3927Research Article

IntroductionThe establishment of axonal-dendritic polarity is a key eventin the development of the nervous system. It has beenextensively studied using isolated hippocampal neurons, whichpolarise to form a single axon from three to five preformedminor neurites (Dotti et al., 1988). A recent report suggestedthat polarised centrosomal-based activities at the immediatepost-mitotic stage are necessary and sufficient to define theposition of the first-formed neurite and its subsequent axonalfate (de Anda et al., 2005). However, since axonal growth doesnot begin until all neurites have formed, this leaves open thequestion of what distinguishes the first and subsequentneurites. An early manifestation of axon formation is aselective bulk-flow of organelles and trans-Golgi-network(TGN)-derived vesicles before elongation can be observedmorphologically (Bradke and Dotti, 1997), and thedevelopment of a more dynamic actin cytoskeleton in thegrowth cone of the presumptive axon (Bradke and Dotti, 1999).However, the spatially and temporally restricted molecularevents that orchestrate these changes are poorly understood.

The localised inhibition of the kinase glycogen synthasekinase-3 (GSK-3) isoform � (GSK-3�) has been reported toplay a key role in the decision to form an axon (Jiang et al.,2005; Shi et al., 2004; Yoshimura et al., 2005). GSK-3 hasmany potential substrates including several microtubule-binding proteins, such as the adenomatous polyposis coli

(APC) tumour suppressor protein, Tau, Crmp-2 and MAP1B.Alterations in GSK-3 activity could, therefore, have multipleeffects on microtubule dynamics. Phosphorylation of Crmp-2by GSK-3, for example, reduces its microtubule-bindingactivity (Yoshimura et al., 2005), whereas GSK-3-mediatedphosphorylation of APC inhibits its ability to bindmicrotubules (Zumbrunn et al., 2001). Interestingly, inmigrating astrocytes, the polarised elongation of microtubulesis mediated by inhibition of GSK-3 leading to the associationof APC with microtubule plus-ends (Etienne-Manneville andHall, 2003).

Several groups have reported that inactivation of GSK-3� inneurons occurs specifically at the tip of the axon and that thisis mediated through a PI 3-kinase and the Akt/PKB pathway,culminating in phosphorylation of GSK-3� at Ser9 (Jiang etal., 2005; Shi et al., 2004). By contrast, we show here that, byusing neurons derived from knock-in mice whose two isoformsof GSK-3 (GSK-3� and GSK-3�) have been rendered non-phosphorylatable by Akt/PKB (McManus et al., 2005), GSK-3 inhibition, which leads to polarised axonal outgrowth, isregulated independently of phosphorylation at Ser9 or Ser21.

ResultsGSK-3 is required for the establishment of polarisedaxonal growthAfter plating, freshly dissociated hippocampal neurons

An essential step during the development of hippocampalneurons is the polarised outgrowth of a single axon.Recently, it has been suggested that inhibition of glycogensynthase kinase-3�� (GSK-3��) via Akt/PKB-dependentphosphorylation of Ser9, specifically at the tip of thepresumptive axon, is required for selective axonaloutgrowth. We now report that, by using neurons fromdouble knock-in mice in which Ser9 and Ser21 of the twoGSK-3�� isoforms have been replaced by Ala, polaritydevelops independently of phosphorylation at these sites.Nevertheless, global inhibition of GSK-3�� disturbs polaritydevelopment by leading to the formation of multiple axon-like processes in both control and knock-in neurons. Thisunpolarised outgrowth is accompanied by the symmetric

delivery of membrane components to all neurites. Finally,the adenomatous polyposis coli (APC) protein accumulatesat the tip of one neurite before and during axon elongation,but global inhibition of GSK-3�� leads to APC proteinaccumulation in all neurites. We conclude that GSK-3��inhibition promotes the development of neuronal polarity,but that this is not mediated by Akt/PKB-dependentphosphorylation.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/119/19/3927/DC1

