Insight in the molecular consequenses of LRRK2 kinase inhibition, a kinase involved in Parkinson's disease.

Parkinson’s disease is the second most common neurodegenerative disorder, after Alzheimer’s disease, and the most common movement disorder. Worldwide, 7 to 10 million people suffer from Parkinson’s disease. It is estimated that this number will only increase due to the general ageing of the population, as the disease primarily affects people older than the age of 60. Current therapies fail to cure, halt or slow down disease progression, partly because the exact etiology of Parkinson’s disease is still poorly understood. However, treatment options that are able to (temporarily) suppress a patient’s symptoms do exist.
Since 1997, different genetic studies revealed that mutations in several genes underlie the development of Parkinson’s disease. To date, mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common known cause of genetic forms of Parkinson’s disease. Moreover, the clinical phenotype of patients carrying LRRK2 mutations strongly resembles the phenotype of patients with a sporadic form of the disease, which might indicate a common etiology. Consequently, more insight in the molecular processes that underlie the development of genetic Parkinson’s disease could provide important insights for the development of potential new therapies, both for genetic and sporadic forms of the disease.
For example, studies have shown that pathogenic mutations in LRRK2 display increased kinase activity. As a result, kinase inhibition was proposed as a potential therapeutic strategy. Studies in cell culture and in animal models, both LRRK2-based and non-LRRK2-associated PD models, have shown the protective effect of LRRK2 kinase inhibition. Regardless, some caution is warranted, as we might not yet fully comprehend all the consequences of LRRK2 kinase inhibition.

First of all, LRRK2 kinase inhibition induces dephosphorylation of the LRRK2 protein, which is also seen in pathogenic LRRK2 variants. This finding indicates a potential contribution of dephosphorylation to LRRK2 pathogenesis in Parkinson’s disease. Signal transduction in a cell or between cells often relies on whether or not certain proteins are phosphorylated. It is of great importance to identify the different phosphatase complexes involved in LRRK2 dephosphorylation after LRRK2 kinase inhibition or in pathogenic conditions. Our lab identified the catalytic subunit of protein phosphatase 1 (PP1) as the phosphatase acting on pathogenic variants of LRRK2 and during LRRK2 kinase inhibition. The regulatory proteins or other phosphatases involved in the process, remain elusive. In the first part of this work, we aimed to identify phosphatases or regulatory proteins involved in LRRK2 dephosphorylation after LRRK2 kinase inhibition. Therefore, a small interfering (si) RNA screen was executed, in which the effects on LRRK2 phosphorylation were investigated after knockdown of specific phosphatases or regulatory proteins. In this project, we aimed to further validate the top hits from the siRNA screen in cells, using lentiviral vector-mediated knockdown. Besides PP1, we found that also the protein phosphatase 2A (PP2A) complex is involved in the LRRK2 dephosphorylation cycle, especially after LRRK2 kinase inhibition. Further identification of the LRRK2-phosphatase complex in pathogenic variants or after kinase inhibition, will provide more insight in potential therapies for Parkinson’s disease.

Besides, we and others found that prolonged treatment with LRRK2 kinase inhibitors causes a decrease in total LRRK2 protein levels. This could be observed both in cell culture and in animals treated with LRRK2 inhibitors. A characteristic pathology has been described in the lung of inhibitor-treated animals. Moreover, this phenotype was also observed in LRRK2 knockout animals. In the next part of this work, we aimed to identify the underlying mechanism of LRRK2 kinase inhibitor-induced destabilization. By studying and/or blocking the different ways for synthesis or degradation of proteins in the cell, we found that LRRK2 is mainly degraded by the proteasomal system after LRRK2 kinase inhibition. Next, we aimed to further characterize LRRK2 kinase inhibitor-induced destabilization in different pathogenic LRRK2 mutants. We observed that not all pathogenic variants of LRRK2 show a decrease in total LRRK2 protein levels after treatment. Remarkably, mutants that are strongly dephosphorylated, do not destabilize after LRRK2 kinase inhibition, in contrast to an only partly dephosphorylated mutant. Consequently, we assumed that the basal phosphorylation levels of the LRRK2 protein could be important in the regulation of protein homeostasis and induction of destabilization. We investigated the importance of already known phosphorylation sites, by mutating these residues. We found that none of the already known phosphorylation sites were crucial residues in the induction of protein degradation. Casein kinase 1α (CK1α) was identified as the kinase phosphorylating these known, and potentially other unknown, phosphorylation sites in LRRK2. After inhibition of CK1, destabilization of the LRRK2 protein still occurs, both in wild type LRRK2, in pathogenic LRRK2 variants, in the LRRK2 variant in which we mutated known phosphorylation sites and in the lung in mice. Consequently, we hypothesize that currently unknown phosphorylation sites, regulated by CK1, could be the primary inductors of LRRK2 kinase inhibitor-induced destabilization of LRRK2. The next crucial step is the further identification of this/these phosphorylation site(s). The second part of the study offers important new insights in the protein homeostasis of LRRK2, which will contribute to a broader knowledge on the working mechanism of LRRK2 kinase inhibitors, one of the prevailing therapeutic strategies for the treatment of Parkinson’s disease nowadays.