Author response: Pharmacological rescue of impaired mitophagy in Parkinson’s disease-related LRRK2 G2019S knock-in mice (2021)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics 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. Introduction Parkinson’s disease (PD) is the second most common neurodegenerative disorder, affecting 1–2% of the population over 60 years old, and 4% above 85 years of age (Coppedè, 2012). The main symptoms of PD are muscle rigidity, bradykinesia, resting tremor, and postural instability and may be accompanied by sleep disorders, anosmia, depression, and dementia (Sironi et al., 2020). It is characterised by a progressive and selective degeneration of dopaminergic (DA) neurons of the substantia nigra pars compacta (SNpc). Currently, there are no treatments available that modify the course of neurodegenerative decline. Although this disease is mostly sporadic, about 15% of cases appear to be inherited and in support of this, 20 genes implicated in PD have been identified from familial genetic studies while ~90 loci have been identified from PD-GWAS (Deng et al., 2018). The exact causes of PD are currently unknown but some evidence strongly links impaired mitochondrial and lysosomal function to disease pathology (Sironi et al., 2020). Mutations in PARK8, encoding for LRRK2 (Leucine-Rich Repeat Kinase 2), are the most frequently reported cause of PD (Bouhouche et al., 2017). The most common mutation associated with PD is the substitution of glycine at position 2019 of LRRK2 to serine (G2019S), representing 4% of familial and 1% of sporadic cases (Lill, 2016). LRRK2 is a large multidomain protein with two catalytic domains: a Ras of complex (ROC) GTPase domain that is able to bind GTP and hydrolyse it, and a kinase domain that utilises a subset of Rab GTPases as substrates (Steger et al., 2016). Importantly, all the segregating mutations associated with PD are located in the catalytic core. A mutation in the ROC/COR domain, such as the R1441C/G/H or the Y1699C mutation, leads to decreased GTPase activity and elevated kinase activity (Rudenko and Cookson, 2014). Mutations in the kinase domain, such as the G2019S or the I2020T, also lead to an elevated kinase activity. Hence, enhanced kinase activity appears to be a common factor in pathogenic LRRK2 mutations. Although the function of LRRK2 within cells is currently unknown, mounting evidence implicates a role in membrane trafficking (Steger et al., 2016; Pfeffer, 2018; Hur et al., 2019). Macroautophagy is a membrane trafficking pathway that delivers intracellular components to the lysosome for degradation (Yu et al., 2018). These components can include whole organelles such as mitochondria. The autophagic turnover of mitochondria is termed mitophagy, which acts as a mitochondrial quality control mechanism that allows the selective degradation of damaged or unnecessary mitochondria (Rodger et al., 2018; Montava-Garriga and Ganley, 2020). Mitophagy itself has strong links to PD following the landmark discoveries that PINK1 and Parkin, two other genes mutated in familial PD, sequentially operate to initiate mitophagy in response to mitochondrial depolarisation in cell lines (Narendra et al., 2008; Koyano et al., 2014; Pickrell and Youle, 2015; Yamano et al., 2018). However, when this pathway becomes relevant in vivo, and under what physiological conditions, is unclear especially given that PINK1 and Parkin are not required for regulation of mitophagy under normal, or basal, conditions (McWilliams et al., 2018a; McWilliams et al., 2018b; Lee et al., 2018). Indeed, our understanding of the detailed mechanisms regulating basal mitophagy remains elusive. In this study, we sought to define the physiological link between mitochondrial turnover and LRRK2 in relation to PD. We utilised our previously published mouse reporter models to study mitophagy (mito-QC) and autophagy (auto-QC; Fig. 1A, D and McWilliams et al., 2018a; McWilliams et al., 2016; McWilliams et al., 2019) in either LRRK2 knockout mice, or knock-in mice harbouring the pathogenic LRRK2 G2019S mutation. Whilst we found minimal impact of LRRK2 on general autophagy (macroautophagy), we observed that the LRRK2 G2019S activation-mutation was associated with reduced mitophagy in specific