Abstract
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.
Highlights
Mutations in leucine-rich repeat kinase 2 (LRRK2) are a known genetic cause of familial Parkinson’s disease (PD) [1,2,3];
Using unsupervised hierarchical clustering we show that the G2019S Merch LRRK2 inhibitor 2 (MLi-2) and LRRK2 KO mice cluster together, suggesting that chronic LRRK2 inhibition mimics a LRRK2 KO effect in kidney. c Bubble plot showing GO:CC term enrichment in the significant total protein hits highlights enrichment for endolysosomal processes and compartments. d Volcano plot showing changes in phospho-proteins in G2019S MLi-2 treated vs G2019S untreated mice, highlighting dysregulation of proteins involved in trafficking, glucose transport and metabolism, and the TCA cycle e. f Bubble plot showing GO:CC term enrichment in the significant phospho-peptide hits highlights enrichment for endolysosomal processes as well as kidney-specific functional processes
Using the commercially available pRab antibodies, we found that S106 Rab12 phosphorylation pattern resembled that of pS1292 LRRK2 most closely across experiments, in that G2019S KI mice displayed hyperphosphorylation that was ameliorated with MLi-2 treatment
Summary
Mutations in leucine-rich repeat kinase 2 (LRRK2) are a known genetic cause of familial Parkinson’s disease (PD) [1,2,3];. Some kinase inhibitors have been shown to reduce cytotoxicity associated with LRRK2 mutations in PD-relevant cell and animal models [10,11,12], supporting the hypothesis that LRRK2 inhibition may be efficacious for PD One such LRRK2-specific inhibitor, MLi-2, has been found to be ~300x more selective for LRRK2 over other kinases and can readily cross the blood-brain barrier [13] demonstrating that it is possible to generate tool compounds that have clinical potential for therapeutic intervention in PD. Preclinical studies have reported macroscopic changes in vivo that include morphological changes in lung from nonhuman primates, and kidney tissue from rats treated with specific LRRK2 inhibitors that are reversible after termination of treatment [14,15,16] Some of these effects overlap with those seen in Lrrk knockout mice [17] demonstrating that they result from on-target effects of inhibitors on LRRK2 itself rather than resulting from inhibition of other kinases
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