Research Report Transient expression of Nxf, a bHLH–PAS transactivator induced by neuronal preconditioning, confers neuroprotection in cultured cells Ian Hester a,1 , Sarah McKee a,1 , Phillip Pelletier b , Charles Thompson b , Christopher Storbeck a , Alan Mears c , Jörg B. Schulz d , Antoine A. Hakim a,b , Luc A. Sabourin a,b, ⁎ a University of Ottawa, Department of Cellular and Molecular Medicine, Ottawa, ON, Canada b Ottawa Health Research Institute, Neuroscience Program, Ottawa, ON, Canada c Ottawa Health Research Institute, Vision Program, Ottawa, ON, Canada d Department of Neurology, University of Tübingen, Hoppe-Seyler-Str. 3, D-72076 Tübingen, Germany ARTICLE INFO ABSTRACT Article history: Accepted 28 November 2006 Available online 9 January 2007 Cortical spreading depression (CSD) induces waves of neuronal depolarization that confer neuroprotection to subsequent ischemic events in the rat brain. To gain insights into the molecular mechanisms elicited by CSD, we used representational difference analysis (RDA) to identify mRNAs induced by potassium depolarization in vivo. Using this approach, we have isolated a cDNA encoding the SIM2-related bHLH–PAS protein Nxf. Our results confirm that Nxf mRNA and protein are rapidly and transiently expressed in cortical neurons following CSD. Reporter assays show that Nxf is a transcriptional activator that associates with the bHLH–PAS sub-class co-factor ARNT2. Adenovirus-mediated expression of epitope- tagged Nxf results in cell death and the direct activation of the Bax gene in cultured cells. However, RNA interference studies show that endogenous Nxf is required for optimal neuroprotection by preconditioning in cultured F-11 cells. Together, our data indicate that Nxf is a novel bHLH–PAS transactivator transiently induced by preconditioning and that its sustained expression is detrimental. The identification of Nxf may represent an important step in our understanding of the molecular mechanisms of brain preconditioning and injury. © 2006 Elsevier B.V. All rights reserved. Keywords: Nxf bHLH–PAS Neuronal preconditioning Spreading depression 1. Introduction One potential approach to the treatment of stroke is to har- ness the molecular events elicited by neuroprotective treat- ments such as cortical spreading depression (CSD), a form of pre-ischemic conditioning. CSD was first described more than 50 years ago as a slowly propagating wave of depressed cortical neuronal activity (Leao, 1944). CSD may be induced experimentally by mechanical trauma, electrical stimulation, the application of high potassium or a variety of other BRAIN RESEARCH 1135 (2007) 1 – 11 ⁎ Corresponding author. Ottawa Health Research Institute, University of Ottawa, 451 Smyth Rd, Ottawa, ON, Canada K1H 8M5. Fax: +1 613 562 5403. E-mail address: lsab@uottawa.ca (L.A. Sabourin). 1 These authors contributed equally to this work. 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.11.083 available at www.sciencedirect.com www.elsevier.com/locate/brainres