Key words: GSK-3, Neuronal polarity, Phosphorylation, APC, Axon

Summary

Neuronal polarity is regulated by glycogen synthasekinase-3 (GSK-3��) independently of Akt/PKB serinephosphorylationAnnette Gärtner1,*,‡, Xu Huang2 and Alan Hall1,§

1MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, London, WC1E 6BT, UK2MRC Protein Phosphorylation Unit, MSI/WTB Complex, Dow Street, University of Dundee, Dundee, DD1 5EH, UK*Author for correspondence (e-mail: a.gaertner@ucl.ac.uk)‡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

Accepted 5 July 2006Journal of Cell Science 119, 3927-3934 Published by The Company of Biologists 2006doi:10.1242/jcs.03159

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initially form 3-5 buds or lamellipodia (stage 1; for definitionof stages in the development of polarity of hippocampalneurons see Dotti et al., 1998). Highly dynamic ‘minorneurites’ 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 ofGSK-3 in the establishment of neuronal polarity, we treatedhippocampal neurons with different small-moleculeinhibitors. Addition of either of the ATP-competitiveinhibitors SB216763 or SB415286 during a 4-48 hour timeperiod induced the symmetric outgrowth (Fig. 1A,D) ofseveral Tau-1-positive axon-like processes, similar to whathas been reported by others (Jiang et al., 2005; Yoshimura etal., 2005). These inhibitors stabilise cytosolic �-catenin,demonstrating efficient GSK-3 inhibition (Fig. 1B). We havealso used two structurally distinct inhibitors of GSK-3 thathave been reported to be even more specific and potenttowards GSK-3, CHIR 99021 and AR-A014418 (Cohen andGoedert, 2004). Their effect in compromising polarity wassimilar to the SB inhibitors (Fig. 1A,C). This ‘depolarizing’effect of GSK-3 inhibition was most effective at the transitionfrom stage 2 to 3 (supplementary material Fig. S1). Thesupernumerary long neurites have clear axonal propertiesbecause they each contain the marker proteins Tau-1 andplasma membrane ganglioside sialidase (PMGS, Fig. 1D) (DaSilva et al., 2005).

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Regulation of GSK-3 activity during polarityestablishmentIt has been reported that inhibition of GSK-3� associated withneuronal polarisation is regulated by phosphorylation at itsserine residue 9 by the kinase Akt/PKB. However, localisedaccumulation of GSK-3� phosphorylated at Ser9 (GSK-3�–Pat Ser9) was described only after the axon had formed raisingthe possibility that this is a consequence rather than a cause ofpolarisation (Jiang et al., 2005). To investigate this further, wemade use of an antibody specific to phosphorylated GSK-3�(anti-GSK-3�-P antibody). The specificity of this antibody wasfirst confirmed using neurons isolated from knock-in mice inwhich the Akt/PKB Ser phosphorylation sites of both GSK-3�alleles had been replaced with Ala (GSK-3�21A/21A/9A/9A,supplementary material Fig. S2). Using this antibody withwild-type neurons and normalising the immunofluorescencesignal to the signal obtained from a purely cytosolic protein,green fluorescent protein (GFP) or a dye staining unspecificallyall proteins, dichlorotriazinylaminofluorescein (DTAF),revealed no significant accumulation of either GSK-3�–P atSer9 or total GSK-3� in neurites at stage 2, or in the axon atstage 3 (Fig. 2A,B).

To assess whether phosphorylation of GSK-3 is at allrequired for axon formation, we used hippocampal neuronsisolated from double knock-in mice, in which the Akt/PKBserine phosphorylation sites of both alleles of both GSK-3� 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) Quantificationof the effects of GSK-3 inhibition on the percentage of neurons bearing one or multiple axon-like neurites (percent of stage-2 neurons notshown). For each treatment, 200-300 neurons of different hippocampal preparations were counted (mean ± s.e.m., **P<0.001; *P<0.002). (D)Control (bottom left two panels) and SB21673-treated (bottom right two panels) neurons were fixed and endogenous PMGS was detected inconjunction with Tau-1. PMGS-fluorescence along the neurites was measured, normalised and sorted according to intensity. In control neuronsthe fluorescence in the axon was set to 100% (mean ± s.e.m., **P<0.001; *P<0.01). Bars, 10 �m.

