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New methods based on capillary electrophoresis for in vitro evaluation of protein tau phosphorylation by glycogen synthase kinase 3-β

Analytical and Bioanalytical Chemistry volume 407, pages 2821–2828 (2015)Cite this article

Abstract

The hyperphosphorylation of tau protein is associated with the development of the neuronal pathology of Alzheimer’s disease. As most conventional methods study only particular phosphorylation sites of tau, it is necessary to develop a simple and reliable assay to determine the phosphorylation of tau at multiple sites. Capillary electrophoresis (CE)-based enzymatic assays are not yet used to monitor tau phosphorylation. The present work aims to develop CE-based assays to evaluate tau phosphorylation by the glycogen synthase kinase 3-β (GSK3β). A novel pre-capillary CE assay was first developed. An in-capillary CE-based enzymatic assay was also used since this approach is known to be time- and cost- effective. The enzymatic reaction was monitored by quantifying the product adenosine 5′- diphosphate (ADP). The influence of two classes of glycosaminoglycan (GAG), namely heparin and heparan sulfate, on the phosphorylation reaction was also assessed. Results obtained by both CE approaches were comparable and in excellent agreement with those reported in the literature using conventional radiometric and immunoblotting methods. In fact, CE results confirmed the inductory effect of the sulfated sugars heparin and heparan sulfate on tau hyperphosphorylation, probably because of the exposition of new sites phosphorylatable by GSK3β. This study shows that simple (no-labeling), rapid (less than 30 min per assay), and eco-friendly (no-radioactivity) CE-based kinase assays can give insight into the abnormal phosphorylation of tau. They can be extended to screen different modulators of tau phosphorylation to highlight their function and to develop effective drugs for neurodegenerative disease treatments.

On-line capillary electrophoresis for the evaluation of glycosaminoglycan (GAG) influence on the phosphorylation of tau protein by GSK3b

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Affiliations

  1. Institut de Chimie Organique et Analytique (ICOA), Université d’Orléans - Centre National de la Recherche Scientifique (CNRS), FR 2708 - UMR 7311, 45067, Orléans, France

    Hala Nehmé, Justine Defert, Philippe Morin & Reine Nehmé

  2. Laboratoire Croissance, Réparation et Régénération Tissulaires (CRRET), Centre National de la Recherche Scientifique (CNRS) EAC 7149, Université Paris Est Créteil, 94000, Créteil, France

    Sandrine Chantepie & Dulce Papy-Garcia

Authors
  1. Hala Nehmé
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  2. Sandrine Chantepie
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  3. Justine Defert
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  4. Philippe Morin
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  5. Dulce Papy-Garcia
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  6. Reine Nehmé
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Correspondence to Dulce Papy-Garcia or Reine Nehmé.

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Nehmé, H., Chantepie, S., Defert, J. et al. New methods based on capillary electrophoresis for in vitro evaluation of protein tau phosphorylation by glycogen synthase kinase 3-β. Anal Bioanal Chem 407, 2821–2828 (2015). https://doi.org/10.1007/s00216-015-8495-7

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  • DOI: https://doi.org/10.1007/s00216-015-8495-7

Keywords

  • Protein tau
  • Glycogen synthase kinase-3β (GSK3β)
  • Heparan sulfates
  • Heparin
  • Transverse diffusion of laminar flow profiles (TDLFP)