Preclinical Modeling of Chronic Inhibition of the Parkinson’s Disease Associated Kinase LRRK2 Reveals Altered Function of the Endolysosomal System in Vivo

Abstract

BackgroundThe 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. To date, the molecular effects of chronic LRRK2 inhibition have not yet been examined in vivo. MethodsWe evaluated the utility of newly available phospho-antibodies for Rab substrates pT73 Rab10, pS106 Rab12, pT71 Rab29 and LRRK2 autophosphorylation to examine the pharmacodynamic response to acute treatment paradigms with the potent and specific LRRK2 inhibitor, MLi-2, in brain and peripheral tissue in G2019S LRRK2 knock-in mice to define the relative target engagement between brain and LRRK2-enriched peripheral tissues. The molecular effects of 10 days and 10 weeks of chronic in-diet dosing were also evaluated using TMTpro reagents for LC-MS/MS total and phospho- proteomics in brain and kidney tissues. ResultsWe 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 alterations in endolysosomal and trafficking pathways in the kidney that were sensitive to MLi-2 treatment and that we validated biochemically. Furthermore, a subtle but distinct biochemical signature affecting mitochondrial proteins was observed in brain tissue in the same animals that was reverted by kinase inhibition. ConclusionsThis 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.

Pathogenic LRRK2 control of primary cilia and Hedgehog signaling in neurons and astrocytes of mouse brain

“…However, levels of pRab10 seemed unchanged (Figure 4A,B) with variability among animals (see also Ianotta et al, 2020). Previous studies have also noted that MLi-2 administration does not markedly reduce pRab10 levels in brain (Kelly et al, 2018;Kalogeropulou et al, 2020;Nirujogi et al, 2021;Kluss et al, 2021). Note that analysis of whole brain in this experiment will not capture the precise status of pRab10 levels in rare cholinergic neurons of the dorsal striatum that show LRRK2 mutation-associated ciliary deficits.…”

Endosomal traffic and glutamate synapse activity are increased in VPS35 D620N mutant knock-in mouse neurons, and resistant to LRRK2 kinase inhibition

“…This residue, considered constitutive, is required for LRRK2 kinase activity but is not an autophosphorylation site and its phosphorylation level does not correlate with LRRK2 kinase enzymatic activity levels [49]; however, S935 is dephosphorylated when LRRK2 is inactivated, and it is widely accepted as indicative of effective LRRK2 kinase inhibition [50]. In mice injected with the highly selective LRRK2 kinase inhibitor MLi-2 [27,41,49,50], pLRRK2 was significantly reduced after 2 h (~ 90%) in all genotypes, relative to vehicle injected controls (Fig. 1C.i-ii; 2-way ANOVA treatment p < 0.0001; Uncorrected Fisher's LSD WT-WTMLi2 ****p < 0.0001; Het-HetMLi2 ***p < 0.0002; Ho-HoMLi2 ****p < 0.0001) demonstrating successful target engagement.…”

LRRK2 and idiopathic Parkinson’s disease

“…A recent study in nonhuman primates examined the pulmonary effects of three structurally distinct LRRK2 kinase inhibitors and found only mild histopathological lung changes that were reversable and without noticeable effect on lung function [96]. Studies in mice and rats also yielded reassuring results [38,[97][98][99]. Importantly, two recent genetic studies demonstrated that LRRK2 loss-of-function variants in humans do not appear to have negative effects on health or survival [100,101], which further argues in favor of the potential safety of LRRK2-targeted therapeutic approaches.…”

“…These phosphorylation sites are currently considered biomarkers for on-target Type I LRRK2 kinase inhibitors, because these inhibitors result in dephosphorylation of Ser910 and Ser935 [35]. This has been validated by several independent groups using kinase inhibitors [21,[36][37][38][39]. Additionally, using overexpression systems and purified LRRK2 phosphopeptides, more recent data have suggested that LRRK2 binding to 14-3-3 proteins also occurs in the ROC-COR GTPase domain at pSer1444 [40,41].…”