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

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.

Lysosomal positioning regulates Rab10 phosphorylation at LRRK2-positive lysosomes

“…We observed a striking difference in colocalization pattern between Rabs, with pT73 Rab10 showing strict perinuclear localization in a subset of lysosomes whereas pS106 Rab12 was found in LRRK2-positive lysosomes throughout the cell. Recently , we proposed that pRab12 more accurately reports the effects of inhibition of LRRK2 kinase activity than pRab10 in vivo (Kluss et al 2021). Our current data suggests that Rab12 is regulated by additional mechanisms distinct from Rab10 (discussed below).…”

“…LRRK2 may also play an important role in regulation of lysosomes in vivo, as lysosomal proteins are dysregulated in LRRK2 knockout mice (Pellegrini et al 2018) or after chronic LRRK2 kinase inhibition (Kluss et al 2021). Intriguingly, the same treatments result in variable effects on Rab phosphorylation with Rab12 being strongly inhibited compared to Rab10 and Rab29 in vivo (Kluss et al 2021).…”

Astrocytes, a Promising Opportunity to Control the Progress of Parkinson’s Disease

“…PD may also be facilitated by changes in the activity of different genes which increase the incidence of PD and are directly involved in the astrocyte biology [137][138][139][140][141][142][143][144][145][146][147][148][149]. This is the case of PARK7 (DJ-1 protein), which is involved in the glutamate uptake, mitochondrial function, oxidative stress, and inflammatory response of astrocytes [150][151][152][153][154]; PARK2 (Parkin), which is involved in the inflammatory response, neuroprotection, proliferation, and mitochondrial functions of astrocytes [149,[155][156][157]; SNCA (α-synuclein), which is involved in glutamate uptake, neurotrophic activity, water transport, and endocytosis functions of astrocytes [158][159][160][161][162]; PINK1 (PTEN-induced putative kinase 1), which is involved in proliferation and mitochondrial function of astrocytes [139,163]; GBA (β-glucorecebrosidase), which is involved in autophagy, lysosome functions, and mitochondrial functions of astrocytes [164,165]; LRRK2 (leucine-rich repeat kinase 2), which is involved in autophagy and lysosome functions of astrocytes [166][167][168]; ATP13A2 (lysosomal type 5 ATPase), which is involved in the neurotrophic activity, inflammatory response, and lysosome functions of astrocytes [169]; and PLA2G6 (group VI Ca 2+ -independent phospholipase A 2 ), which is involved in inflammatory response and calcium signaling functions of astrocytes [170,…”

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

“…This residue is widely used as a proxy for LRRK2 activation (49). In mice injected with the highly selective LRRK2 kinase inhibitor MLi-2 (27,41,49,50), pLRRK2 was significantly reduced after 2h (~90%) in all genotypes, relative to vehicle injected controls (Fig. 1 C.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.…”