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GSK3 and β-catenin determines functional expression of sodium channels at the axon initial segment

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Abstract

Neuronal action potentials are generated through voltage-gated sodium channels, which are tethered by ankyrinG at the membrane of the axon initial segment (AIS). Despite the importance of the AIS in the control of neuronal excitability, the cellular and molecular mechanisms regulating sodium channel expression at the AIS remain elusive. Our results show that GSK3α/β and β-catenin phosphorylated by GSK3 (S33/37/T41) are localized at the AIS and are new components of this essential neuronal domain. Pharmacological inhibition of GSK3 or β-catenin knockdown with shRNAs decreased the levels of phosphorylated-β-catenin, ankyrinG, and voltage-gated sodium channels at the AIS, both “in vitro” and “in vivo”, therefore diminishing neuronal excitability as evaluated via sodium current amplitude and action potential number. Thus, our results suggest a mechanism for the modulation of neuronal excitability through the control of sodium channel density by GSK3 and β-catenin at the AIS.

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Acknowledgments

The authors would like to acknowledge Hector Diez, Sylvain Rama, Alfonso Araque, and Michael Seagar for their advice and critical reading of the manuscript. This work was supported by grants SAF2009-12249-C02-02 and SAF2009-12249-C02-01 from the Ministerio de Ciencia e Innovacion (Spain), by the Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED, Spain), INSERM and Agence National de la Recherche (EXCION, EPISOM). Mónica Tapia is supported by a CSIC-JAE fellowship.

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Authors and Affiliations

  1. Department of Cellular, Molecular and Developmental Neurobiology, Instituto Cajal, CSIC, Avenida Doctor Arce, Madrid, 28002, Spain

    Mónica Tapia, Ana Del Puerto, Diana Sánchez-Ponce, Noemí Pallas-Bazarra & Juan José Garrido

  2. Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain

    Mónica Tapia, Ana Del Puerto, Diana Sánchez-Ponce, Noemí Pallas-Bazarra, Francisco Wandosell & Juan José Garrido

  3. Departamento de Anatomía Patológica, Capio Fundación Jiménez Díaz, Madrid, 28040, Spain

    Alberto Puime

  4. INSERM U1072, Marseille, 13344, France

    Laure Fronzaroli-Molinieres, Edmond Carlier, Pierre Giraud & Dominique Debanne

  5. Aix-Marseille University, U1072, Marseille, 13344, France

    Laure Fronzaroli-Molinieres, Edmond Carlier, Pierre Giraud & Dominique Debanne

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  1. Mónica Tapia
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18_2012_1059_MOESM1_ESM.pdf

