Pharmacological rescue of impaired mitophagy in Parkinson’s disease-related LRRK2 G2019S knock-in mice

Abstract

Parkinson’s disease (PD) is a major and progressive neurodegenerative disorder, yet the biological mechanisms involved in its aetiology are poorly understood. Evidence links this disorder with mitochondrial dysfunction and/or impaired lysosomal degradation – key features of the autophagy of mitochondria, known as mitophagy. Here, we investigated the role of LRRK2, a protein kinase frequently mutated in PD, in this process in vivo. Using mitophagy and autophagy reporter mice, bearing either knockout of LRRK2 or expressing the pathogenic kinase-activating G2019S LRRK2 mutation, we found that basal mitophagy was specifically altered in clinically relevant cells and tissues. Our data show that basal mitophagy inversely correlates with LRRK2 kinase activity in vivo. In support of this, use of distinct LRRK2 kinase inhibitors in cells increased basal mitophagy, and a CNS penetrant LRRK2 kinase inhibitor, GSK3357679A, rescued the mitophagy defects observed in LRRK2 G2019S mice. This study provides the first in vivo evidence that pathogenic LRRK2 directly impairs basal mitophagy, a process with strong links to idiopathic Parkinson’s disease, and demonstrates that pharmacological inhibition of LRRK2 is a rational mitophagy-rescue approach and potential PD therapy.

Impact of Type II LRRK2 inhibitors on signaling and mitophagy

“…This effect was specific for inhibition of LRRK2, as neither GZD-824 or GSK3357679A impacted basal mitophagy in LRRK2 knock-out cells. Consistent with previous work [ 25 ], basal mitophagy was lower in LRRK2[G2019S] MEFs compared with wild-type MEFs and treatment with Type I or Type II inhibitors increased basal mitophagy to levels similar to those observed in LRRK2 knock-out cells ( Figure 8A,B ).…”

“…Previous work revealed that pathogenic LRRK2[G2019S] mutation significantly reduces basal mitophagy, but not general autophagy, in primary MEFs and various mouse tissues in a manner that is rescued by inhibiting LRRK2 with structurally distinct and selective Type I inhibitors, GSK3357679A and MLi-2 [ 25 ]. We therefore monitored the impact that 10–300 nM GZD-824 had on basal mitophagy in previously generated wild type, LRRK2[G2019S] knock-in and LRRK2 knock-out MEFs [ 7 ] stably expressing the mito -QC mitophagy reporter [ 25 ]. The mito -QC reporter is localized to mitochondria via an outer mitochondrial membrane targeting sequence derived from the protein FIS1 and bears a tandem GFP and mCherry tag.…”

“…GSK3357679A [ 25 ]was synthesized at GlaxoSmithKline. MLi-2 was synthesized by Natalia Shpiro (University of Dundee) as described previously [ 16 ].…”

“…To assess mitophagy, wild type, G2019S knock in, and LRRK2 knock out primary mouse embryonic fibroblasts (MEFs) were derived, from time-mated pregnant females at E12.5 as previously described [ 25 ]. Briefly, cells were plated on glass coverslips and incubated with DMEM (Gibco, 11960-044) supplemented with 10% FBS, 2 mM l -Glutamine (Gibco, 2503-081), 1% non-essential amino acids (Gibco, 11140-035), 1% Antibiotics (Penicillin/Streptomycin 100 U/ml penicillin and 100 µg/ml streptomycin; Gibco), and 150 µM β-Mercaptoethanol (Gibco, 21985-023) at 37°C under a humidified 5% CO 2 atmosphere.…”

“…Evidence points towards LRRK2 controlling mitochondrial [ 18 , 19 ] and lysosomal [ 20–24 ] homeostasis. Recent work has revealed that LRRK2 inhibits basal mitophagy in a variety of cells including dopaminergic neurons and that LRRK2 inhibitors promote mitophagy in LRRK2[G2019S] knock-in mice [ 25 ]. LRRK2 has also been shown to control repair of damaged endomembranes through its ability to phosphorylate Rab8A [ 26 , 27 ].…”