Impact of LRRK2 kinase activation and inhibition in vivo and in vitro

Parkinson’s Disease (PD) is a progressive neurodegenerative disorder that is characterized in part by a loss of dopamine neurons in the Substantia Nigra and affects the nigrostriatal pathway. Mutations in the gene encoding leucine�rich repeat kinase 2 (LRRK2) have been found to cause late onset PD through a gain of function of its kinase domain. Thus, LRRK2 has become an intriguing candidate for therapeutic intervention by kinase inhibition. While preclinical studies have shown that ablating the kinase activity of wildtype LRRK2 is safe with a mild and reversible lung phenotype, the molecular effects of chronic LRRK2 inhibition have not been examined in the context of mutant LRRK2. Using the potent LRRK2 kinase-specific inhibitor, Merck LRRK2 inhibitor 2 (MLi�2), hyperactive G2019S LRRK2 was reduced to wildtype levels chronically in G2019S knock-in (KI) mice and autophosphorylation of LRRK2 and phosphorylation of direct substrates Rab10, Rab12, and Rab29 was assessed. Unbiased total and phospho-proteomics revealed alterations in endolysosomal proteins similar to those found in LRRK2 knockout (KO) animals after 10 weeks of treatment. LRRK2 has been shown to play roles in a number of pathways within the endolysosomal system and studies have reported LRRK2 presence on many organelles from the trans-Golgi network (TGN) to the lysosome. Based on the current study’s proteomic results, eight different trap plasmids were generated to evaluate LRRK2 kinase activity at distinct endolysosomal membranes. As a result, LRRK2 kinase activity was found to be enhanced after being trapped to all membranes and downstream Rab10 and Rab12 phosphorylation were increased in vitro, but recruitment of these Rabs revealed differential patterns in localization specifically when targeting LRRK2 on lysosomes compared to other membranes. Evaluation of lysosomal position through manipulation of various motor proteins showed phosphorylated Rab10 was preferentially restricted to a subset of perinuclear lysosomes, whereas pRab12 was present at most LRRK2-positive lysosomes regardless of their position. This is the first study to examine the molecular underpinnings of chronic LRRK2 inhibition in a preclinical in vivo PD model and highlights cellular pathways that may be influenced by therapeutic strategies aimed at restoring LRRK2 physiological activity in PD patients. Complementary in vitro data provides novel insight into the differences in LRRK2-dependent Rab localization that can help elucidate the role of LRRK2 at the lysosome which may be relevant to PD pathogenesis. The work presented in this thesis additionally contributes to our knowledge on the utility of pS1292 LRRK2 and pS106 Rab12 as robust biomarkers of both kinase hyperactivity and inhibition in G2019S LRRK2 KI mice in brain and peripheral tissues that is worth assessment in patients with PD harboring the G2019S mutation

Leucine-rich repeat kinase 2 (LRRK2): an update on the potential therapeutic target for Parkinson’s disease

Mutations in Leucine-rich repeat kinase 2 (LRRK2) are a risk factor for and a cause of sporadic and familial Parkinson’s disease (PD), respectively. These mutations are some of the most common genetic contributors to PD and render the kinase hyperactive. Increasingly within the past decade, there has been substantial effort investigating LRRK2 as a target for therapeutics in preclinical studies, and currently, small-molecule inhibitors and antisense oligonucleotides are being assessed in clinical trials as therapies to reduce the toxic hyperactivity of its kinase and/or reduce total levels of the protein in healthy individuals and people with PD

Leucine-rich repeat kinase 2 (LRRK2): an update on the potential therapeutic target for Parkinson's disease.

In this review, we will provide an update on the current status of drugs and other technologies that have emerged in recent years and provide an overview of their efficacy in ameliorating LRRK2 kinase activity and overall safety in animal models and humans. The growth of both target discovery and innovative drug design has sparked a lot of excitement for the future of how we treat Parkinson's disease. Given the immense focus on LRRK2 as a therapeutic target, it is expected within the next decade to determine its therapeutic properties, or lack thereof, for PD

Directing LRRK2 to membranes of the endolysosomal pathway triggers RAB phosphorylation and JIP4 recruitment

Coding mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene, which are associated with dominantly inherited Parkinson's disease (PD), lead to an increased activity of the encoded LRRK2 protein kinase. As such, kinase inhibitors are being considered as therapeutic agents for PD. It is therefore of interest to understand the mechanism(s) by which LRRK2 is activated during cellular signaling. Lysosomal membrane damage represents one way of activating LRRK2 and leads to phosphorylation of downstream RAB substrates and recruitment of the motor adaptor protein JIP4. However, it is unclear whether the activation of LRRK2 would be seen at other membranes of the endolysosomal system, where LRRK2 has also shown to be localized, or whether these signaling events can be induced without membrane damage. Here, we use a rapamycin-dependent oligomerization system to direct LRRK2 to various endomembranes including the Golgi apparatus, lysosomes, the plasma membrane, recycling, early, and late endosomes. Irrespective of membrane location, the recruitment of LRRK2 to membranes results in local accumulation of phosphorylated RAB10, RAB12, and JIP4. We also show that endogenous RAB29, previously nominated as an activator of LRRK2 based on overexpression, is not required for activation of LRRK2 at the Golgi nor lysosome. We therefore conclude that LRRK2 signaling to RAB10, RAB12, and JIP4 can be activated once LRRK2 is accumulated at any cellular organelle along the endolysosomal pathway

