UNCORRECTED PROOF 1 Posttranscriptional regulation of SOD1 gene expression under oxidative 2 stress: Potential role of ELAV proteins in sporadic ALS 3 Pamela Q1 Milani a,b, ⁎ ,1 , Marialaura Amadio c, ⁎⁎ ,1 , Umberto Laforenza d , Michela Dell'Orco a,b , Luca Diamanti b,e , 4 Valentina Sardone a,b , Stella Gagliardi a , Stefano Govoni c , Mauro Ceroni b,e , Alessia Pascale c , Cristina Cereda a 5 a Laboratory of Experimental Neurobiology, “C. Mondino” National Institute of Neurology Foundation, IRCCS, Via Mondino 2, 27100 Pavia, Italy 6 b Department of Public Health, Neuroscience, Experimental and Forensic Medicine, University of Pavia, Via Ferrata 9, 27100 Pavia, Italy 7 c Department of Drug Sciences, Pharmacology Section, University of Pavia, Viale Taramelli 14, 27100 Pavia, Italy 8 d Department of Molecular Medicine, University of Pavia, Via Forlanini 6, 27100 Pavia, Italy 9 e Division of General Neurology, “C. Mondino” National Institute of Neurology Foundation, IRCCS, Via Mondino 2, 27100 Pavia, Italy 10 11 abstract article info 12 Article history: 13 Received 4 March 2013 14 Revised 5 July 2013 15 Accepted 7 August 2013 16 Available online xxxx 17 18 19 20 Keywords: 21 Sporadic amyotrophic lateral sclerosis 22 SOD1 23 mRNA 24 Posttranscriptional regulation 25 ELAV proteins 26 Peripheral blood mononuclear cells 27 Motor cortex 28 Spinal cord 29 Increased levels of SOD1 mRNA have been observed in sporadic ALS patients (SALS) compared to controls. Hence, 30 the understanding of the mechanisms by which SOD1 gene expression is modulated may shed new light on SOD1 31 involvement in ALS. Of interest, some adenine/uracil-rich elements ( Q2 AREs) in SOD1 3′-untranslated region have 32 been identified. These sequences represent the docking sites for several RNA-binding proteins such as ELAV pro- 33 teins (ELAVs), positive regulators of gene expression. We first investigated in SH-SY5Y cells whether SOD1 mRNA 34 represents a target of ELAVs. Results from RNA Electrophoretic Mobility Shift and RNA-immunoprecipitation 35 assays showed a molecular interaction between ELAVs and SOD1 mRNA. We also observed that the treatment 36 with H 2 O 2 induced a significant increase of the amount of SOD1 mRNA bound by ELAVs and an up-regulation 37 of SOD1 protein levels. We found a specific increase in ELAV/HuR phosphorylation, suggesting activation of 38 this protein, in peripheral blood mononuclear cells from SALS patients compared to controls. Finally, we found 39 increased levels of ELAV proteins in the motor cortex and spinal cord from SALS patients compared to controls, 40 in parallel with SOD1 up-regulation in the same areas. This study suggests, for the first time, that ELAVs are 41 involved in the regulation of SOD1 gene expression at post-transcriptional level and that these proteins are 42 more activated in ALS pathology. The link between ELAVs and SOD1 may open novel perspectives for ALS 43 research, paving the way for new therapeutic options. 44 © 2013 Published by Elsevier Inc. 45 46 47 48 49 Introduction Q4 50 Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenera- 51 tive disease clinically characterized by progressive muscular paralysis 52 reflecting the degeneration of both upper and lower motor neurons. 53 The majority (90%) of cases is sporadic (SALS), while the remainder 54 presents a family history (FALS). As recently reviewed by Cozzolino 55 et al. (2012), ALS is a complex and multifactorial disease characterized 56 by the involvement of several pathogenic conditions, including oxida- 57 tive stress. In particular, the occurrence of distinctive oxidation markers, 58 such as elevated protein carbonyl levels, increased lipid peroxidation 59 and DNA/RNA oxidative modifications, has been reported in the ner- 60 vous and peripheral tissues in both sporadic and familial ALS patients 61 (Babu et al., 2008; Q5 Barber and Shaw, 2010; Bogdanov et al., 2000; 62 Bonnefont-Rousselot et al., 2000; Chang et al., 2008; Cova et al., 2010; 63 Robberecht, 2000; Shaw et al., 1995; Simpson et al., 2003). An important 64 contribution towards the understanding of ALS pathogenesis came from 65 the discovery of mutations within the gene encoding for Cu/Zn superox- 66 ide dismutase (SOD1) which accounts for the 15–20% of FALS patients. 67 In addition, there is increasing evidence that SOD1 protein is also 68 involved in sporadic ALS, since the wild-type form of the protein un- 69 dergoes conformational changes due to either age- or environmental- 70 dependent posttranslational modifications, such as oxidation, rendering 71 its behavior similar to mutSOD1 (Bosco et al., 2010; Ezzi et al., 2007; 72 Guareschi et al., 2012). Furthermore, we reported that also the increase 73 of SOD1 mRNA expression is a distinctive feature of sporadic ALS pathol- 74 ogy compared to other neurodegenerative diseases and healthy controls 75 (Gagliardi et al., 2010). Notably, SOD1 mRNA level is elevated in both 76 nervous areas typically affected by ALS disease (i.e., the brain stem and 77 spinal cord) and peripheral blood mononuclear cells (PBMCs) from 78 SALS patients compared to control population. The increased SOD1 79 mRNA expression in lymphocytes of SALS patients has been recently Neurobiology of Disease xxx (2013) xxx–xxx ⁎ Corresponding author. ⁎⁎ Corresponding author. Fax: +39 0382 987405. E-mail addresses: pame.milani@gmail.com (P. Milani), amadio@unipv.it (M. Amadio). Available online on ScienceDirect (www.sciencedirect.com). 1 These authors equally contributed to the work. YNBDI-03022; No. of pages: 10; 4C: 4, 5, 7, 8 0969-9961/$ – see front matter © 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.nbd.2013.08.005 Contents lists available at ScienceDirect Neurobiology of Disease journal homepage: www.elsevier.com/locate/ynbdi Please cite this article as: Milani, P., et al., Posttranscriptional regulation of SOD1 gene expression under oxidative stress: Potential role of ELAV proteins in sporadic ALS, Neurobiol. Dis. (2013), http://dx.doi.org/10.1016/j.nbd.2013.08.005