Previously we reported a chemical genetic screen that reveal

Previously, we reported a chemical genetic screen that revealed that several statins activate a β-catenin-responsive luciferase reporter (BAR) in a cell-based assay (Biechele et al., 2010). This result supports prior in vitro studies that have shown that statins modulate Wnt/β-catenin signaling (henceforth referred to as Wnt signaling) in human neuronal cells (Salins et al., 2007), in rat mesangial cells (Lin et al., 2008), and in mouse embryonic stem cells (Qiao et al., 2011). Given that Wnt signaling is a key regulator of adult hippocampal neurogenesis (Jang et al., 2013; Kuwabara et al., 2009; Lie et al., 2005; Luo et al., 2010; Mao et al., 2009; Seib et al., 2013), we sought to determine whether statin-mediated enhancement of the Wnt pathway can occur in this region of the cholecystokinin receptor and to characterize any downstream effects on neurogenesis. We chose to focus on simvastatin (simva), as it is a lipophilic statin capable of crossing the blood-brain barrier (Tamai and Tsuji, 2000) and is commonly studied in neural contexts.
Results
Discussion Simva is under investigation for its potential therapeutic effects outside of hyperlipidemia treatment. While statins have been reported to enhance Wnt signaling in vitro, it was heretofore not known whether statins can enhance this pathway in vivo and in the context of neurogenesis. Here we provide evidence that oral simva treatment enhances Wnt signaling in the mammalian adult hippocampus. This is significant in that aside from lithium, no other clinically approved compound has been demonstrated to enhance Wnt signaling in the brain (Zimmerman et al., 2012). The observations in this study are consistent with reports of increased hippocampal neurogenesis due to both simva treatment (Chen et al., 2003; Lu et al., 2007; Wu et al., 2008) and increased Wnt signaling (as cited in introduction). Importantly, we demonstrate a link between these phenomena by probing Wnt’s role in simva enhancement of neurogenesis in vitro, and subsequently investigating the effect of enhanced Wnt signaling during multiple stages of in vivo adult hippocampal neurogenesis. To help map the biological connection between enhanced Wnt signaling and enhanced neurogenesis, we examined costaining of BAT-GAL with various cell-type-specific markers following simva treatment. However, a comparison showing differences in temporal expression patterns between different in vivo Wnt reporters presents a potential caveat to this approach (Garbe and Ring, 2012). The mechanism underlying statin enhancement of Wnt signaling had not been previously reported. Providing initial insight, we show that HMGCR loss of function is sufficient to enhance the Wnt pathway. Furthermore, we demonstrate that simva acts on Wnt signaling by depleting isoprenoids, rather than through a cholesterol-dependent mechanism. Prenylation guides membrane localization of small GTPases such as RAS and RHO-associated kinases and other signaling proteins (Zhang and Casey, 1996), and serves as a regulatory mechanism for these enzymes that can be targeted therapeutically (Gelb et al., 2006). To this point, recent studies have measured an age-dependent increase of isoprenoid levels in brains of mice (Hooff et al., 2012) and have identified an overabundance of isoprenoids in the brains of AD patients (Eckert et al., 2009). The identity of the specific prenylated protein or proteins responsible for the effect of simva on the Wnt pathway remains elusive. However, there are a number of prenylated proteins known to regulate Wnt signaling (e.g., RAC1 and RHOA) that may serve as candidates for future studies (Schlessinger et al., 2009).
Experimental Procedures
Acknowledgments
Introduction Phosphorylation is a pervasive form of cell signaling that orchestrates numerous processes, including metabolism, cell mobility, cell cycle, and differentiation (Brumbaugh et al., 2011; Van Hoof et al., 2009; Xu and Fisher, 2012). Mass spectrometry has revealed the complexity of the phosphoproteome in pluripotent cells with great detail (Muñoz and Heck, 2011; Phanstiel et al., 2011; Rigbolt et al., 2011; Swaney et al., 2009; Van Hoof et al., 2009); however, determining the biological relevance of such data remains a major challenge. Mapping the kinases responsible for a specific phosphorylation event is instructive because it places that information in the context of signaling molecules that direct biological function. Traditionally, kinase assays are performed by detecting the transfer of a radioactive phosphoryl group to a given substrate following an in vitro reaction. This method provides a direct measure of phosphorylation but necessitates the use of hazardous materials, cannot directly localize phosphorylation to a single amino acid when more than one potential site is present, and cannot multiplex kinases and substrates. Recently, a mass-spectrometric method was introduced to profile phosphorylation on synthetic peptides treated with cell lysates (Yu et al., 2009). This method is ideal for profiling cell-type-specific phosphorylation, but does not directly determine the kinase responsible for phosphorylation. Another method assesses kinase activity but relies upon heavy isotope-labeled amino acids and is limited to testing one or two kinases at a time (Singh et al., 2012b). Several other methods have been developed to identify kinase consensus motifs or test a single kinase, but are not capable of multiplexed analysis (Hennrich et al., 2013; Kettenbach et al., 2012; Songyang et al., 1994; Xue et al., 2012). Thus, there remains a pressing need for a high-throughput method to screen for kinase(s) that phosphorylate a protein of interest.