Inhibition of LRRK2 or Casein Kinase 1 Results in LRRK2 Protein Destabilization.

Mutations and variations in the leucine-rich repeat kinase 2 (LRRK2) gene are strongly associated with an increased risk to develop Parkinson's disease (PD). Most pathogenic LRRK2 mutations display increased kinase activity, which is believed to underlie LRRK2-mediated toxicity. Therefore, major efforts have been invested in the development of potent and selective LRRK2 kinase inhibitors. Several of these compounds have proven beneficial in cells and in vivo, even in a LRRK2 wild-type background. Therefore, LRRK2 kinase inhibition holds great promise as disease-modifying PD therapy, and is currently tested in preclinical and early clinical studies. One of the safety concerns is the development of lung pathology in mice and non-human primates, which is most likely related to the strongly reduced LRRK2 protein levels after LRRK2 kinase inhibition. In this study, we aimed to better understand the molecular consequences of chronic LRRK2 kinase inhibition, which may be pivotal in the further development of a LRRK2 kinase inhibitor-based PD therapy. We found that LRRK2 protein levels are not restored during long-term LRRK2 kinase inhibition, but are recovered upon inhibitor withdrawal. Interestingly, LRRK2 kinase inhibitor-induced destabilization does not occur in all pathogenic LRRK2 variants and the N-terminal part of LRRK2 appears to play a crucial role in this process. In addition, we identified CK1, an upstream kinase of LRRK2, as a regulator of LRRK2 protein stability in cell culture and in vivo. We propose that pharmacological LRRK2 kinase inhibition triggers a cascade that results in reduced CK1-mediated phosphorylation of yet unidentified LRRK2 phosphorylation sites. This process involves the N-terminus of LRRK2 and ultimately leads to LRRK2 protein degradation.

Pharmacological LRRK2 kinase inhibition induces LRRK2 protein destabilization and proteasomal degradation.

Leucine-rich repeat kinase 2 (LRRK2) kinase activity is increased in several pathogenic mutations, including the most common mutation, G2019S, and is known to play a role in Parkinson's disease (PD) pathobiology. This has stimulated the development of potent, selective LRRK2 kinase inhibitors as one of the most prevailing disease-modifying therapeutic PD strategies. Although several lines of evidence support beneficial effects of LRRK2 kinase inhibitors, many questions need to be answered before clinical applications can be envisaged. Using six different LRRK2 kinase inhibitors, we show that LRRK2 kinase inhibition induces LRRK2 dephosphorylation and can reduce LRRK2 protein levels of overexpressed wild type and G2019S, but not A2016T or K1906M, LRRK2 as well as endogenous LRRK2 in mouse brain, lung and kidney. The inhibitor-induced reduction in LRRK2 levels could be reversed by proteasomal inhibition, but not by lysosomal inhibition, while mRNA levels remained unaffected. In addition, using LRRK2 S910A and S935A phosphorylation mutants, we show that dephosphorylation of these sites is not required for LRRK2 degradation. Increasing our insight in the molecular and cellular consequences of LRRK2 kinase inhibition will be crucial in the further development of LRRK2-based PD therapies.