HALO, a novel bHLH-PAS protein induced by neuronal preconditioning and ischemia, mediates cytotoxicity through BAX gene upregulation

Abstract

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 mRNA induced by potassium depolarization in vivo. We have isolated a cDNA encoding a novel bHLH-PAS protein distantly related to SIM2, termed HALO. Our results confirm that HALO mRNA and protein are rapidly and transiently expressed in cortical neurons following CSD but not following short duration ischemia, another form of pre-ischemic conditioning. In the untreated adult brain, HALO is expressed at low levels but is highly expressed during embryonic development in neuronal lineages. Surprisingly, delayed HALO expression is also observed following middle cerebral artery occlusion (MCAO) in rats. Reporter assays show that HALO is a transcriptional activator that associates with the bHLH-PAS sub-class co-factor ARNT2. Adenovirus-mediated expression of epitope-tagged HALO results in the direct induction of the Bax gene and sensitization of cultured cells to cytotoxic stress. Together, our data indicate that HALO is a novel bHLH-PAS transactivator transiently induced by preconditioning and that its sustained expression is detrimental. The identification of HALO may represent an important step in our understanding of the molecular mechanisms of brain preconditioning and injury.