Michela Dell'Orco1, Pamela Milani2, Laura Arrigoni3, Orietta Pansarasa4, Valentina Sardone5, Elisa Maffioli6, Francesca Polveraccio7, Matteo Bordoni8, Luca Diamanti9, Mauro Ceroni10, Fiorenzo A Peverali11, Gabriellla Tedeschi12, Cristina Cereda13. 1. Laboratory of Experimental Neurobiology, "C. Mondino" National Institute of Neurology Foundation, IRCCS, Via Mondino 2, 27100 Pavia, Italy; Department of Brain and Behavioural Sciences, University of Pavia, Via Forlanini 9, 27100 Pavia, Italy. Electronic address: michela.dellorco@outlook.com. 2. Laboratory of Experimental Neurobiology, "C. Mondino" National Institute of Neurology Foundation, IRCCS, Via Mondino 2, 27100 Pavia, Italy. Electronic address: pame.milani@gmail.com. 3. Institute of Molecular Genetics (IGM) CNR, Via Abbiategrasso 207, 27100 Pavia, Italy. Electronic address: arrigoni@ie-freiburg.mpg.de. 4. Laboratory of Experimental Neurobiology, "C. Mondino" National Institute of Neurology Foundation, IRCCS, Via Mondino 2, 27100 Pavia, Italy. Electronic address: orietta.pansarasa@mondino.it. 5. Laboratory of Experimental Neurobiology, "C. Mondino" National Institute of Neurology Foundation, IRCCS, Via Mondino 2, 27100 Pavia, Italy. Electronic address: valentina.sardone@gmail.com. 6. Filarete Foundation, Viale Ortles 22/4, 20139 Milan, Italy; Department of Veterinary Science and Public Health, University of Milan, via Celoria 10, 20133 Milano, Italy. Electronic address: elisa.maffioli@guest.unimi.it. 7. Laboratory of Experimental Neurobiology, "C. Mondino" National Institute of Neurology Foundation, IRCCS, Via Mondino 2, 27100 Pavia, Italy. Electronic address: francesca.polveraccio@gmail.com. 8. Laboratory of Experimental Neurobiology, "C. Mondino" National Institute of Neurology Foundation, IRCCS, Via Mondino 2, 27100 Pavia, Italy; Department of Brain and Behavioural Sciences, University of Pavia, Via Forlanini 9, 27100 Pavia, Italy. Electronic address: matteo.bordoni02@universitadipavia.it. 9. Department of Brain and Behavioural Sciences, University of Pavia, Via Forlanini 9, 27100 Pavia, Italy; Division of General Neurology, "C. Mondino" National Institute of Neurology Foundation, IRCCS, Via Mondino 2, 27100, Pavia, Italy. Electronic address: luca.diamanti@mondino.it. 10. Department of Brain and Behavioural Sciences, University of Pavia, Via Forlanini 9, 27100 Pavia, Italy; Division of General Neurology, "C. Mondino" National Institute of Neurology Foundation, IRCCS, Via Mondino 2, 27100, Pavia, Italy. Electronic address: mauro.ceroni@mondino.it. 11. Institute of Molecular Genetics (IGM) CNR, Via Abbiategrasso 207, 27100 Pavia, Italy. Electronic address: peverali@igm.cnr.it. 12. Filarete Foundation, Viale Ortles 22/4, 20139 Milan, Italy; Department of Veterinary Science and Public Health, University of Milan, via Celoria 10, 20133 Milano, Italy. Electronic address: gabriella.tedeschi@unimi.it. 13. Laboratory of Experimental Neurobiology, "C. Mondino" National Institute of Neurology Foundation, IRCCS, Via Mondino 2, 27100 Pavia, Italy. Electronic address: cristina.cereda@mondino.it.
Abstract
BACKGROUND: It is still unclear whether oxidative stress (OS) is a disease consequence or is directly involved in the etiology of neurodegenerative disorders (NDs) onset and/or progression; however, many of these conditions are associated with increased levels of oxidation markers and damaged cell components. Previously we demonstrated the accumulation of reactive oxygen species (ROS) and increased SOD1 gene expression in H2O2-treated SH-SY5Y cells, recapitulating pathological features of Amyotrophic Lateral Sclerosis (ALS). Since we observed a post-transcriptional regulation of SOD1 gene in this cellular model, we investigated the transcriptional regulation of SOD1 mRNA under oxidative stress (OS). RESULTS: In response to H2O2 treatment, PolII increased its association to SOD1 promoter. Electrophoretic mobility shift assays and mass spectrometry analyses on SOD1 promoter highlighted the formation of a transcriptional complex bound to the ARE sequences. Western Blotting experiments showed that in our in vitro model, H2O2 exposure increases Nrf2 expression in the nuclear fraction while immunoprecipitation confirmed its phosphorylation and release from Keap1 inhibition. However, H2O2 treatment did not modify Nrf2 binding on SOD1 promoter, which seems to be regulated by different transcription factors (TFs). CONCLUSIONS: Although our data suggest that SOD1 is transcriptionally regulated in response to OS, Nrf2 does not appear to associate with SOD1 promoter in this cellular model of neurodegeneration. Our results open new perspectives in the comprehension of two key antioxidant pathways involved in neurodegenerative disorders.
BACKGROUND: It is still unclear whether oxidative stress (OS) is a disease consequence or is directly involved in the etiology of neurodegenerative disorders (NDs) onset and/or progression; however, many of these conditions are associated with increased levels of oxidation markers and damaged cell components. Previously we demonstrated the accumulation of reactive oxygen species (ROS) and increased SOD1 gene expression in H2O2-treated SH-SY5Y cells, recapitulating pathological features of Amyotrophic Lateral Sclerosis (ALS). Since we observed a post-transcriptional regulation of SOD1 gene in this cellular model, we investigated the transcriptional regulation of SOD1 mRNA under oxidative stress (OS). RESULTS: In response to H2O2 treatment, PolII increased its association to SOD1 promoter. Electrophoretic mobility shift assays and mass spectrometry analyses on SOD1 promoter highlighted the formation of a transcriptional complex bound to the ARE sequences. Western Blotting experiments showed that in our in vitro model, H2O2 exposure increases Nrf2 expression in the nuclear fraction while immunoprecipitation confirmed its phosphorylation and release from Keap1 inhibition. However, H2O2 treatment did not modify Nrf2 binding on SOD1 promoter, which seems to be regulated by different transcription factors (TFs). CONCLUSIONS: Although our data suggest that SOD1 is transcriptionally regulated in response to OS, Nrf2 does not appear to associate with SOD1 promoter in this cellular model of neurodegeneration. Our results open new perspectives in the comprehension of two key antioxidant pathways involved in neurodegenerative disorders.
Authors: Maida De Bortoli; Elena Taverna; Elisa Maffioli; Patrizia Casalini; Francesco Crisafi; Vikas Kumar; Claudio Caccia; Dario Polli; Gabriella Tedeschi; Italia Bongarzone Journal: J Exp Clin Cancer Res Date: 2018-04-03