GE , biotinylated IB4 (1:100, Sigma) 48 2 1 , mouse anti-NeuN (1:600, EMD) , mouseanti–,

32 P-labeled double-stranded DNA probe The homogenization solution from solutionhomogenization The β -actin (1:2,000;Santa-Cruz-actin µ 4 g) was incubated with 9 0 48 , mouse anti-Kcna2 mouse , , mouse anti-Scn10a , rabbit anti-P2X3 rabbit , 48 48 48 , rabbit anti-rabbit , overnight at , rabbit anti- 48 9 , mouse , rabbit µ g) at ­ - - - -

W were performed. to activity firefly (Promega).relativeThe reporter activityobtainedwas bynormalization ofthe assayedDual-LuciferaseAssayfortheluciferaseReporter withSystem activity transfectedwerecellspassivewith lysed lysisbuffer and40 midsusing Lipofectamine 2000 (Invitrogen). After another 48ofh culture, the 1 plus or alone WI) Madison, Promega, control; normalizing (a plasmid pRL-TK the of ng 40 with transfected were cells the Supplementary Table 2 in shown are sequences primer All sequencing. DNA by verified were clones recombinant of sequences The WI). Madison, the (Promega, vector of pGL3-Basic sites restriction HindIII and SmaI the into subcloned were products PCR The DNA. genomic from PCR by motif) MZF1-binding the (including the from fragment 633-bp the and region the fromfragment 1,268-bp the amplified we plasmids, L used are listed in containingprimers All predicted binding theMZF1by PCR. detectedsite was positivecontrol.a as nt)fragmentof(156The protein(10 (5 immunoprecipitated with 5 room temperature. atexperimentswereperformed All softwarepackage(Molecular9.2Devices). on computer by a DigiData 1322A interface and were analyzed by the pCLAMP AP overshoot was measured between the AP peak and 0 mV.between the maximum hyperpolarization The and the final plateaudata voltage, wereand the stored from 200 pA to −2,000 pA. The after-hyperpolarization amplitude was measured series of hyperpolarizing currents, 200-ms duration delivered in steps of 100 pA each cell was obtained from the slope of a steady-state for resistance input membrane The baseline. the and peak the between ured which the change in voltage exceeded 50 mV/ms. The AP amplitude was meas threshold was defined as the first point on the rapid rising phase of the spike at at the existing resting membrane potential during the current injection. The AP mumcurrent required to evoke the first AP. The membrane potential was held (AP) was tial evoked. The injection current threshold was asdefinedthemini (200-ms duration) were delivered in incrementspA 100–1,400 of of 100 currents pA Depolarizing untilobtained. anfirst actionwas recordingpoten stable a 300 mOsm with sucrose. The resting membrane potential was taken 3 min after toadjusted osmolarityKOH and with 7.38 toadjusted was pH 5; glucose and Mg-ATPNaKCl135, 3, mM) contained(in tion and glucose 5, with pH adjusted to 7.38 by NaOH. The intracellular pipette solu CaCl KClsolutionNaCl4,140, mM)consisting (in of extracellular with perfused andchamber the clamp.in Coverslipsplacedwere analyzed by the pCLAMP 9.2 software package (Molecular Devices). tion. The data were stored on computer by a DigiData 1322A interface and were onlineP forcapacitancewhole-cellvalue compensationamplifier. thefrom directly An pensated by 60–80%. Cell membrane capacitances were acquired by reading the 1322A with pClamp 9.2 software (MolecularDigiData a using by Devices).digitized and kHz Series1 at resistancefiltered were Signals mOsm). was320 com KCl 5, CdCl 150, chloridecholine mM) (in ofsolution composedextracellular an usingby mized the Na Wemini mOsm). 310 KOH, with 7.3 (pH 4 Mg-ATP 10, HEPES 10, EGTA pipette solution contained (in mM) potassium gluconate 120, KCl 20, MgCl Axopatch-700B amplifier (Molecular Devices, M Sunnyvale, 4 CA). to The intracellular 2 from ranged micropipettes of resistances electrode The recorded. were neurons green-labeled Only CT). Coverslips were placed in the perfusion chamber (Warner Instruments,above. Whole-cellHamden, patch clamp recording was carried out 4 to 24described h after as plating.neurons DRG rat dissociated freshly prepared first we neurons, uciferase assay. hole-cell patch clamp recording. µ HEK-293T cells were prepared as described above. After 24 h of culture, of h 24 After above. described as prepared were cells HEK-293T To record the action potential, we switched the recording mode into current g) or rabbit anti-MZF1 (5 / 4 leak subtraction was performed to eliminatecurrentsubtractiontoperformedcontribu wasleak leak 4 µ 2 + 1, CaCl g). Input (10% of the sampleimmunoprecipitation)forthe Inputof g). (10% used was and Ca To construct the Supplementary Table 2 Renilla 2 2+ 2, MgCl component in voltage-gated potassium current recording . activity.experimentstransfectionindependent Three µ g of rabbit anti-MZF1 (ref. µ 2 g) after preabsorption with excess MZF-1 fusion 1, HEPES 10, glucose 10 (pH 7.4 with Tris base, Kcna2 To record total potassium current in DRG Ω gene and . Cells were voltage-clamped with an with voltage-clamped were Cells . . Kcna2 Kcna2 2 Kcna2 ATPCaCl 0.5, µ nies gn promoter gene antisense g of the constructed plas constructed the of g antisensegenepromoter I–V 2 2 2, MgCl 2, 1 antisense gene reporter ), normal rabbit serum Kcna2 plot in response to a doi: µ l supernatantl was 10.1038/nn.3438 gene promotergene 2 2 2, HEPES 10 HEPES 2, 1.1, EGTA1.1, 2 2 2, ------