tissues, including dopaminergic neurons and microglia within the brain. In contrast, knockout of LRRK2 resulted in increased mitophagy. Taken together, these data imply that LRRK2 kinase activity inversely correlates with basal mitophagy levels. In support of this, we found that that treatment of cells or animals with the potent and selective CNS penetrant LRRK2 kinase inhibitor, GSK3357679A (Tasegian et al., 2021; Ding, 2021, in preparation), rescued these LRRK2 G2019S-associated mitophagy defects and enhanced mitophagy in dopamine neurons and microglia in the brains of genotypically normal mice. Our results identify a physiological role for LRRK2 in the regulation of basal mitophagy in vivo and underline the potential value of pharmacological inhibition of LRRK2 as a potential therapeutic strategy to ameliorate aspects of Parkinson’s disease driven by mitochondrial dysfunction. Results The pathogenic G2019S LRRK2 mutation impairs basal mitophagy but not autophagy in vitro To investigate the physiological role of LRRK2 in regulating autophagy we utilised two previously validated and highly similar mouse reporter models (McWilliams et al., 2018a; McWilliams et al., 2016; McWilliams et al., 2019). These transgenic reporter models rely on constitutive expression of a tandem mCherry-GFP tag from the Rosa26 locus. In the mito-QC model, which monitors mitophagy, the tandem tag is localised to mitochondria (by an outer mitochondrial targeting sequence derived from residues 101–152 of the protein FIS1). In the auto-QC model, which monitors general (macro)autophagy, the tandem tag is localised to autophagosomes (by conjugation to the N-terminus of MAP1LC3b). For both models, when a mitochondrion or autophagosome is delivered to lysosomes, the low lysosomal luminal pH is sufficient to quench the GFP signal, but not that from mCherry. Hence, the degree of mitophagy or general autophagy can be determined by the appearance of mCherry-only puncta, which represent mito/autolysosomes (Figure 1A and D). Given that mitophagy is a form of autophagy, the use of both models allows us to monitor the specificity of autophagy in vivo. A large disruption of autophagy in general will also influence mitophagy, whereas a block in mitophagy, which likely represents a small fraction of the total autophagy occurring at any one time, will tend to have little influence on the total autophagic levels. Figure 1 with 1 supplement see all Download asset Open asset LRRK2 kinase activity impairs basal mitophagy in vitro. (A) Schematics of the mito-QC reporter in mouse model. (B) Representative images of mito-QC primary MEF cultures established from LRRK2 WT, LRRK2 G2019S, and LRRK2 KO embryos. Boxed area is magnified on the right and arrowheads indicate examples of mitophagy (mCherry-only mitolysosomes). (C) Mitophagy quantitation of data shown in B from six to nine independent experiments, as described in Materials and methods. Below is representative immunoblot showing LRRK2 protein expression. (D) Schematics of the auto-QC reporter in mouse model. (E) Representative images of auto-QC primary MEF cultures established from LRRK2 WT, LRRK2 G2019S, and LRRK2 KO embryos. Boxed area is magnified on the right and arrowheads indicate examples of autolysosomes and arrows highlight autophagosomes. (F) Quantitation of data shown in E from four to six independent experiments. (G) Representative images of mito-QC primary MEFs treated with control (DMSO) or 10 nM GSK3357679A. Boxed area is magnified on bottom left and arrowheads indicate examples of mitolysosomes. (H) Quantitation of mitophagy shown in G from three to seven independent experiments. Corresponding immunoblot of indicated proteins is shown below. Scale bars, 10 μm. Overall data is represented as mean +/- SEM. Statistical significance is displayed as *p<0.05, and ***p<0.001. Figure 1—source data 1 Numerical data for Figure 1. https://cdn.elifesciences.org/articles/67604/elife-67604-fig1-data1-v1.xlsx Download elife-67604-fig1-data1-v1.xlsx Figure 1—source data 2 Western blot raw data files for Figure 1C. https://cdn.elifesciences.org/articles/67604/elife-67604-fig1-data2-v1.zip Download elife-67604-fig1-data2-v1.zip Figure 1—source data 3 Western blot raw data files for Figure 1F. https://cdn.elifesciences.org/articles/67604/elife-67604-fig1-data3-v1.zip