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(GSK3�/�21A/21A/9A/9A), and from littermate mice(GSK3�/�+/+/+/+) (McManus et al., 2005). The levels of GSK-3� and GSK-3� protein were similar in the two types ofneurons but, as expected, there was no detectable GSK-3phosphorylation in neurons from the knock-in mouse using the

anti-GSK-3 � and �-P antibodies (Fig. 3A). As shown in Fig.3B,C, the development of neurons from the double knock-inneurons was indistinguishable from that of wild-type neurons.After 48 hours in culture, fully polarised neurons with only asingle Tau-1-positive axon and minor neurites of equal lengthdeveloped in both cases (Fig. 3B,C,H). During 48-70 hours inculture, the percentage of polarised stage-3 neurons increasedsimilarly in both types of neurons (Fig. 3G). Importantly, an invivo analysis of knock-in neurons showed axonal and dendriticprojections indistinguishable from wild-type littermates (Fig.3D). Thus, we conclude that Ser9 or Ser21 phosphorylation ofGSK-3 isoforms is not required for the establishment of apolarised neuron in vivo or in vitro.

To determine whether GSK-3 inhibition can still inducemultiple axon-like projections in knock-in mice, we made useof the GSK-3 inhibitors. After 70 hours in the presence ofSB216763, about one third of wild-type neurons and knock-inneurons developed multiple, axon-like processes (Fig. 3E-G).

GSK-3� accumulates in the Golgi region and is involvedin the regulation of polarised trafficSince we (Fig. 2B) and others (Jiang et al., 2005) could notfind any specific accumulation of GSK-3� in the axonal tip ofstage-3 neurons, or in a single neurite of stage-2 neurons, weanalysed its intracellular localisation. At the light-microscopylevel, GSK-3� is enriched in the Golgi-centrosome region ofhippocampal neurons (Fig. 4A). Even after extraction of livecells with Triton X-100 (Fig. 4B) or saponin (data not shown)GSK-3� remains associated with the Golgi region, suggestingthat it is membrane bound (supplementary material Fig. S3B).This is supported by biochemical experiments, in which GSK-3� was found in the same fraction as a Golgi marker in acontinuous sucrose gradient (supplementary material Fig.S3A) and in the membrane pellet (Fig. 4C). This prominentlocalisation of GSK-3� raises the possibility that GSK-3 isinvolved in regulating polarised traffic to the axon. Indeed,GSK-3 inhibition leads to symmetric outgrowth from allneurites at a similar rate (22 �m/24 hours) (Fig. 4D), which isaround three times slower than normal axonal outgrowth (69�m/24 hours), but three times faster than that of minor neurites(8 �m/24 hours) in control neurons. The sum of overall neuritelength, therefore, remains unchanged (supplementary materialFig. S3C).

To determine whether GSK-3 controls polarised, axonalmembrane transport, we analysed the level of membranetrafficking along a selected neurite segment by using phase-contrast microscopy (supplementary material Fig. S4 andMovies 1-4) as described by Bradke and Dotti (Bradke and Dotti,1997). After GSK-3 inhibition, membrane traffic – which isnormally directed preferentially to axons – becomessymmetrically distributed towards all neurites (Fig. 4E).Moreover, new membrane carriers exiting from the TGN (whichare concentrated in the axon of control cells) werehomogeneously distributed in all processes after treatment witha GSK-3 inhibitor (supplementary material Fig. S3D). This wasnot due to a dispersal of the Golgi itself, which stayed intact intreated neurons (data not shown). The overall velocity ofanterograde and retrograde membrane transport was not changedby GSK-3 inhibition – as can be seen by the overlapping peaksof membrane carrier velocities in the frequency-distributiondiagrams in Fig. 4F – but only the polarity of the transport, which

