European Journal of Neuroscience, Vol. 11, pp. 1545–1553, 1999 © European Neuroscience Association ARNT2, a transcription factor for brain neuron survival? Guillaume Drutel,* Anne He ´ ron, 1, * Markus Kathmann,* Claude Gros, Se ´ verine Mace ´ , Michel Plotkine, 2 Jean-Charles Schwartz and Jean-Michel Arrang Unite ´ de Neurobiologie et Pharmacologie Mole ´ culaire (U.109) INSERM, Centre Paul Broca, 2ter rue d’Ale ´ sia, 75014 Paris, France 1 Laboratoire de Physiologie and 2 Laboratoire de Pharmacologie, Faculte ´ des Sciences Pharmaceutiques et Biologiques, 4 Avenue de l’Observatoire, 75006 Paris, France Keywords: antisense, apoptosis, cell proliferation, PC12 cell, rat brain Abstract The processes responsible for the limited ability to divide and long survival of neurons are not well understood but may involve aryl hydrocarbon receptor nuclear translocator 2 (ARNT2), a recently identified protein, apparently belonging to the basic helix– loop–helix superfamily of transcription factors, which is expressed almost exclusively in brain during the whole lifetime. In agreement, we show, in the rat, that ARNT2 immunoreactivity could be observed only within nuclei of brain neurons and of dividing and neuronal PC12 cells, a localization consistent with a role in transcription regulation. Cell death elicited either by focal ischaemia in brain or oxidative stress in PC12 cells was largely preceded by an almost complete suppression of ARNT2 expression. In contrast, when PC12 cell cycle progression was impaired, ARNT2 expression was enhanced. Finally, the downregulation of ARNT2 levels induced by antisense oligonucleotides prevented PC12 cell proliferation and induced apoptosis. These observations support the hypothesis that ARNT2 is a neuronal transcription factor, regulating cell cycle progression and preventing cell death, whose sustained expression might ensure brain neuron survival. Introduction Within each lineage, the control of cell number is determined by a balance between the two highly regulated processes of cell prolifera- tion and cell death, which occurs generally by apoptosis (Raff, 1992; Wyllie, 1993; Thompson, 1995). Adult neural cells are almost unique by their very limited capacity for self-renewal, associated with a high survival capacity, so that the lifetime of most neurons is the same as that of the organism. Whereas an increasing number of effectors of the apoptotic cell death program (e.g. proteins such as caspases or proteins related to the BCL-2 oncoprotein) are being identified (Chinnaiyan & Dixit, 1996; Peter et al., 1997; Wallach, 1997), little is known about the mechanisms through which their, presumably coordinated, gene expression is triggered, and the processes involved in the long survival of neurons are almost unknown. Arnt2 is a recently identified gene which is expressed in kidney and brain, throughout development and adult life. Its sequence suggests that it belongs to the basic helix–loop–helix (bHLH) superfamily of transcription factors, but the 712-amino acid protein, aryl hydrocarbon receptor nuclear translocator 2 (ARNT2), that it encodes was neither characterized nor localized within brain cells and its function is unknown (Drutel et al., 1996; Hirose et al., 1996). ARNT2 deduced sequence contains a conserved domain designated PER-ARNT-SIM (PAS), shared by the period protein (PER) and the single-minded protein (SIM). It is homologous (63% identity) to that of ARNT (now designated ARNT1), a nuclear translocator which heterodimerizes with other bHLH-PAS-containing partners such as Correspondence: Dr J.-M. Arrang, as above. E-mail: arrang@broca.inserm.fr *These authors contributed equally to this study. Received 16 June 1998, revised 19 October 1998, accepted 3 December 1998 the aryl hydrocarbon receptor (AHR), SIM or the hypoxia-inducible factor-1α (HIF-1α) (Hoffman et al., 1991; Sogawa et al., 1995; Swanson et al., 1995; Wang et al., 1995); the ARNT1-containing dimers appear to bind to and regulate the expression of genes involved in the control of biological responses to xenobiotics or hypoxia (Hankinson, 1995; Semenza, 1996). In contrast to arnt1, which is expressed in an ubiquitous fashion in the whole body, arnt2 mRNA was only detected in embryo and adult brain and kidney in rodents (Drutel et al., 1996; Hirose et al., 1996). The continuous and heterogeneous expression of arnt2 in the brain, throughout development and adult life, suggested to us the presence of the protein it encodes in neurons and its role in the control of the related processes of cell division and/or death. We have presently explored these hypotheses by establishing the cellular and intracellular localizations of ARNT2, evaluating the changes in its expression during cell cycle progression and death and the effects of inhibiting selectively its synthesis on these processes. Materials and methods Northern blot analysis Poly(A) + mRNAs (8 μg) isolated from various brain regions of male Wistar rats or from treated or untreated PC12 cells were run on a 1% agarose gel containing 1 M formaldehyde, blotted overnight onto nitrocellulose filters (BAS 85, Schleicher and Schuell, Dassel, Germany) and immobilized by heating at 80 °C for 2 h. The blots were first probed with a 32 P-labelled arnt2 probe (Drutel et al., 1996) using QuickHyb (Stratagene, La Jolla, CA, USA) and rehybridized with a 32 P-labelled fragment of a rat β-actin cDNA. Autoradiograms were scanned with an absorbance detector (ISCO UA-5; Roucaire,