TorsinA protects against oxidative stress in COS-1 and PC12 cells Rohini Kuner a , Peter Teismann b , Annette Trutzel c , Jomana Naim c , Angelika Richter d , Nicole Schmidt b , Oliver von Ahsen c , Alfred Bach c , Boris Ferger e , Armin Schneider c, * a Pharmacology Institute, University of Heidelberg, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany b Institute for Pharmacology and Toxicology, University of Marburg, Ketzerbach 63, 35037 Marburg, Germany c Axaron Bioscience AG, Im Neuenheimer Feld 515, 69120 Heidelberg, Germany d Institute of Pharmacology and Toxicology, School of Veterinary Medicine, FU Berlin, Koserstrasse 20, 14195 Berlin, Germany e Behavioural Neurobiology Laboratory, Swiss Federal Institute of Technology, Schorenstrasse 16, CH-8603 Schwerzenbach, Switzerland Received 3 July 2003; received in revised form 18 July 2003; accepted 21 July 2003 Abstract Dystonia is a highly frequent movement disorder, the pathogenesis of which remains unclear. The cloning of TorsinA, the gene responsible for early-onset dystonia, was a major breakthrough. However, the function of this protein remains unclear. By sequence homology, TorsinA belongs to the ATPases associated with diverse cellular activities-family, many of whose members are chaperones and/or proteases. We report here that in an in vitro model for oxidative stress, H 2 O 2 treatment, overexpression of TorsinA was protective against cell death. COS-1 cells overexpressing TorsinA demonstrated drastically reduced terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling- staining following exposure to H 2 O 2 . Furthermore, transfection with TorsinA significantly increased survival of PC12 after H 2 O 2 treatment. To our knowledge, this is the first demonstration that TorsinA protects against oxidative stress. We speculate that a loss of this cellular function in mutant TorsinA may be linked to the pathogenesis of early-onset dystonia. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Dystonia; TorsinA; Oxidative stress Dystonia is one of the most frequent movement disorders, which is characterized by twisting movements and abnor- mal postures, and causes severe disability and social stigmatization [3]. The cloning of the gene mutated in early-onset dystonia, TorsinA, represented a major break- through in dystonia research [11]. Apart from early-onset dystonia, the glutamate deletion near the carboxy-terminus of the TorsinA protein can apparently cause different dystonic phenotypes [8]. The TorsinA protein belongs to the AAA (ATPases associated with diverse cellular activities) family, many of whose members are chaperones and/or proteases [10]. We hypothesized, therefore, that TorsinA might function as a stress-related protein in neurons involved in dystonic pathophysiology. Thus, TorsinA may perform a cytoprotective function in refolding and trafficking proteins denatured by stressful stimuli. Therefore, we utilized the H 2 O 2 -model for in vitro oxidative stress and studied the effects of overexpression of TorsinA on cellular survival following H 2 O 2 treatment. The results of the above experiments provide evidence for a protective role for TorsinA in cellular stress. For expression in mammalian cells, the open reading frame of TorsinA and Bax was cloned into a cytomegalo- virus (CMV) promoter-containing vector. The construct was fully sequenced on both strands. For COS-1 cell transfec- tions, cells were cultured in Dulbecco’s Modified Earle Medium (DMEM) containing 10% fetal calf serum and 1% Penicillin-Streptamycin (all reagents from Life technol- ogies, Karlsruhe, Germany) at 378C and 5% CO 2 and transfected using Lipofectamine Plus reagent (Life Tech- nologies) according to the manufacturer’s instructions. Typically, a transfection efficiency of approximately 40% was reached. Expression of constructs after transfection in COS-1 cells was confirmed by reverse-transcriptase (RT)- polymerase chain reaction analysis on cDNAs synthetized by using MMLV-reverse transcriptase and intron-spanning, gene-specific primers (sense-1: ATA GGT GCG GTT CCG 0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00904-2 Neuroscience Letters 350 (2003) 153–156 www.elsevier.com/locate/neulet * Corresponding author. Tel.: þ49-6221-454-713; fax: þ 49-6221-454- 700. E-mail address: schneider@axaron.de (A. Schneider).