VCP binding influences intracellular distribution of the slow Wallerian degeneration protein, Wld S Anna L. Wilbrey, a Jane E. Haley, b Thomas M. Wishart, b Laura Conforti, a Giacomo Morreale, a Bogdan Beirowski, a Elisabetta Babetto, a Robert Adalbert, a Thomas H. Gillingwater, b Trevor Smith, a David J.A. Wyllie, b Richard R. Ribchester, b and Michael P. Coleman a, ⁎ a The Babraham Institute, Babraham Research Campus, Cambridge, CB22 3AT, UK b Centre for Neuroscience Research, University of Edinburgh, 1 George Square, Edinburgh, EH8 9JZ, UK Received 13 October 2007; revised 5 March 2008; accepted 14 March 2008 Available online 25 March 2008 Wallerian degeneration slow (Wld S ) mice express a chimeric protein that delays axonal degeneration. The N-terminal domain (N70), which is essential for axonal protection in vivo, binds valosin-containing protein (VCP) and targets both Wld S and VCP to discrete nuclear foci. We characterized the formation, composition and localization of these potentially important foci. Missense mutations show that the N-terminal sixteen residues (N16) of Wld S are essential for both VCP binding and targeting Wld S to nuclear foci. Removing N16 abolishes foci, and VCP binding sequences from ataxin-3 or HrdI restore them. In vitro, these puncta co-localize with proteasome subunits. In vivo, Wld S assumes a range of nuclear distribution patterns, including puncta, and its neuronal expression and intranuclear distribution is region-specific and varies between spontaneous and transgenic Wld S models. We conclude that VCP influences Wld S intracellular distribution, and thus potentially its function, by binding within the N70 domain required for axon protection. © 2008 Elsevier Inc. All rights reserved. Introduction Axonal degeneration contributes to many neurodegenerative disorders including amyotrophic lateral sclerosis (ALS), Alzhei- mer's disease and multiple sclerosis and often precedes cell death (Ferguson et al., 1997; Ferri et al., 2003; Fischer et al., 2005; Stokin et al., 2005). Hence, understanding its molecular basis could lead to new treatment strategies. An excellent model for studying mechanisms regulating axonal breakdown after toxic or physical injury is the Wld S mouse, which carries a spontaneous mutation causing a tenfold delay in Wallerian degeneration; the process by which the severed distal stump of an injured axon dies (Lunn et al., 1989; Mack et al., 2001). Furthermore, Wld S can delay axon degeneration and attenuate symptoms in mouse models of some ‘dying-back’ disorders (Ferri et al., 2003; Mi et al., 2005; Sajadi et al., 2004; Samsam et al., 2003). Thus, axon degeneration in both injury and disease is a highly regulated process, potentially amenable to therapeutic intervention. The remarkable delay in axon degeneration in the Wld S mouse results from a tandem triplication, which produces a novel chimeric gene at its internal boundaries (Coleman et al., 1998). The resulting in-frame fusion protein derives its N-terminal 70 amino acids (N70) from the E4 ubiquitin ligase Ube4b, followed by the full coding sequence of Nmnat1, an NAD + synthesizing enzyme. The two domains are separated by eighteen amino acids (Wld18) from the Nmnat1 5′ UTR that becomes translated. This cDNA dose- dependently delays Wallerian degeneration when expressed in transgenic mice, rats and flies and also in virally transduced dorsal root ganglion (DRG) explant cultures (Adalbert et al., 2005; Hoopfer et al., 2006; Macdonald et al., 2006; Mack et al., 2001; Wang et al., 2001). Exactly how Wld S delays Wallerian degeneration remains controversial. Several reports have suggested that increased NAD + synthesis is sufficient in vitro and strong overexpression of Nmnat1 also confers a degree of axo-protective phenotype in Drosophila in vivo (Araki et al., 2004; Macdonald et al., 2006; Wang et al., 2005). However, Nmnat1 alone is not sufficient for any detectable effect in mice, so more N-terminal sequences are also required (Conforti et al., 2007). Nmnat1 and Ube4b portions also appear to act together in modulating potential downstream transcripts or proteins (Gillingwater et al., 2006; Wishart et al., 2007). Wld S requires its N-terminal domain to bind VCP as well as for phenotype. VCP is an AAA-ATPase with many functions including a critical role in the ubiquitin proteasome system (UPS) (Laser et al., 2006). VCP is partially redistributed by Wld S into discrete nuclear foci (Laser et al., 2006) suggesting a role for the N-terminal sequence in subcellular targeting of Wld S . Neither parent protein alone produce these foci; Nmnat1 has a diffuse nuclear distribution while Ube4b is www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 38 (2008) 325 – 340 ⁎ Corresponding author. B501, The Babraham Institute, Babraham Research Campus, Cambridge, CB22 4AT, UK. Fax: +44 1223 496348. E-mail address: michael.coleman@bbsrc.ac.uk (M.P. Coleman). Available online on ScienceDirect (www.sciencedirect.com). 1044-7431/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2008.03.004