Download elife-67604-fig1-data3-v1.zip Figure 1—source data 4 Western blot raw data files for Figure 1H. https://cdn.elifesciences.org/articles/67604/elife-67604-fig1-data4-v1.zip Download elife-67604-fig1-data4-v1.zip To investigate the effect of LRRK2 kinase activity on mitophagy, we first isolated and cultured primary mouse embryonic fibroblasts (MEFs) derived from wild-type mice (WT), mice homozygous for the Parkinson’s disease-associated LRRK2 G2019S mutant, or mice homozygous for a LRRK2 knockout (KO) variant; all of which were on a homozygous mito-QC reporter background (Figure 1B and C). A small degree of basal mitophagy was evident in all cell lines. However, we observed that LRRK2 G2019S KI mutant cells displayed significantly lower basal mitophagy levels, whereas the absence of LRRK2 (KO) led to an increase of this process. Interestingly, our data suggests that LRRK2 predominantly influences basal mitophagy as deferiprone (DFP) and long-term amino acid starvation in Earls Balanced Salt Solution (EBSS), both strong mitophagy inducers (Allen et al., 2013), increased mitophagy to a similar level across all genotypes (Figure 1—figure supplement 1A and B). We next investigated general autophagy using the LRRK2 mouse lines mentioned above on the homozygous auto-QC background. In contrast to mitophagy, in isolated primary MEFs we noticed no significant difference in the number of mCherry-only autolysosomes across all the Lrrk2 genotypes under basal conditions (Figure 1E and F). We also analysed amino acid starvation-induced autophagy, by EBSS incubation. A robust autophagy response was observed in all cells and as with basal autophagy, the Lrrk2 genotype failed to significantly alter this large increase in autolysosomes (Figure 1—figure supplement 1C). LRRK2 kinase inhibitors correct the G2019S mitophagy defects in vitro Using genetics, our observations show that LRRK2 kinase activity inversely correlates with mitophagy in vitro. If this is the case, then pharmacological inhibition of LRRK2 kinase activity should also increase mitophagy. Therefore, we aimed to investigate if the mitophagy deficit observed in the G2019S cells could be rescued with LRRK2-selective kinase inhibitors. To that end, we turned to GSK3357679A, a novel pyrrolopyrimidine LRRK2 kinase inhibitor that exhibits excellent cellular potency, selectivity, oral bioavailability and pharmacokinetics/pharmacodynamics correlation in animal studies (Tasegian et al., 2021; Ding, 2021, in preparation). We tested GSK3357679A in primary mito-QC MEFs and observed a dose-dependent effect on mitophagy with a maximal stimulation achieved at a concentration of 10 nM in WT cells (Figure 1G and H). The level of stimulation was equivalent to the level of mitophagy observed in LRRK2 KO cells. Importantly, GSK3357679A failed to alter mitophagy in the absence of LRRK2, demonstrating that its mitophagy-enhancing properties are dependent on LRRK2. In the G2019S cells, we observed a reduced response, with a maximal effect on mitophagy reached at 100 nM and this may reflect the increased kinase activity of this mutant (Nichols et al., 2009). Western blotting analysis confirmed that GSK3357679A potently inhibited LRRK2 kinase activity in a dose-dependent manner, as indicated by decreased phosphorylation of its substrate Rab10 at threonine 73 (Steger et al., 2016), as well as reduced LRRK2 S935 phosphorylation (an indirect measure of LRRK2 activity (Ito et al., 2016), Figure 1H). To further support a role for LRRK2 kinase activity in negatively regulating basal mitophagy, we utilised two additional and structurally distinct tool LRRK2 kinase inhibitors, GSK2578215A (Reith et al., 2012) and MLi-2 (Fell et al., 2015), in primary mito-QC MEFs. As with GSK3357679A, both these compounds were able to inhibit LRRK2 in cells and increase mitophagy (Figure 1—figure supplement 1D and E). We do note that at high concentrations, MLi2 failed to stimulate mitophagy and this may be due to off-target effects, as at 20 nM it also inhibited mitophagy in the LRRK2 KO cells (Figure 1—figure supplement 1D). Thus, genetically and chemically, the data show that LRRK2 inhibition enhances basal mitophagy in cells and in these assays, GSK3357679A displayed a superior