Fig. 2. GSK-3� phosphorylated at Ser9 does not accumulate at thetip of neurites in stage-2 neurons or in the axon of stage-3 neurons.(A) Confocal single planes of an individual neuron transfected withGFP and stained for phosphorylated Ser9 GSK-3�. The ratio imageof the two is shown in the right picture. Bar, 10 �m.(B) Quantification of the results of A. Hippocampal neurons werefixed at different stages of development and total GSK-3� or GSK-3� phosphorylated at Ser9 visualised using specific primary andfluorescent secondary antibodies. The fluorescence signal wasdivided by the signal obtained from the soluble cytoplasmic markerGFP or the DTAF signal, and the ratio of intensities in the tips wasdetermined, normalised and sorted according to intensity. As controlthe fluorescent intensity of a soluble protein that does not accumulatein individual tips (p38) was used (mean ± s.e.m., *P<0.005).

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means the number of carriers travelling towards the axon (Fig.4E) with respect to minor neurites.

Finally, ablation experiments (Goslin and Banker, 1989)suggest that activities at the axonal tip are not required for the

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maintenance of axonal outgrowth. Thus, when axons of stage-2+ or stage-3 neurons are cut such that the axonal stumpremains longer than the remaining neurites, 81% of the axonsre-grow (Fig. 4G).

Fig. 3. GSK-3 activity leading to polarised axonal outgrowth is regulated independently of phosphorylation at Ser9 or Ser21. (A) Western blotof lysates form cortical neurons showing total and phosphorylated forms of GSK-3� and GSK-3� of mice in which the Ser21 and Ser9phosphorylation sites of GSK3� and GSK3� had been replaced with Ala (GSK-3�/�21A/21A/9A/9A) compared with wild-type littermates (GSK-3�/�+/+/+/+). (B,C) Hippocampal neurons from wild-type and double knock-in mice were grown to low density and fixed at 48 hours. Bars, 10�m. (D) Coronal sections of embryonal (E18) wild-type and double knock-in mice brains. Axonal projections are visualised with antibodyagainst Tau-1, dendritic projections with antibody against Map-2. Single confocal sections are shown. In the developing cortex, the uppercortical plate (cp) contains more dendrites and, thus, more Map-2; the intermediate zone (iz) contains more Tau-1-positive axons but fewdendrites. As shown here, this polarity is not disturbed in developing double knock-in brains. Also, staining the axons in the iz for anothermarker, synaptobrevin, resulted in the same pattern as shown here for the double knock-in mice (right panels). Bars, 25 �m. (E,F) Hippocampalneurons from the same cultures as shown in B and C incubated with the GSK-3 inhibitor SB216763. Bars, 10 �m. (G) Quantification of theexperiments. At least 300 neurons from each condition were analysed and the number with one or multiple axons counted based on the lengthof Tau-1-positive neurites (mean ± s.e.m.). (H) Total length of axons and minor neurites after 70 hours in culture in wild-type and doubleknock-in neurons (mean ± s.e.m.).

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3931GSK-3 and neuronal polarity

Fig. 4. GSK-3� is membrane bound and accumulates in the Golgi region. (A) Hippocampal neurons were fixed and the localisation ofendogenous GSK-3� was detected by immunofluorescence. The Golgi was labelled by WGA-FITC. Single confocal sections are shown.(B) Neurons were solubilised for 5 minutes on ice in 0.1% Triton X-100, fixed in 4% PFA and GSK-3� was visualised by immunofluorescence.(C) Cytosolic and membrane fractions were prepared by ultra-centrifugation at 100,000 g and analysed by western blot comparing equalamounts of total protein. N-cadherin and p38 MAP kinase were used as markers for membrane and cytosolic fractions, respectively. (D) Thedevelopment of individual neurons (untreated control or treated from 24-48 hours with SB216763) was followed on cellocate (Eppendorf)coverslips. (E) Traffic of vesicles and membrane compartments in all neurites was imaged by phase-contrast microscopy in control andSB216763-treated hippocampal neurons. The number of membrane carriers travelling within 1 minute through a defined proximal and distalneurite segment (see Materials and Methods) were counted for all neurites. Numbers were sorted according the amount of membrane carrierscounted. In control neurons, the first column represents the traffic in the axon, which was always the neurite with the most intense traffic (mean± s.e.m., **P<0.001). Images of the experiment are shown in supplementary material, Fig. S4 and Movies 1-4. (F) Frequency distribution of thevelocities of the membrane carriers measured in Fig. 4E. Fifty membrane carriers were tracked for each condition and the speed of anterogradeor retrograde transport was measured. The overlapping peaks in the frequency-distribution diagrams show that there is no significant change inthe distribution of velocities measured in control neurons and neurons treated for 48 hours with the GSK-3 inhibitor. (G) The growing axon wasablated and re-growth followed over 24 hours. The axon was marked by anti-Tau-1 antibody. Bars, 10 �m.

