Axotomy-Induced miR-21 Promotes Axon Growth in Adult Dorsal Root Ganglion Neurons

et al. (2011) Axotomy-Induced miR-21 Promotes Axon Growth in Adult Dorsal Root Ganglion Neurons. PLoS ONE 6(8): e23423. doi:10.1371/journal.pone.0023423 Axotomy-Induced miR-21 Promotes Axon Growth in Adult Dorsal Root Ganglion Neurons Iain T. Strickland 0 Louise Richards 0 Fiona E. Holmes 0 David Wynick 0 James B. Uney 0 Liang-Fong Wong 0 Stephen D. Ginsberg, Nathan Kline Institute and New York University, United States of America 0 1 School of Clinical Sciences, University of Bristol , Bristol , United Kingdom , 2 School of Physiology and Pharmacology, University of Bristol , Bristol , United Kingdom Following injury, dorsal root ganglion (DRG) neurons undergo transcriptional changes so as to adopt phenotypic changes that promote cell survival and axonal regeneration. Here we used a microarray approach to profile changes in a population of small noncoding RNAs known as microRNAs (miRNAs) in the L4 and L5 DRG following sciatic nerve transection. Results showed that 20 miRNA transcripts displayed a significant change in expression levels, with 8 miRNAs transcripts being altered by more than 1.5-fold. Using quantitative reverse transcription PCR, we demonstrated that one of these miRNAs, miR-21, was upregulated by 7-fold in the DRG at 7 days post-axotomy. In dissociated adult rat DRG neurons lentiviral vectormediated overexpression of miR-21 promoted neurite outgrowth on a reduced laminin substrate. miR-21 directly downregulated expression of Sprouty2 protein, as confirmed by Western blot analysis and 39 untranslated region (UTR) luciferase assays. Our data show that miR-21 is an axotomy-induced miRNA that enhances axon growth, and suggest that miRNAs are important players in regulating growth pathways following peripheral nerve injury. - Funding: This work was supported by the Medical Research Council (grant code G0700986) and Research Councils UK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Sciatic nerve injury triggers gene expression changes in the dorsal root ganglion (DRG) of transected nerves and in the microenvironment of the nerve stumps. These transcriptional alterations translate into phenotypic changes that enable the damaged DRG neurons to adapt to the injury, for example by promoting stress response and cell survival pathways as well as growth programs to regenerate severed axons. In order to elucidate the molecular pathways that contribute to neural regeneration a number of laboratories have undertaken microarray and proteomic approaches to identify differentially expressed genes and proteins in DRG neurons following nerve axotomy [19]. A large number of genes and proteins were found to be regulated; these were diverse and distinct, comprising members of several classes such as neuropeptides, receptors, ion channels, signal transduction molecules, synaptic vesicle proteins, cell cytoskeletal components, extracellular matrices and inflammatory mediators. While these studies have provided an insight into the molecular changes that occur in the injured nerve and its environs, it is still unknown how these global changes are regulated in a coordinated fashion. One possibility is transcriptional regulation by signal transduction molecules or transcription factors such as cAMP [10,11], c-Jun [12] or retinoic acid receptor b2 [13,14]. Another possible mechanism of coordinated control can occur at the post-transcriptional level, for example regulation by microRNAs (miRNAs). miRNAs have recently emerged as important post-transcriptional regulators in several developmental and physiological processes. In the nervous system, miRNAs have been implicated in cell specification [15,16], neurite outgrowth [17], dendritic spine development [1820] and disease [21,22]. More recently it has been demonstrated that abolition of the miRNA pathway in the Nav1.8 population of nociceptive neurons attenuated inflammatory pain [23]. We postulated that altered miRNA levels after peripheral nerve injury can contribute to growth programs that promote axonal regeneration. Here we show that an axotomyregulated miRNA, miR-21, promotes neurite growth from injured adult DRG neurons by targeting the Sprouty2 protein. Our results uncover a role for miRNAs in regulating axonal regeneration following peripheral nerve injury. miRNA regulation after sciatic nerve transection We carried out a microarray screen to determine miRNA changes in adult rodent DRG after sciatic nerve injury. After transection, injured peripheral nerves initially undergo Wallerian degeneration before regrowing. The timepoint of 7 days postaxotomy was chosen to capture miRNA expression profiles at a time when the injured neurons were beginning to regenerate. Total RNA was extracted from axotomised and control contralateral DRGs and simultaneously hybridised to microarrays that contained probes from all mouse mature miRNAs listed in the Sanger database (Sanger version 9.0). Statistical analyses revealed that 20 miRNA transcripts were differentially expressed in axotomised DRG compared to the non-axotomised contralateral DRG; with 8 being upregulated and 12 down-regulated (p,0.05; Fig. 1A, Table S1). Using a 1.5-fold cut-off, 8 candidate miRNAs remained for further investigation (Fig. 1B). miR-21, miR-223, miR-455-5p, miR-431 and miR-18 were significantly increased, while miR-138, miR-483 and miR-383 were significantly decreased following nerve transection. These observed changes were further validated by quantitative real-time reverse transcription PCR (qRT-PCR) and from these candidates we chose to further examine miR-21 (Fig. 1C). Following axotomy, miR-21 expression increased 7-fold and 3fold in the mouse and rat DRG respectively (Fig. 2A), indicating that injury-induced upregulation of miR-21 was replicated in both rodent models. qRT-PCR analysis also indicated that miR-21 increased significantly as early as 2 days post-injury, which was sustained 28 days post-injury (Fig. 2B). In situ hybridisation studies confirmed that the upregulation of miR-21 occurred in rat DRG neurons at 7 days post-injury (Fig. 2C). Analysis of miR-21 neuronal profiles indicated that increased miR-21 expression occurred in neurons of all sizes, with 29.0 6 4.1%, 40.5 6 4.2% and 29.6 6 2.6% of all miR-21 expressing neurons found to be in small (,30 mm), medium (3040 mm) and large (.40 mm) diameter neurons respectively (Fig. 2D). Furthermore, co-localisation experiments demonstrated that miR-21 was detected in large diameter neurons expressing neurofilament 200kD (NF200) as well as small and medium diameter neurons expressing Calcitonin gene-related peptide (CGRP, Fig. 2E). miR-21 increases neurite outgrowth from DRG neurons In order to determine the effects of miR-21 on neuronal growth, we overexpressed miR-21 in dissociated adult rat DRG neurons that were plated on a red (...truncated)