performance compared to other available LRRK2 kinase inhibitors. LRRK2 inhibition activates conventional mitophagy independently of the PINK1 pathway Although mito-QC monitors the delivery of mitochondria to lysosomes, it does not give any mechanistic insight as to how this occurs. Given that multiple mitophagy-like pathways exist, which can be dependent or independent of the canonical macroautophagy machinery (Montava-Garriga and Ganley, 2020), we decided to investigate this further. LC3 is the classical autophagosome marker and if mitophagy is occurring via canonical autophagy then LRRK2 inhibition should result in increased co-localisation between mitochondria and LC3. To test this, we took advantage of our primary auto-QC MEFs and co-stained these for the mitochondrially localised ATP synthase subunit beta (ATPB, Figure 2A). Treatment of cells with GSK3357679A significantly increased co-localisation of mCherry-GFP-LC3 with ATPB, both at the autophagosome stage (GFP and mCherry-positive, Figure 2B) and autolysosome stage (mCherry-only positive, Figure 2C). This implies LRRK2 inhibition uses a canonical autophagy pathway to drive mitophagy. To confirm the requirement for the LC3 conjugation machinery in this form of mitophagy, we expressed the mito-QC reporter in the previously reported macroautophagy-deficient ATG5 KO MEFs (Kuma et al., 2004). In the matched WT MEFs, GSK3357679A was able to induce mitophagy in a similar manner to the primary mito-QC MEFs; however, mitophagy induction was blocked in the ATG5 KO MEFs (Figure 2D). As a final, reporter-independent method to confirm mitophagy, we carried out transmission electron microscopy (TEM, Figure 2E and F). To aid in this process, we first immortalised the primary mito-QC MEFs, to remove their time limitation, and confirmed that LRRK2 inhibition could still induce mitophagy (Figure 2—figure supplement 1A–C). Partially degraded mitochondrial structures could clearly be identified within autolysosomes (representative images shown in Figure 2E) and when quantified, a significant 1.3-fold increase was detected in the GSK3357679A-treated samples. Taken together, the data show that LRRK2 inhibition stimulates mitophagy through canonical ATG5-dependent autophagy and confirms that the mito-QC reporter is measuring a conventional mitophagy pathway in this instance. Figure 2 with 1 supplement see all Download asset Open asset LRRK2-dependent mitophagy relies on the general autophagy machinery but is independent of the PINK1 pathway. (A) Representative images of auto-QC primary MEF cultures treated with 10 nM GSK3357679A for 24 hr and immunolabelled with ATPB. Arrows show examples of autophagosomes containing mitochondria, and arrowheads show examples of autolysosomes containing mitochondria. Scale bars, 10 μm. (B) Quantitation of autophagosomes containing mitochondria shown in (B) or autolysosomes containing mitochondria shown in (C) from three independent experiments. (D) Representative images of WT and ATG5 KO mito-QC immortalised MEF cultures treated with 10 nM GSK3357679A for 24 hr. Corresponding quantitation from three independent experiments is shown below. Scale bars, 10 μm. (E) Representative transmission electron microscopy images from immortalised MEFs treated with 10 nM GSK3357679A for 24 hr. MLy: Mitolysosome; M: Mitochondria; A: autophagosome. Scale bars, 500 nm. (F) Quantitation of data in E from three independent experiments. (G) Representative images of PINK1 KO mito-QC primary MEF cultures treated with/without with 10 nM GSK3357679A for 24 hr. Scale bars, 10 μm. (H) Quantitation of the of mitophagic cells from data shown in G from 3 (4 for KO) independent experiments. (I) Immunoblots of the indicated proteins from WT and PINK1 KO primary MEFs (KO-1 and KO-3 are derived from different embryos) treated with/without 10 nM GSK3357679A or 10 μM CCCP for 24 hr. Overall data is represented as mean +/- SEM. Statistical significance is displayed as *p<0.05 and **p<0.01. Figure 2—source data 1 Numerical data for Figure 2. https://cdn.elifesciences.org/articles/67604/elife-67604-fig2-data1-v1.xlsx Download elife-67604-fig2-data1-v1.xlsx Figure 2—source data 2 Western blot raw data files for Figure 2I. https://cdn.elifesciences.org/articles/67604/elife-67604-fig2-data2-v1.zip Download