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APC accumulation at neurite tips is promoted by GSK-3inhibitionThe ability of APC to bind and stabilise MTs is influenced byGSK-3 activity (Zumbrunn et al., 2001) and APC has beenlocalised to axon tips in polarised neurons (Zhou et al., 2004)and before axon formation (Votin et al., 2005). To examine theimportance of APC in regulating polarity, we analysed itsdistribution in stage-2 neurons before any morphological signs

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of axon elongation could be detected. As shown in Fig. 5A,APC accumulation could be detected specifically in one of theminor neurites in stage 2 neurons (quantification: Fig. 5E). Tosee whether this early accumulation correlates with the futureaxon, we localised APC during the course of axon formation.In late stage-2+ neurons, APC strongly accumulates inpresumptive axonal tips, which can be identified by a largegrowth cone (Bradke and Dotti, 1997). In 70% of stage-2+

Fig. 5. Adenomatous polyposis coli (APC) accumulates at the tip of the presumptive axon. (A) Endogenous APC was visualised in stage-2neurons transfected with a GFP expression construct. Phase-contrast, APC-immuno-reactivity, GFP fluorescence and the resulting ratio imageare shown. (B) Localisation of endogenous APC in stage-2+ neurons in the largest and longest tip. (C) Preferential axonal localisation ofendogenous APC in stage-3 neurons is shown in a representative neuron. (D) In the same neuron as shown in C, the intensity of APCfluorescence along the axon (labelled 1) and two minor neurites (labelled 2 and 3) was measured to demonstrate that APC accumulates stronglyin the axonal tip but not in the tips of minor neurites. (E) Quantification of the preferential APC accumulation in one neurite tip. The fluorescentAPC signal was divided by the signal obtained in the same cell from a soluble cytoplasmic marker GFP or from DTAF, and the ratio ofintensities at neurite tips was measured, normalised and sorted in descending order according to intensity. As a control for the specificity ofAPC accumulation, the fluorescence intensity of a soluble protein that does not accumulate in individual tips (p38) was used (mean ±s.e.m.,**P<0.001, *P<0.01). (F) A neuron 48 hours after GSK-3 inhibition is shown. Here, endogenous APC is distributed symmetrically in thetips of all long neurites. (G) In the same neurons as shown in F, the signal of APC fluorescence intensity was measured along the three longestneurites. Here, APC accumulates in the tip of three neurites. (H) Quantification of the results shown in F and G, comparing the accumulation ofAPC in neurites of control stage-3 neurons versus neurons in which GSK-3 was inhibited for 48 hours. The APC signal was normalised againstGFP or DTAF, and the ratio intensity at neurite tips calculated, normalised and sorted in descending according to intensity (mean ± s.e.m.,**P<0.001). (I) Localisation of endogenous APC in neurons isolated from GSK–3�/�21A/21A/9A/9A mice is preferentially restricted to oneneurite, similar to wild type neurons. (J) Localisation of APC and EB1 in an individual neuron. Bars, 10 �m.

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3933GSK-3 and neuronal polarity

neurons, the largest growth cone correlated with the highestAPC accumulation in the tip (Fig. 5B). Moreover, in fullypolarised (stage 3) neurons, APC was found at the axonal tipin 100% of neurons (Fig. 5C,E). The average normalisedintensity in the axonal tip is five times higher than the averageintensity of all the minor neurite tips and 2.5 times higher thanthe mostly intensely stained of the minor neurites (Fig. 5E).APC can also be found along the length of neurites but isstrongly accumulated in the axonal tip, as demonstrated bymeasuring the profile of APC staining intensity along the axonand neurites (Fig. 5D).

