Research Article
3927
Neuronal polarity is regulated by glycogen synthase ) independently of Akt/PKB serine kinase-3 (GSK-3 phosphorylation Annette Gärtner1,*,‡, Xu Huang2 and Alan Hall1,§ 1
MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, London, WC1E 6BT, UK MRC Protein Phosphorylation Unit, MSI/WTB Complex, Dow Street, University of Dundee, Dundee, DD1 5EH, UK
2
*Author for correspondence (e-mail:
[email protected]) ‡ Present address: Cavalieri Ottolenghi Scientific Institute, University of Turin, Regione Gonzole 10, 10043 Orbassano, Turin, Italy § Present address: Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA
Journal of Cell Science
Accepted 5 July 2006 Journal of Cell Science 119, 3927-3934 Published by The Company of Biologists 2006 doi:10.1242/jcs.03159
Summary An essential step during the development of hippocampal neurons is the polarised outgrowth of a single axon. Recently, it has been suggested that inhibition of glycogen  (GSK-3 ) via Akt/PKB-dependent synthase kinase-3 phosphorylation of Ser9, specifically at the tip of the presumptive axon, is required for selective axonal outgrowth. We now report that, by using neurons from double knock-in mice in which Ser9 and Ser21 of the two  isoforms have been replaced by Ala, polarity GSK-3 develops independently of phosphorylation at these sites.  disturbs polarity Nevertheless, global inhibition of GSK-3 development by leading to the formation of multiple axonlike processes in both control and knock-in neurons. This unpolarised outgrowth is accompanied by the symmetric
Introduction The establishment of axonal-dendritic polarity is a key event in the development of the nervous system. It has been extensively studied using isolated hippocampal neurons, which polarise to form a single axon from three to five preformed minor neurites (Dotti et al., 1988). A recent report suggested that polarised centrosomal-based activities at the immediate post-mitotic stage are necessary and sufficient to define the position of the first-formed neurite and its subsequent axonal fate (de Anda et al., 2005). However, since axonal growth does not begin until all neurites have formed, this leaves open the question of what distinguishes the first and subsequent neurites. An early manifestation of axon formation is a selective bulk-flow of organelles and trans-Golgi-network (TGN)-derived vesicles before elongation can be observed morphologically (Bradke and Dotti, 1997), and the development of a more dynamic actin cytoskeleton in the growth cone of the presumptive axon (Bradke and Dotti, 1999). However, the spatially and temporally restricted molecular events that orchestrate these changes are poorly understood. The localised inhibition of the kinase glycogen synthase kinase-3 (GSK-3) isoform  (GSK-3) has been reported to play a key role in the decision to form an axon (Jiang et al., 2005; Shi et al., 2004; Yoshimura et al., 2005). GSK-3 has many potential substrates including several microtubulebinding proteins, such as the adenomatous polyposis coli
delivery of membrane components to all neurites. Finally, the adenomatous polyposis coli (APC) protein accumulates at the tip of one neurite before and during axon elongation,  leads to APC protein but global inhibition of GSK-3  accumulation in all neurites. We conclude that GSK-3 inhibition promotes the development of neuronal polarity, but that this is not mediated by Akt/PKB-dependent phosphorylation. Supplementary material available online at http://jcs.biologists.org/cgi/content/full/119/19/3927/DC1 Key words: GSK-3, Neuronal polarity, Phosphorylation, APC, Axon
(APC) tumour suppressor protein, Tau, Crmp-2 and MAP1B. Alterations in GSK-3 activity could, therefore, have multiple effects on microtubule dynamics. Phosphorylation of Crmp-2 by GSK-3, for example, reduces its microtubule-binding activity (Yoshimura et al., 2005), whereas GSK-3-mediated phosphorylation of APC inhibits its ability to bind microtubules (Zumbrunn et al., 2001). Interestingly, in migrating astrocytes, the polarised elongation of microtubules is mediated by inhibition of GSK-3 leading to the association of APC with microtubule plus-ends (Etienne-Manneville and Hall, 2003). Several groups have reported that inactivation of GSK-3 in neurons occurs specifically at the tip of the axon and that this is mediated through a PI 3-kinase and the Akt/PKB pathway, culminating in phosphorylation of GSK-3 at Ser9 (Jiang et al., 2005; Shi et al., 2004). By contrast, we show here that, by using neurons derived from knock-in mice whose two isoforms of GSK-3 (GSK-3␣ and GSK-3) have been rendered nonphosphorylatable by Akt/PKB (McManus et al., 2005), GSK3 inhibition, which leads to polarised axonal outgrowth, is regulated independently of phosphorylation at Ser9 or Ser21. Results GSK-3 is required for the establishment of polarised axonal growth After plating, freshly dissociated hippocampal neurons