elife-67604-fig2-data2-v1.zip The most widely studied mitophagy pathway involves the activation of PINK1 and Parkin that occurs following mitochondrial depolarisation (Montava-Garriga and Ganley, 2020). Given that mutations in PINK1, Parkin and LRRK2 can all lead to PD, it was important to determine if LRRK2 inhibition resulted in PINK1-dependent mitophagy. To test this, we used previously generated primary MEFs derived from mito-QC x PINK1 KO mice (McWilliams et al., 2018a). Treatment of litter-matched WT and PINK1 KO mito-QC MEFs with GSK3357679A resulted in a comparable increase in mitophagy regardless of the presence or absence of PINK1 (Figure 2G and H). To support the PINK1-independent nature of this pathway we immunoblotted for PINK1-dependent phospho-ubiquitin. We observed no detectible increase in phospho-ubiquitin levels following LRRK2 inhibition, which contrasted with that observed following mitochondrial depolarisation with CCCP treatment (Figure 2I). To further examine the relationship between PINK1/Parkin-dependent mitophagy and LRRK2 inhibition, we sought to directly monitor Parkin-dependent mitophagy. As high levels of Parkin are needed to observe PINK1/Parkin-dependent mitophagy, we next overexpressed HA-tagged Parkin and induced mitophagy with CCCP treatment (Figure 2—figure supplement 1D–F). mito-QC clearly detected a large and significant increase in mitophagy following mitochondrial depolarisation, but this was unaffected by GSK3357679A, as were phospho-ubiquitin levels. The lack of involvement of PINK1 and Parkin in the observed mitophagy could imply that mitochondrial depolarisation is not a major trigger for this pathway. We thus explored mitochondrial function in general in response to LRRK2 inhibition and GSK3357679A treatment. Firstly, we noticed no obvious changes to mitochondrial morphology and ultrastructure, as observed using TEM (Figure 2—figure supplement 1G). Secondly, using high-resolution respirometry, we measured mitochondrial oxygen consumption. We first investigated if GSK3357679A treatment directly affected the mitochondrial respiratory chain. Thus, we activated the NADH- and succinate-linked pathway and injected sequentially incremental doses of GSK3357679A. This had no direct effect on the mitochondrial respiratory chain (Figure 2—figure supplement 1H). Secondly, we evaluated the chronic effects of LRRK2 inhibition by incubating immortalised MEFs for 24 hr with 10 nM GSK3357679A (Figure 2—figure supplement 1I). Despite an increase in mitophagy (Figure 2—figure supplement 1C), we did not observe any difference in mitochondrial respiration. Furthermore, we did not observe any change in substrate preference. Thus, LRRK2 inhibition does not globally impact mitochondrial form or function at this timepoint. However, given that the fraction of mitochondria targeted for mitophagy is likely small compared to the total pool, we cannot rule out that this population is functionally impaired. Mutation of LRRK2 in vivo alters mitophagy in specific cell populations within the brain Given the effects of LRRK2 kinase activity on mitophagy in vitro, we next sought to use our mouse lines to investigate this in vivo. PD is primarily a neurodegenerative disorder, so we first explored mitophagy in the brain. We focussed on four cell populations: two neuronal populations linked to movement – dopaminergic (DA) neurons of the substantia nigra pars compacta (SNpc) and neurons of the as well as in two cell populations – microglia and In we identified neurons using and found no difference in the number of neurons across the Lrrk2 genotypes (Figure and B). These cells are the mouse equivalent of the population of neurons that in PD and we have previously found that mitophagy (McWilliams et al., 2018a). mitophagy was significantly enhanced in the LRRK2 KO neurons compared to WT, and not mitophagy reduced in neurons of LRRK2 G2019S KI mice compared to WT (Figure and C). we cannot that mitophagy is significantly impaired in the G2019S neurons from this of experiments using nine mice, we did a significant mitophagy in G2019S neurons in a of experiments, with 10 mice (Figure and B). Taken together, these observations are similar to our results in MEFs and that the presence of LRRK2 can impact mitophagy in this