Measurement of LRRK2 Kinase Activity by Proximity Ligation Assay

Background

Parkinson’s disease (PD) is the second most common neurodegenerative disease (Dorsey and Bloem, 2018), which is caused by both genetic and environmental factors. Approximately 10% of cases are caused by single gene mutations, while 90% are due to generally unknown causes and are termed idiopathic PD (iPD). Missense mutations in leucine rich-repeat kinase 2 (LRRK2) are the most frequent cause of autosomal dominant PD (Paisan-Ruiz et al., 2004; Zimprich et al., 2004). These mutations result in a toxic gain of function related to elevated LRRK2 kinase activity (West et al., 2005; Greggio et al., 2006). The LRRK2 gene locus is also a risk factor for iPD (Simon-Sanchez et al., 2009), and increased kinase activity has been implicated in the pathogenesis of iPD (Fraser et al., 2016; Di Maio et al., 2018).

Assessment of LRRK2 kinase activity has been challenging until recently. Phosphorylation of the LRRK2 substrate Rab10 has been used as an indicator of elevated kinase activity (Steger et al., 2016). There has been growing consensus that phosphoserine 1292 (pS1292) is an effective surrogate for LRRK2 kinase activity, constituting a reliable readout of kinase activity (Sheng et al., 2012). Several LRRK2 mutations (i.e., G2019S and R1441G) that result in elevated kinase activity also display an increase in pS1292 levels (Sheng et al., 2012). Kinase activity is typically measured by immunoblotting for pS1292, which negates anatomical and cellular resolution. LRRK2 is believed to be a relatively low-abundance protein, and detection of pS1292 with traditional immunocytochemistry is difficult due to problems inherent in detecting a fraction of a sparse protein, such as the need for high antibody concentrations that result in nonspecific signals. Signal amplification, an important feature of the proximity ligation assay (PLA), provides higher sensitivity and allows the use of low concentrations of antibodies, which minimizes nonspecific signals. Additionally, the requirement for molecular proximity of the antibody binding sites to generate a signal effectively filters out off-target or nonspecific antibody binding.

We employed the PLA technique (Weibrecht et al., 2010) to amplify the signal of pS1292 (PLA LRRK2) (Figure 1) which, in turn, allows us to visualize kinase active LRRK2 in a cellular context (Di Maio et al., 2018). This approach relies on two antibodies, one that binds to total LRRK2 protein and another antibody from a different species that binds to the pS1292 residue. Next, species-specific oligonucleotide-labeled secondary antibodies (PLA probes) bind to the respective primary antibody (i.e., anti-mouse PLA Probe Minus binds to mouse anti-LRRK2/N241A and anti-rabbit PLA Probe Plus binds to rabbit anti-LRRK2 pS1292). If both primary antibodies are bound to LRRK2 (in close proximity), the PLA probes will be connected and ligated together into circular DNA. This newly ligated circular DNA is used as template for a rolling circle amplification reaction that amplifies the signal several hundred-fold. Then, fluorescently labeled complementary detection oligonucleotides are hybridized to the amplified signal. The resulting fluorescent product can be visualized by confocal microscopy. We validated PLA LRRK2 in CRISPR/Cas9 genome-edited human embryonic kidney 293 (HEK293) cells LRRK2^{G2019S/G2019S} (kinase active) and LRRK2^-/- (kinase deficient). We also used PLA to look at LRRK2 kinase activity in rat brain and post-mortem human brain tissues (Di Maio et al., 2018). Importantly, independent research groups have used our PLA LRRK2 assay to measure LRRK2 kinase activity (Fernandez et al., 2019; Obergasteiger et al., 2020; Bucher et al., 2020). Here, we describe in detail our method for how to investigate LRRK2 kinase activity in situ via PLA LRRK2.

Figure 1. PLA LRRK2 Schematic. In its kinase active form, S1292 undergoes autophosphorylation (pS1292). Therefore, both pS1292 (blue) and total LRRK2 (red) antibodies will bind to LRRK2. The species-specific PLA probes coupled with oligonucleotides recognize a primary antibody (pS1292 or total LRRK2). As both pS1292 and total LRRK2 antibodies are bound to the LRRK2 protein and are in close proximity, the PLA probes are connected and ligated together. This is amplified by a rolling circle amplification reaction, and fluorescent detection oligonucleotides (red dots) are hybridized to this amplified product. The fluorescent signal is visualized by confocal microscopy. ARM, armadillo; ANK, ankyrin; LRR, leucine-rich repeat; ROC, ras of complex; COR, c-terminal of ROC; KIN, kinase.