Journal of Cell Science
3928
Journal of Cell Science 119 (19)
initially form 3-5 buds or lamellipodia (stage 1; for definition of stages in the development of polarity of hippocampal neurons see Dotti et al., 1998). Highly dynamic ‘minor neurites’ emerge from these buds within 24 hours (stage 2). By 48 hours, 70% of neurons have developed a clear, polarised phenotype with a single, long process (stage 3; Fig. 1A,C) that will become the axon. To examine the role of GSK-3 in the establishment of neuronal polarity, we treated hippocampal neurons with different small-molecule inhibitors. Addition of either of the ATP-competitive inhibitors SB216763 or SB415286 during a 4-48 hour time period induced the symmetric outgrowth (Fig. 1A,D) of several Tau-1-positive axon-like processes, similar to what has been reported by others (Jiang et al., 2005; Yoshimura et al., 2005). These inhibitors stabilise cytosolic -catenin, demonstrating efficient GSK-3 inhibition (Fig. 1B). We have also used two structurally distinct inhibitors of GSK-3 that have been reported to be even more specific and potent towards GSK-3, CHIR 99021 and AR-A014418 (Cohen and Goedert, 2004). Their effect in compromising polarity was similar to the SB inhibitors (Fig. 1A,C). This ‘depolarizing’ effect of GSK-3 inhibition was most effective at the transition from stage 2 to 3 (supplementary material Fig. S1). The supernumerary long neurites have clear axonal properties because they each contain the marker proteins Tau-1 and plasma membrane ganglioside sialidase (PMGS, Fig. 1D) (Da Silva et al., 2005).
Regulation of GSK-3 activity during polarity establishment It has been reported that inhibition of GSK-3 associated with neuronal polarisation is regulated by phosphorylation at its serine residue 9 by the kinase Akt/PKB. However, localised accumulation of GSK-3 phosphorylated at Ser9 (GSK-3–P at Ser9) was described only after the axon had formed raising the possibility that this is a consequence rather than a cause of polarisation (Jiang et al., 2005). To investigate this further, we made use of an antibody specific to phosphorylated GSK-3 (anti-GSK-3-P antibody). The specificity of this antibody was first confirmed using neurons isolated from knock-in mice in which the Akt/PKB Ser phosphorylation sites of both GSK-3 alleles had been replaced with Ala (GSK-321A/21A/9A/9A, supplementary material Fig. S2). Using this antibody with wild-type neurons and normalising the immunofluorescence signal to the signal obtained from a purely cytosolic protein, green fluorescent protein (GFP) or a dye staining unspecifically all proteins, dichlorotriazinylaminofluorescein (DTAF), revealed no significant accumulation of either GSK-3–P at Ser9 or total GSK-3 in neurites at stage 2, or in the axon at stage 3 (Fig. 2A,B). To assess whether phosphorylation of GSK-3 is at all required for axon formation, we used hippocampal neurons isolated from double knock-in mice, in which the Akt/PKB serine phosphorylation sites of both alleles of both GSK3 and GSK-3␣ had been replaced with alanine
Fig. 1. Inhibition of GSK-3 leads to the formation of multiple axon-like processes. (A) Hippocampal neurons were incubated without (control) or with different GSK-3 inhibitors from the time of cell attachment up to 48 hours. Axons were visualised using an anti-Tau-1 antibody. (B) Western blot showing cytoplasmic -catenin accumulation in dissociated hippocampal neurons after GSK-3 inhibition. (C) Quantification of the effects of GSK-3 inhibition on the percentage of neurons bearing one or multiple axon-like neurites (percent of stage-2 neurons not shown). For each treatment, 200-300 neurons of different hippocampal preparations were counted (mean ± s.e.m., **P