clinically relevant population of neurons within the To determine if this effect is of neurons in we investigated mitophagy in neuronal population involved in the These cells were identified in using the These cells are in mitochondria and as shown previously (McWilliams et al., 2016), also significant mitophagy (Figure to what we observed in no difference in mitophagy in cells was found between any (Figure Figure 3 with 1 supplement see all Download asset Open asset Mutation of LRRK2 in vivo alters brain mitophagy. (A) Representative of immunolabelled dopaminergic neurons within the substantia nigra pars compacta (SNpc) basal mitophagy in LRRK2 WT, LRRK2 G2019S, and LRRK2 KO mito-QC mice. show examples of and arrows indicate mitochondria. (B) Quantitation of the number of cells of using a (C) Quantitation of basal mitophagy (Sironi et al., of data represent from mice. (D) Representative maximal and of immunolabelled neurons in mito-QC basal mitophagy. (E) Quantitation of basal mitophagy (Sironi et al., of data represent from mice. (F) Representative and of immunolabelled microglia basal mitophagy in from LRRK2 WT, LRRK2 G2019S, and LRRK2 KO mito-QC mice. highlight mitolysosomes. (G) Quantitation of the number of cells in the brain of using a (H) Quantitation of basal mitophagy cell data represent from mice. (I) Representative maximal and of immunolabelled basal mitophagy in mito-QC Quantitation of basal mitophagy (Sironi et al., of data represent from mice. Scale bars, 10 μm. Overall data is represented as mean +/- SEM. Statistical significance is displayed as *p<0.05, and ***p<0.001. Figure data 1 Numerical data for Figure Download As we observed of mitophagy, we next the effect of Lrrk2 genotype in two distinct populations cells within the microglia were identified by Figure we observed an enhanced presence of cells in the of G2019S animals when compared to WT or KO mice (Figure We do not yet the nature of this increase and further will be needed to determine if there are microglia in the G2019S mice, or an increased movement of cells to this area of the brain. when mitophagy quantitation was for cell number cell we found a significant in basal mitophagy in G2019S microglia compared to WT, as well as an increase in mitophagy levels in KO cells (Figure Thus, as with LRRK2 can basal mitophagy in In contrast, with protein Figure did not show any difference in mitophagy across Lrrk2 genotypes (Figure In contrast to the Lrrk2 genotype effects on mitophagy in neurons and analysis of auto-QC mouse brains indicated no change in general macroautophagy in these cell (Figure supplement Thus, the LRRK2 G2019S mutation is not a major disruption in neuronal autophagy but does influence basal mitophagy levels. Mutation of LRRK2 in vivo also alters mitophagy in with high LRRK2 expression We next mitophagy levels in the a in which the levels of LRRK2 are known to be elevated et al., et al., mitophagy across the whole was evident in all genotypes and in a similar to MEFs, neurons and mitophagy was reduced in G2019S mice and enhanced to in KO mice (Figure and with this, mitochondrial was decreased when KO to G2019S no significant increase of this was detected compared to WT, see Figure supplement As previously reported et al., 2018; et al., we observed with structures in all the animals of the LRRK2 KO that is to the of large which are for We confirmed the nature of these structures as by for a found in (Figure supplement Figure 4 with 1 supplement see all Download asset Open asset of Lrrk2 genotype on basal mitophagy and macroautophagy in tissues. (A) Representative and images of mito-QC from LRRK2 WT, LRRK2 G2019S, and LRRK2 KO mice. Arrows on the highlight show in the images indicate examples of and cells to and (B) Quantitation of basal mitophagy from data shown in data represent from mice. (C) Representative images of mito-QC from LRRK2 WT, LRRK2 G2019S, and LRRK2 KO mice. and (D) Quantitation of basal mitophagy from data shown in data represent from mice. (E) Representative images of auto-QC from LRRK2 WT, LRRK2 G2019S, and LRRK2 KO mice. highlight arrows indicate and (F) Quantitation of autophagy from data shown in data represent from mice. (G) Representative images of auto-QC from LRRK2 WT, LRRK2 G2019S, and LRRK2 KO mice. arrows as in