Lysosomal positioning regulates Rab10 phosphorylation at LRRK2+ lysosomes

Genetic variation at the leucine-rich repeat kinase 2 (LRRK2) locus contributes to an enhanced risk of familial and sporadic Parkinson’s disease. Previous data have demonstrated that recruitment to various membranes of the endolysosomal system results in LRRK2 activation. However, the mechanism(s) underlying LRRK2 activation at endolysosomal membranes and the cellular consequences of these events are still poorly understood. Here, we directed LRRK2 to lysosomes and early endosomes, triggering both LRRK2 autophosphorylation and phosphorylation of the direct LRRK2 substrates Rab10 and Rab12. However, when directed to the lysosomal membrane, pRab10 was restricted to perinuclear lysosomes, whereas pRab12 was visualized on both peripheral and perinuclear LRRK2+ lysosomes, suggesting that lysosomal positioning provides additional regulation of LRRK2-dependent Rab phosphorylation. Anterograde transport of lysosomes to the cell periphery by increasing the expression of ARL8B and SKIP or by knockdown of JIP4 blocked the recruitment and phosphorylation of Rab10 by LRRK2. The absence of pRab10 from the lysosomal membrane prevented the formation of a lysosomal tubulation and sorting process we previously named LYTL. Conversely, overexpression of RILP resulted in lysosomal clustering within the perinuclear area and increased LRRK2-dependent Rab10 recruitment and phosphorylation. The regulation of Rab10 phosphorylation in the perinuclear area depends on counteracting phosphatases, as the knockdown of phosphatase PPM1H significantly increased pRab10 signal and lysosomal tubulation in the perinuclear region. Our findings suggest that LRRK2 can be activated at multiple cellular membranes, including lysosomes, and that lysosomal positioning further provides the regulation of some Rab substrates likely via differential phosphatase activity or effector protein presence in nearby cellular compartments

Preclinical modeling of chronic inhibition of the Parkinson’s disease associated kinase LRRK2 reveals altered function of the endolysosomal system in vivo

The most common mutation in the Leucine-rich repeat kinase 2 gene (LRRK2), G2019S, causes familial Parkinson’s Disease (PD) and renders the encoded protein kinase hyperactive. While targeting LRRK2 activity is currently being tested in clinical trials as a therapeutic avenue for PD, to date, the molecular effects of chronic LRRK2 inhibition have not yet been examined in vivo. We evaluated the utility of newly available phospho-antibodies for Rab substrates and LRRK2 autophosphorylation to examine the pharmacodynamic response to treatment with the potent and specific LRRK2 inhibitor, MLi-2, in brain and peripheral tissue in G2019S LRRK2 knock-in mice. We report higher sensitivity of LRRK2 autophosphorylation to MLi-2 treatment and slower recovery in washout conditions compared to Rab GTPases phosphorylation, and we identify pS106 Rab12 as a robust readout of downstream LRRK2 activity across tissues. The downstream effects of long-term chronic LRRK2 inhibition in vivo were evaluated in G2019S LRRK2 knock-in mice by phospho- and total proteomic analyses following an in-diet administration of MLi-2 for 10 weeks. We observed significant alterations in endolysosomal and trafficking pathways in the kidney that were sensitive to MLi-2 treatment and were validated biochemically. Furthermore, a subtle but distinct biochemical signature affecting mitochondrial proteins was observed in brain tissue in the same animals that, again, was reverted by kinase inhibition. Proteomic analysis in the lung did not detect any major pathway of dysregulation that would be indicative of pulmonary impairment. This is the first study to examine the molecular underpinnings of chronic LRRK2 inhibition in a preclinical in vivo PD model and highlights cellular processes that may be influenced by therapeutic strategies aimed at restoring LRRK2 physiological activity in PD patients

Correction: Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia

[This corrects the article DOI: 10.1371/journal.pbio.3001480.]

Evaluation of Current Methods to Detect Cellular Leucine-Rich Repeat Kinase 2 (LRRK2) Kinase Activity

Background: Coding variation in the Leucine rich repeat kinase 2 gene linked to Parkinson’s disease (PD) promotes enhanced activity of the encoded LRRK2 kinase, particularly with respect to autophosphorylation at S1292 and/or phosphorylation of the heterologous substrate RAB10. Objective: To determine the inter-laboratory reliability of measurements of cellular LRRK2 kinase activity in the context of wildtype or mutant LRRK2 expression using published protocols. Methods: Benchmark western blot assessments of phospho-LRRK2 and phospho-RAB10 were performed in parallel with in situ immunological approaches in HEK293T, mouse embryonic fibroblasts, and lymphoblastoid cell lines. Rat brain tissue, with or without adenovirus-mediated LRRK2 expression, and human brain tissues from subjects with or without PD, were also evaluated for LRRK2 kinase activity markers. Results: Western blots were able to detect extracted LRRK2 activity in cells and tissue with pS1292-LRRK2 or pT73-RAB10 antibodies. However, while LRRK2 kinase signal could be detected at the cellular level with over-expressed mutant LRRK2 in cell lines, we were unable to demonstrate specific detection of endogenous cellular LRRK2 activity in cell culture models or tissues that we evaluated. Conclusion: Further development of reliable methods that can be deployed in multiple laboratories to measure endogenous LRRK2 activities are likely required, especially at cellular resolution