If APC is involved in axon formation, then inhibition ofGSK-3 should affect APC localisation. Indeed, the addition ofthe GSK-3 inhibitor SB216763 dramatically changes thedistribution of APC. Concomitant with the induction ofmultiple axon-like extensions, we observe the accumulation ofAPC at the tips of all the long, axon-like processes aftertreatment with inhibitor (Fig. 5F-H). This effect is independentof GSK-3 phosphorylation, because the accumulation ofAPC at the axon tip can also be seen in neurons fromGSK3�/�21A/21A/9A/9A knock-in mice (Fig. 5I). We did notobserve a similar selective axonal accumulation for other plus-end microtubule-binding proteins, including EB1 (Fig. 5J),CLIP170 or CLIP115 (data not shown). When axons aresevered, as in Fig. 4G, APC accumulated again in the axonaltip during re-growth in the majority of neurons (>80%).

DiscussionWe show here that, GSK-3 inhibition in hippocampal neuronsleads to the symmetric outgrowth of multiple axon-likeprocesses (Fig. 1A,C,D). The axon-like nature of theseprocesses was evident by the accumulation of several markerproteins such as dephosphorylated Tau (Tau-1) and PMGS (DaSilva et al., 2005), (Fig. 1D). Underlying this symmetricneurite outgrowth is a symmetric trafficking of membranecarriers (Fig. 4E, supplementary material Fig. S3D). However,since the net outgrowth of all neurites stays unchanged, wesuggest that GSK-3 activity does not regulate the levels ofmembrane trafficking but rather its directionality.

Although the mechanism linking GSK-3 inhibition to axonaloutgrowth is not clear, the protein APC seems to play animportant role. This plus-end microtubule-binding proteinshows polarised accumulation at the tip of one neurite even instage-2 neurons and preceding fast axonal outgrowth (Fig.5A,E) (Votin et al., 2005). That this early accumulation of APCdepends on GSK-3 inhibition is suggested by the observationthat global GSK-3 inhibition leads to the appearance of APCat the tips of all neurites (Fig. 5F-H). A role for APC in axonoutgrowth has been reported by others (Shi et al., 2004) and,furthermore, a dominant-negative mutant of APC preventsaxon outgrowth. This suggests that APC is, at least in part,responsible for the GSK-3 effect. The localised accumulationof APC in the presumptive axon could act by conferring earlyasymmetry on microtubule dynamics, because APC binds andstabilises microtubules and can promote their capture bymembrane-associated complexes (Zumbrunn et al., 2001). Oneconsequence of GSK-3 inhibition could be a change inmicrotubule-mediated membrane trafficking, an event knownto be crucial during the course of polarisation (Bradke andDotti, 1997; Martinez-Arca et al., 2001; Tang, 2001). In fact,we have found that inhibition of GSK-3 activity results in equal

flow of membrane traffic (Fig. 4E) and the equal distributionof TGN-derived vesicles in all neurites (supplementary Fig.S3D), rather than polarised flow to a single axon. Interestingly,a large pool of GSK-3 is localised in the centrosome/Golgi area(Fig. 4A, supplementary Fig. S3A), but whether this pool ofGSK-3 facilitates polarised trafficking or whether polarisedtrafficking is a consequence of polarity establishment is notclear.

GSK-3 activity can be regulated in many ways. In the Wntsignalling pathway, GSK-3 inhibition is essential forstabilisation of �-catenin and is thought to occur throughprotein-protein interactions. Interestingly, it was shown that,Wnt signalling is involved in the regulation of the anteriorposterior polarity of neurons in C. elegans (Hilliard andBargmann, 2006). However, in contrast to our observations,disturbance of Wnt signalling in C. elegans leads to aninversion of the polarity axis rather than loss of polarity.

