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

• Published in: Journal of pharmaceutical and biomedical analysis Vol. 76; pp. 49 - 58
• Main Authors: Delbroek, Lore, Van Kolen, Kristof, Steegmans, Liesbeth, da Cunha, Raquel, Mandemakers, Wim, Daneels, Guy, De Bock, Pieter-Jan, Zhang, Jinwei, Gevaert, Kris, De Strooper, Bart, Alessi, Dario R., Verstreken, Patrik, Moechars, Diederik W.
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 25.03.2013
• Subjects: Animals; antibodies; biomarkers; Blotting, Western; drugs; ELISA; enzyme-linked immunosorbent assay; Enzyme-Linked Immunosorbent Assay - methods; genetically modified organisms; HEK293 Cells; Humans; in vivo studies; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Leukocytes, Mononuclear - metabolism; LRRK2; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; mononuclear leukocytes; mutation; Parkinson disease; Parkinson's disease; Phosphorylation; Protein Serine-Threonine Kinases - metabolism; Rats; Rats, Long-Evans; S935 phosphorylation; screening; Sensitivity and Specificity; signal transduction; Western blotting; Parkinson's disease; KO; LRRK2; MOI; NT; S935 phosphorylation; HRP; TMB; S910/935; PBMC; LC–MS/MS; HEK293; Tg; PD; FCS; WT; ELISA
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2012.12.002

General outline and example of two ELISAs to study expression and activity of overexpressed GFP-LRRK2 (A, B) and endogenous LRRK2 (C, D). In panel (B), the effect of indicated concentrations of a LRRK2 inhibitor on relative S935 phosphorylation is measured by ELISA (A) while in (D) this is determined in rodent tissue by using the assay indicated in (C). [Display omitted] ► We established two LRRK2 ELISA's. ► Serine 935 phosphorylation is used as readout for LRRK2 activity. ► One assay can be used to screen for cellular active LRRK2 inhibitors. ► We present an ELISA to measure in vivo pS935 LRRK2 levels. ► With this assay we demonstrate in vivo efficacy of a LRRK2 inhibitor. After the discovery 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.