© 2013 Nature America, Inc. All rights reserved. 40. 39. reported previously in the fields were used to predetermine sample sizes, but our sample sizes are similar to those by the showed significant difference, pairwise comparisons between means were tested or unpaired Student’s not formally tested. The data were statistically analyzed with two-tailed, paired given as means mals were distributed into various treated groups randomly. All of the results are then randomly distributed in each well tested. For Statistical analysis. from viral injection were therefore minimal. structural integrity and contained no visible leukocytes. The immune responses malities. The injected DRGs, stained with hematoxylin and eosin, retained their woundwith clips. injectedTherats showed signsofnoparesis orotherabnor pipetteremovedwas Afterinjection,min.afterincisionskin10 the closedwas site in the L5 DRG with a glass micropipette connectedviral tosolution a (2 Hamilton syringe. The vertebral body was exposed. After the lamina was removed andBriefly, thea midline DRGincision was exposed,made in the lower lumbar back region and the L5 D doi: R

G Singh, O.V. of expression up-regulates injury nerve Peripheral Y.X. Tao, & X. Zhu, X., Guan, horn after peripheral nerve injury. nerve peripheral after horn ganglion. root dorsal rat in mRNA (IPCEF1) 1 factor exchange cytohesin for protein interactor 10.1038/nn.3438 microinjection. post hoc Naunyn Schmiedebergs Arch. Pharmacol. Arch. Schmiedebergs Naunyn et al. Tukey method (SigmaStat, San Jose, CA). No statistical methods ± µ s.e.m. Data distribution was assumed to be normal, but this was l) was injected into two sites in the L4 and L5 DRGs or into one Proteome of synaptosome-associated proteins in spinal cord dorsal For t -test and a one-way or two-way ANOVA. When ANOVA DRG microinjection was carried out as described as out carried was microinjection DRG in vitro experiments, the cells were evenly suspended and 39– 4 Proteomics 1 . Significance was set at

9 , 1241–1253 (2009). 1241–1253 , in vivo

380 , 459–463 (2009). 459–463 , experiments, the ani P < 0.05. 51 , 5 2 - - . 46. 45. 44. 43. 42. 41. 52. 51. 50. 49. 48. 47.

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