In the insulin pathway, GSK-3 is inactivated byphosphorylation of Ser9 (GSK-3�) or Ser21 (GSK-3�) (Dobleand Woodgett, 2003). Recently, there have been two reportsthat GSK-3�–P at Ser9 accumulates in the axon of stage-3hippocampal neurons (Jiang et al., 2005; Shi et al., 2004) ordorsal root ganglion neurons (Zhou et al., 2004). The authorsconcluded that this spatial accumulation was promoted by thelocal axon-specific activation of Akt/PKB (Jiang et al., 2005)or ILK (Zhou et al., 2004). Our data do not support this model:First, we could not detect any localised accumulation of totalor phosphorylated GSK-3 either in the axonal tip or in neuritesof stage-2 neurons (Fig. 2A,B). These differences could beowing to the experimental conditions or to the different waywe have evaluated specific axonal accumulation. Second, andmore importantly, we have found that neurons isolatedfrom double knock-in mice, in which the Akt/PKB Serphosphorylation sites of GSK-3� and GSK-3� had beenreplaced with Ala (McManus et al., 2005), develop normalpolarity indistinguishable from wild type neurons (Fig. 3).Finally, phosphorylation of GSK-3� at Ser9 is also thought tobe involved at later stages in neuronal development duringaxon outgrowth and guidance in peripheral neurons (Eickholtet al., 2002; Zhou et al., 2004). Although we have not directlyinvestigated this for hippocampal neurons, the lack of anyobvious neuronal defects in the knock-in mice suggests thatphosphorylation is not a major mechanism of regulating GSK-3 during neuronal development in the CNS.

We conclude that, GSK-3 inhibition is an important signalin the establishment of neuronal polarity and GSK-3 mediatesits effects, in part at least, through the polarised accumulationof APC. GSK-3 inhibition regulating these processes is not,however, initiated by phosphorylation through Akt/PKB.

Materials and MethodsMaterialsAntibodies used were against APC (I. Näthke), EB1, GSK-3� (BD), Tau-1(Chemicon), GSK-3� (Upstate), GSK-3� phosphorylated on Ser9, GSK-3�phosphorylated on Ser21 (Cell Signalling), �-tubulin (YOL1/34, Serotec), PMGS(J. Abad-Rodriguez), GFP (Molecular Probes), synaptobrevin (Synaptic Systems)and MAP-2 (Peninsula Laboratories). Inhibitors: SB216763 and SB415286 (Tocris),used at a final concentration of 20 �M or 40 �M. AR-A014418 and CHIR99021were kindly provided by P. Cohen (U. Dundee, UK) and used at a concentration of10 �M and 2 �M, respectively.

CulturesHippocampal or cortical neurons from embryonic stage (E)18 or E19 embryos were

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prepared according to (Banker and Goslin, 1988). In brief, hippocampi weredissected and cells dissociated by trypsin (15 minutes, 37°C) and mechanicaltrituration using a fire polished Pasteur pipette. Neurons were plated immediatelyin dishes containing CO2 and temperature-equilibrated Dulbecco’s modified Eagle’smedium (DMEM) with 10% horse serum at a density of 2500 cells per cm2. After4 hours incubation neurons were sufficiently attached and coverslips were placedinverted, separated by paraffin dots, onto astrocyte feeder-cell layers equilibrated ina serum-free complete medium (Brewer and Cotman, 1989). Hippocampal neuronswere transfected before plating using the rat neuron nucleofector kit (Amaxa)according to the manufacturer’s instructions.

ImagingNeurons were fixed for 15 minutes at room temperature in 4% paraformaldehyde(PFA), 4% sucrose, 2 mM MgCl2, 3 mM EGTA-PBS, or for 3 minutes in MeOH at–20°C, washed in PBS, quenched in 50 mM NH4Cl-PBS and permeabilised in 0.1%Triton X-100 in PBS. After blocking with 20% normal goat serum, the first antibodywas added and incubated overnight at 4°C in 2% normal goat serum in PBS. Thesecond antibody was added for 1 hour at room temperature and cells were embeddedin mounting medium (Dako). To detect cytosolic proteins, 0.001% DTAF(Molecular Probes) was used. Fluorescence was monitored using conventionalfluorescence microscopy or confocal microscopy (Biorad Radiance) and imageswere analysed by Image J.

