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First published online 5 September 2006
doi: 10.1242/jcs.03159

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Neuronal polarity is regulated by glycogen synthase kinase-3 (GSK-3ß) independently of Akt/PKB serine phosphorylation

Annette Gärtner^1,*,, Xu Huang² and Alan Hall^1,

¹ MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, London, WC1E 6BT, UK
² MRC Protein Phosphorylation Unit, MSI/WTB Complex, Dow Street, University of Dundee, Dundee, DD1 5EH, UK

View larger version (34K): [in a new window]   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 ß-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<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 in conjunction with Tau-1. PMGS-fluorescence along the neurites was measured, normalised and sorted according to intensity. In control neurons the fluorescence in the axon was set to 100% (mean ± s.e.m., **P<0.001; *P<0.01). Bars, 10 µm.

View larger version (29K): [in a new window]   Fig. 2. GSK-3ß phosphorylated at Ser9 does not accumulate at the tip of neurites in stage-2 neurons or in the axon of stage-3 neurons. (A) Confocal single planes of an individual neuron transfected with GFP and stained for phosphorylated Ser9 GSK-3ß. The ratio image of the two is shown in the right picture. Bar, 10 µm. (B) Quantification of the results of A. Hippocampal neurons were fixed at different stages of development and total GSK-3ß or GSK-3ß phosphorylated at Ser9 visualised using specific primary and fluorescent secondary antibodies. The fluorescence signal was divided by the signal obtained from the soluble cytoplasmic marker GFP or the DTAF signal, and the ratio of intensities in the tips was determined, normalised and sorted according to intensity. As control the fluorescent intensity of a soluble protein that does not accumulate in individual tips (p38) was used (mean ± s.e.m., *P<0.005).

View larger version (63K): [in a new window]   Fig. 3. GSK-3 activity leading to polarised axonal outgrowth is regulated independently of phosphorylation at Ser9 or Ser21. (A) Western blot of lysates form cortical neurons showing total and phosphorylated forms of GSK-3 and GSK-3ß of mice in which the Ser21 and Ser9 phosphorylation sites of GSK3 and GSK3ß had been replaced with Ala (GSK-3/ß21A/21A/9A/9A) compared with wild-type littermates (GSK-3/ß+/+/+/+). (B,C) Hippocampal neurons from wild-type and double knock-in mice were grown to low density and fixed at 48 hours. Bars, 10 µm. (D) Coronal sections of embryonal (E18) wild-type and double knock-in mice brains. Axonal projections are visualised with antibody against Tau-1, dendritic projections with antibody against Map-2. Single confocal sections are shown. In the developing cortex, the upper cortical plate (cp) contains more dendrites and, thus, more Map-2; the intermediate zone (iz) contains more Tau-1-positive axons but few dendrites. As shown here, this polarity is not disturbed in developing double knock-in brains. Also, staining the axons in the iz for another marker, synaptobrevin, resulted in the same pattern as shown here for the double knock-in mice (right panels). Bars, 25 µm. (E,F) Hippocampal neurons from the same cultures as shown in B and C incubated with the GSK-3 inhibitor SB216763. Bars, 10 µm. (G) Quantification of the experiments. At least 300 neurons from each condition were analysed and the number with one or multiple axons counted based on the length of 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 double knock-in neurons (mean ± s.e.m.).

View larger version (62K): [in a new window]   Fig. 4. GSK-3ß is membrane bound and accumulates in the Golgi region. (A) Hippocampal neurons were fixed and the localisation of endogenous GSK-3ß 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-3ß was visualised by immunofluorescence. (C) Cytosolic and membrane fractions were prepared by ultra-centrifugation at 100,000 g and analysed by western blot comparing equal amounts of total protein. N-cadherin and p38 MAP kinase were used as markers for membrane and cytosolic fractions, respectively. (D) The development 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 and SB216763-treated hippocampal neurons. The number of membrane carriers travelling within 1 minute through a defined proximal and distal neurite segment (see Materials and Methods) were counted for all neurites. Numbers were sorted according the amount of membrane carriers counted. 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 the velocities of the membrane carriers measured in Fig. 4E. Fifty membrane carriers were tracked for each condition and the speed of anterograde or retrograde transport was measured. The overlapping peaks in the frequency-distribution diagrams show that there is no significant change in the distribution of velocities measured in control neurons and neurons treated for 48 hours with the GSK-3 inhibitor. (G) The growing axon was ablated and re-growth followed over 24 hours. The axon was marked by anti-Tau-1 antibody. Bars, 10 µm.

View larger version (61K): [in a new window]   Fig. 5. Adenomatous polyposis coli (APC) accumulates at the tip of the presumptive axon. (A) Endogenous APC was visualised in stage-2 neurons transfected with a GFP expression construct. Phase-contrast, APC-immuno-reactivity, GFP fluorescence and the resulting ratio image are shown. (B) Localisation of endogenous APC in stage-2+ neurons in the largest and longest tip. (C) Preferential axonal localisation of endogenous APC in stage-3 neurons is shown in a representative neuron. (D) In the same neuron as shown in C, the intensity of APC fluorescence along the axon (labelled 1) and two minor neurites (labelled 2 and 3) was measured to demonstrate that APC accumulates strongly in the axonal tip but not in the tips of minor neurites. (E) Quantification of the preferential APC accumulation in one neurite tip. The fluorescent APC signal was divided by the signal obtained in the same cell from a soluble cytoplasmic marker GFP or from DTAF, and the ratio of intensities at neurite tips was measured, normalised and sorted in descending order according to intensity. As a control for the specificity of APC 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 the tips of all long neurites. (G) In the same neurons as shown in F, the signal of APC fluorescence intensity was measured along the three longest neurites. Here, APC accumulates in the tip of three neurites. (H) Quantification of the results shown in F and G, comparing the accumulation of APC in neurites of control stage-3 neurons versus neurons in which GSK-3 was inhibited for 48 hours. The APC signal was normalised against GFP 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/ß21A/21A/9A/9A mice is preferentially restricted to one neurite, similar to wild type neurons. (J) Localisation of APC and EB1 in an individual neuron. Bars, 10 µm.