Capturing Differences in the Regulation of LRRK2 Dynamics and Conformational States by Small Molecule Kinase Inhibitors.

Mutations in the human leucine rich repeat protein kinase-2 (LRRK2) create risk factors for Parkinson's disease, and pathological functions of LRRK2 are often correlated with aberrant kinase activity. Past research has focused on developing selective LRRK2 kinase inhibitors. In this study, we combined enhanced sampling simulations with HDX-MS to characterize the inhibitor-induced dynamic changes and the allosteric communications within the C-terminal domains of LRRK2, LRRK2RCKW. We find that the binding of MLi-2 (a type I kinase inhibitor) stabilizes a closed kinase conformation and reduces the global dynamics of LRRK2RCKW, leading to a more compact LRRK2RCKW structure. In contrast, the binding of Rebastinib (a type II kinase inhibitor) stabilizes an open kinase conformation, which promotes a more extended LRRK2RCKW structure. By probing the distinct effects of the type I and type II inhibitors, key interdomain interactions are found to regulate the communication between the kinase domain and the GTPase domain. The intermediate states revealed in our simulations facilitate the efforts toward in silico design of allosteric modulators that control LRRK2 conformations and potentially mediate the oligomeric states of LRRK2 and its interactions with other proteins.

The industrial solvent trichloroethylene induces LRRK2 kinase activity and dopaminergic neurodegeneration in a rat model of Parkinson's disease.

Gene-environment interaction is implicated in the majority of idiopathic Parkinson's disease (PD) risk, and some of the most widespread environmental contaminants are selectively toxic to dopaminergic neurons. Pesticides have long been connected to PD incidence, however, it has become increasingly apparent that other industrial byproducts likely influence neurodegeneration. For example, organic solvents, which are used in chemical, machining, and dry-cleaning industries, are of growing concern, as decades of solvent use and their effluence into the environment has contaminated much of the world's groundwater and soil. Like some pesticides, certain organic solvents, such as the chlorinated halocarbon trichloroethylene (TCE), are mitochondrial toxicants, which are collectively implicated in the pathogenesis of dopaminergic neurodegeneration. Recently, we hypothesized a possible gene-environment interaction may occur between environmental mitochondrial toxicants and the protein kinase LRRK2, mutations of which are the most common genetic cause of familial and sporadic PD. In addition, emerging data suggests that elevated wildtype LRRK2 kinase activity also contributes to the pathogenesis of idiopathic PD. To this end, we investigated whether chronic, systemic TCE exposure (200 mg/kg) in aged rats produced wildtype LRRK2 activation and caused nigrostriatal dopaminergic dysfunction. Interestingly, we found that TCE not only induced LRRK2 kinase activity in the brain, but produced a significant dopaminergic lesion in the nigrostriatal tract, elevated oxidative stress, and caused endolysosomal dysfunction and α-synuclein accumulation. Together, these data suggest that TCE-induced LRRK2 kinase activity contributed to the selective toxicity of dopaminergic neurons. We conclude that gene-environment interactions between certain industrial contaminants and LRRK2 likely influence PD risk.

Roles of lysosomotropic agents on LRRK2 activation and Rab10 phosphorylation.

Leucine-rich repeat kinase 2 (LRRK2), the major causative gene product of autosomal-dominant Parkinson's disease, is a protein kinase that phosphorylates a subset of Rab GTPases. Since pathogenic LRRK2 mutations increase its ability to phosphorylate Rab GTPases, elucidating the mechanisms of how Rab phosphorylation is regulated by LRRK2 is of great importance. We have previously reported that chloroquine-induced lysosomal stress facilitates LRRK2 phosphorylation of Rab10 to maintain lysosomal homeostasis. Here we reveal that Rab10 phosphorylation by LRRK2 is potently stimulated by treatment of cells with a set of lysosome stressors and clinically used lysosomotropic drugs. These agents commonly promoted the formation of LRRK2-coated enlarged lysosomes and extracellular release of lysosomal enzyme cathepsin B, the latter being dependent on LRRK2 kinase activity. In contrast to the increase in Rab10 phosphorylation, treatment with lysosomotropic drugs did not increase the enzymatic activity of LRRK2, as monitored by its autophosphorylation at Ser1292 residue, but rather enhanced the molecular proximity between LRRK2 and its substrate Rab GTPases on the cytosolic surface of lysosomes. Lysosomotropic drug-induced upregulation of Rab10 phosphorylation was likely a downstream event of Rab29 (Rab7L1)-mediated enzymatic activation of LRRK2. These results suggest a regulated process of Rab10 phosphorylation by LRRK2 that is associated with lysosomal overload stress, and provide insights into the novel strategies to halt the aberrant upregulation of LRRK2 kinase activity.

LRRK2 in Parkinson disease: challenges of clinical trials.

One of the most common monogenic forms of Parkinson disease (PD) is caused by mutations in the LRRK2 gene that encodes leucine-rich repeat kinase 2 (LRRK2). LRRK2 mutations, and particularly the most common mutation Gly2019Ser, are observed in patients with autosomal dominant PD and in those with apparent sporadic PD, who are clinically indistinguishable from those with idiopathic PD. The discoveries that pathogenic mutations in the LRRK2 gene increase LRRK2 kinase activity and that small-molecule LRRK2 kinase inhibitors can be neuroprotective in preclinical models of PD have placed LRRK2 at the centre of disease modification efforts in PD. Recent investigations also suggest that LRRK2 has a role in the pathogenesis of idiopathic PD and that LRRK2 therapies might, therefore, be beneficial in this common subtype of PD. In this Review, we describe the characteristics of LRRK2-associated PD that are most relevant to the development of LRRK2-targeted therapies and the design and implementation of clinical trials. We highlight strategies for correcting the effects of mutations in the LRRK2 gene, focusing on how to identify which patients are the optimal candidates and how to decide on the timing of such trials. In addition, we discuss challenges in implementing trials of disease-modifying treatment in people who carry LRRK2 mutations.

New Era in disease modification in Parkinson's disease: Review of genetically targeted therapeutics.

Disease modification remains a major unmet need in Parkinson's disease (PD) therapeutics. Despite multiple attempts, not a single study has yet been successful, perhaps due to our incomplete understanding of the underlying disease mechanisms. Genetic and epidemiologic studies of the last decade have substantially increased our comprehension of the etiology of PD. Once considered a pure sporadic disease, the discovery of familial mutations provided the initial paradigm shift and it is now widely accepted that PD has a substantial genetic component. These genetic discoveries have allowed the development of novel therapeutics aimed at halting or slowing the underlying disease process, rather than just ameliorating symptoms. Here, we discuss the latest advances in therapeutics based on three genetic discoveries (SNCA, LRRK2 and GBA) that are currently reaching the clinical arena and outline the challenges of therapeutic development of genetically targeted therapeutics.