For immunofluorescence analyses of embryonic brain sections, embryonic brainswere fixed for 4-6 hours in 4% PFA and equilibrated in 30% sucrose. 50-�m to 100-�m coronal sections were cut with a vibratome. Staining was performed as abovewith the difference that the blocking was done overnight at 4°C on a shaker, in PBScontaining 0.5% Triton X-100 and 3% normal goat serum, and the first antibodywas incubated overnight in 0.3% Triton X-100 at 4°C.

Analysis of membrane traffic was performed on a heated stage in a closedaluminium chamber. Images were captured in 1-second to 2-second intervals for100 seconds. Membrane traffic was analysed by measuring the number of membranecarriers flowing through a proximally (immediately at the neurite shaft) and adistally (approximately half way along the nerurite length) located neurite segmentof 10 �m. Values for the mean were calculated and sorted in descending order. Incontrol neurons, axonal traffic (which is always higher than in neurites) isrepresented in the first column. The speed of membrane carriers was measured usingImage J by following single vesicles for 4-20 seconds. For each condition (Fig. 4F),50 vesicles were tracked.

Axon ablationAxons were cut by rapidly drawing a microinjection needle over the glass surfaceperpendicular to the axon length using a microinjection device (Eppendorf).

Evaluation of protein accumulation at the axonal tipTo quantify the accumulation of proteins at neurite or axon tips, neurons were eithernucleofected with GFP or cytoplasmic proteins visualised with DTAF (Schindelholzand Reber, 1999). Neurons were immunolabelled for the molecule of interest andimages were taken for the respective molecule (GSK-3, APC) and in parallel forGFP or DTAF. Using Image J, the ratio of the two images was calculated, thebackground subtracted and the mean ratio intensities in the neurite tips measured.Owing to the different architecture and volume of the different neurites, GFP orDTAF often accumulate non-specifically in the axon, and thus our measurementsreflect the true accumulation of the protein of interest. As a control, we used theratio between p38 and DTAF. p38 is a soluble protein (Fig. 4C) and is not locatedin axonal tips. The ratio intensities were sorted according to their strength and thennormalised. The graphs represent sorted means of normalised ratio intensities atneurite tips. Since every neuron measured contained at least three neurites, only thefirst three neurites were shown. The slope of the three values represents the strengthof accumulation. No accumulation represents a slope of zero, 100% accumulationin one neurite represents a slope of 1.

Separation of cytosolic and membrane proteinsNeurons were harvested in hypo-osmotic buffer (25 mM Tris, 2 mM EDTA, 1 mMDDT, protease inhibitors; pH 7.0) and cells disrupted by dispensing them througha 22G needle. Nuclei were removed by centrifugation for 5 minutes at 4°C at 600g and membranes were harvested by centrifugation at 100,000 g. Cytosolic proteinsremained in the supernatant, the membrane pellet was solubilised in 0.1% SDS.

Western blotNeurons were lysed in 50 mM Hepes pH 7.4, 150 mM NaCl, 10% glycerol,1%Triton X-100, protease inhibitors. Proteins were separated by SDS-PAGE,transferred to nitrocellulose membranes and western blotted. Blots were incubated

overnight with the first antibody in 5% milk-TBST and then with horseradishperoxidase (HRP)-coupled secondary antibody and detected by the ECL reagent(Amersham).

We are very grateful to Carlos Dotti for important suggestions andcomments on the manuscript and technical help from Etienne Cassinand Bianca Hellias. We also thank Dario Alessi for his help andcomments on this work. We thank David Becker (UCL, London, UK)for supporting the performance of some experiments, and Inke Näthke(University of Dundee, UK), Philip Cohen (MRC Unit, Dundee, UK)and Jose Abad-Rodriguez (Fondazione Ottolenghi, Turin, Italy) forreagents. This work was supported by a programme grant from CancerResearch UK and by a DFG fellowship (A.G.).

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