Supplementary material 1 Fig. S1. β-catenin interference shRNAs suppress β-catenin-pS33/37/T41 expression at the AIS (A) Endogenous β-catenin and β-catenin-pS33/37/T41 in N2a cells is suppressed by the expression of two different β-catenin shRNAs. β-catenin shRNA1 reduced endogenous β-catenin and β-catenin-pS33/37/T41 expression approximately 70 %, while β-catenin shRNA2 reduced them only by 50 %. Graph represents the mean and SD of β-catenin expression levels normalized to β-actin levels in 3 different experiments. ***p < 0.001, **p < 0.01, t test. (B,C) Representative images of hippocampal neurons nucleofected with β-catenin shRNAs or scrambled shRNA plasmids. Neurons were fixed after 3 days and stained with β-catenin (B) or pS33/37T41-β-catenin (C) antibodies as indicated. (D) 6-DIV hippocampal neurons nucleofected with β-catenin shRNA 1 or scrambled shRNA plasmids. Neurons were stained with anti-MAP2, and β-catenin-pS33/37/T41 or β-catenin antibodies. Nucleofected neurons were identified by their RFP fluorescence. High magnification at the level of AIS (boxes) of non-nucleofected, β-catenin shRNA 1 or scrambled shRNA nucleofected neurons is shown. White arrows indicate the soma of non-transfected neurons and red arrows indicate transfected neurons. Arrowheads indicate the position of axons in neurons stained with β-catenin and AIS in neurons stained with pS33/37T41-β-catenin. Scale bar = 50 µm.Fig. S2. β-catenin-pS33/37/T41 staining is also localized at the centrosome in early development neurons.24-h neurons were fixed and stained with β-catenin-pS33/37/T41 (green) and γ-tubulin (red) antibodies. The arrows indicate centrosome position. Scale bar = 25 μm.Fig. S3. Quantification of β-catenin-pS33/37/T41 fluorescence intensity along neurons at different developmental stagesImages of different developmental stages hippocampal neurons stained with anti-β-catenin-pS33/37/T41 antibody (green), as shown in Fig. 1. Graphs represent the β-catenin-pS33/37/T41 fluorescence intensity along a line designed through dendrites-soma and axon. Fluorescence intensity was quantified in gray scale images. Scale bar = 50 μm.Fig. S4. β-catenin-pS45 is located at the nucleus but not at the AIS in hippocampal neurons.Hippocampal neurons from different stages were stained with an antibody that recognizes β-catenin when is phosphorylated at serine 45 and anti-tyrosinated-α-tubulin (top panels, 24-h neurons) or anti-PanNaCh (bottom panels). Note that β-catenin-pS45 is concentrated in nucleus at both stages, but not at the AIS.Fig. S5. Proteasome inhibition does not alter pS33/37T41-β-catenin expression at the AIS, but increases its expression at neuronal soma.(A) β-catenin and β-catenin-pS33/37/T41 expression in cortical neurons treated or not with MG132 50 nM for 24 h. Graph represents the mean ± SEM protein expression levels normalized to β-actin levels in three different experiments. **p < 0.01, *p < 0.05, t test. (B) Hippocampal neurons treated with proteasome inhibitor MG132 from 5 DIV to 6 DIV and stained with anti-β-catenin-pS33/37/T41 and anti-MAP2 antibodies. Note the increased fluorescence intensity of β-catenin-pS33/37/T41 at the neuronal soma. Arrows indicate AIS position and arrowheads soma position. All images were acquired in same intensity fluorescence conditions. Scale bar = 50 µmFig. S6. β-catenin related proteins, APC and N-cadherin, are not localized at the axon initial segment.Hippocampal neurons cultured for 2 DIV (A) and 6 DIV (B and C) were stained with pS33/37T41-β-catenin antibody (green), and APC (B) or N-cadherin (C) antibodies (red). Note that pS33/37T41-β-catenin colocalizes with APC along axonal microtubules and growth cone at 2 DIV, while neither APC nor N-cadherin expression is detected at the AIS of 6-DIV hippocampal neurons. Insets show enlarged views of axon initial segments. Scale bar = 50µm.Fig. S7. Percentage of β-catenin shRNAs nucleofected neurons at different developmental stages, and with different β-catenin-pS33/37/T41, ankyrinG and sodium channels intensities at the AIS.Summarized results of three categories (high expression, weak expression, and absence of expression) of neurons established in function of the staining intensity compared to control neurons mean staining. Results are the mean ± SEM percentage of neurons in each category from three independent experiments (100 neurons/experimental condition and experiment). ***p < 0.001 and **p < 0.01 compared to scrambled shRNA, t test. Neurons showing total fluorescence intensity over 50 % of control neurons (scrambled shRNA) were considered as high intensity category, and neurons with total fluorescence intensity equal or fewer than 50 % of control neurons were considered in the weak intensity category. Sc = scramble shRNA, Sh1 = β-catenin shRNA 1, Sh2 = β-catenin shRNA 2.Fig. S8. β-catenin suppression slows down axonal elongation and dendritic maturation.(A) 3-DIV neurons stained for MAP2 and tau-1 to identify the axon and somatodendritic compartment. The graph represents the axonal length of neurons nucleofected with scrambled and β-catenin shRNAs. Both shRNAs did not impair axon formation, but slightly reduced the length of the axon, identified by the axonal marker Tau-1 (165.58 ± 7.55 µm or 186.28 ± 7.09 µm compared to 227.75 ± 8.92 µm in scrambled shRNA nucleofected neurons). Data represent the mean ± SEM of three independent experiments (50 neurons/experimental condition and experiment). (B) 6-DIV neurons stained with MAP2 and βIII-tubulin antibodies to show dendritic development of β-catenin shRNA 1, β-catenin shRNA 2, or scrambled shRNA-nucleofected neurons. Nucleofected neurons were identified by their RFP fluorescence. Graph represents the mean ± SEM of mean dendritic length from three independent experiments (25 neurons/experimental condition in each experiment). Scale bar = 50 µm. ***p < 0.001, **p < 0.01, paired t test.(PDF 2387 kb)

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Tapia, M., Del Puerto, A., Puime, A. et al. GSK3 and β-catenin determines functional expression of sodium channels at the axon initial segment. Cell. Mol. Life Sci. 70, 105–120 (2013). https://doi.org/10.1007/s00018-012-1059-5

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