Development of an enzyme-linked immunosorbent assay for detection of cellular and in vivo LRRK2 S935 phosphorylation

Lore Delbroek, Kristof Van Kolen, Liesbeth Steegmans, Raquel da Cunha, Wim Mandemakers, Guy Daneels, Pieter-Jan De Bock, Jinwei Zhang, Kris Gevaert, Bart De Strooper, Dario R. Alessi, Patrik Verstreken, Diederik W. Moechars

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    18 Citations (Scopus)

    Abstract

    After the discoveiy of kinase activating mutations in leucine-rich repeat kinase 2 (LRRK2) as associated with autosomal dominant forms of Parkinson's disease, inhibition of the kinase is being extensively explored as a disease modifying strategy. As signaling properties and substrate(s) of LRRK2 are poorly documented, autophosphorylation has been an important readout for the enzyme's activity. Western blotting using anti-phospho-S910 or S935 LRRK2 antibodies showed effectiveness in demonstrating inhibitory effects of compounds.

    In this communication we describe two types of enzyme-linked immunosorbent assays (ELISA) to determine LRRK2 protein levels and kinase activity. Both assays take advantage of the sensitivity of the earlier described total and pS935 antibodies for detection (Nichols et al., Biochem. J. 2010) [10]. The first assay is based on anti-GFP-based capturing of overexpressed LRRK2 and is highly suitable to show cellular effects of kinase inhibitors in a 96-well format. In the other platform anti-LRRK2-based capturing allows detection of endogenously expressed LRRK2 in rat tissue with no significant signal in tissue from LRRK2 knockout rats. Furthermore, both assays showed a significant reduction in pS935 levels on cellular and transgenic R1441C/G LRRK2. With the anti-LRRK2 ELISA we were able to detect LRRK2 phosphorylation in human peripheral blood mononuclear cells (PBMC).

    To conclude, we report two sensitive assays to monitor LRRK2 expression and kinase activity in samples coming from cellular and in vivo experimental settings. Both can show their value in drug screening and biomarker development but will also be useful in the elucidation of LRRK2-mediated signaling pathways. (c) 2012 Elsevier B.V. All rights reserved.

    Original languageEnglish
    Pages (from-to)49-58
    Number of pages10
    JournalJournal of Pharmaceutical and Biomedical Analysis
    Volume76
    DOIs
    Publication statusPublished - 25 Mar 2013

    Keywords

    • Animals
    • Blotting, Western
    • Enzyme-Linked Immunosorbent Assay
    • HEK293 Cells
    • Humans
    • Leukocytes, Mononuclear
    • Male
    • Mice
    • Mice, Inbred C57BL
    • Mice, Transgenic
    • Phosphorylation
    • Protein-Serine-Threonine Kinases
    • Rats
    • Rats, Long-Evans
    • Sensitivity and Specificity

    Cite this

    Delbroek, L., Van Kolen, K., Steegmans, L., da Cunha, R., Mandemakers, W., Daneels, G., De Bock, P-J., Zhang, J., Gevaert, K., De Strooper, B., Alessi, D. R., Verstreken, P., & Moechars, D. W. (2013). Development of an enzyme-linked immunosorbent assay for detection of cellular and in vivo LRRK2 S935 phosphorylation. Journal of Pharmaceutical and Biomedical Analysis, 76, 49-58. https://doi.org/10.1016/j.jpba.2012.12.002