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Parkinson’s disease (PD) is a progressive late-onset neurodegenerative disease leading to physical and cognitive decline. Mutations of leucine-rich repeat kinase 2 ( LRRK2 ) are the most common genetic cause of PD. LRRK2 is a complex scaffolding protein with known regulatory roles in multiple molecular pathways. Two prominent examples of LRRK2-modulated pathways are Wingless/Int (Wnt) and nuclear factor of activated T-cells (NFAT) signaling. Both are well described key regulators of immune and nervous system development as well as maturation. The aim of this study was to establish the physiological and pathogenic role of LRRK2 in Wnt and NFAT signaling in the brain, as well as the potential contribution of the non-canonical Wnt/Calcium pathway. In vivo cerebral Wnt and NFATc1 signaling activity was quantified in LRRK2 G2019S mutant knock-in (KI) and LRRK2 knockout (KO) male and female mice with repeated measures over 28 weeks, employing lentiviral luciferase biosensors, and analyzed using a mixed-effect model. To establish spatial resolution, we investigated tissues, and primary neuronal cell cultures from different brain regions combining luciferase signaling activity, immunohistochemistry, qPCR and western blot assays. Results were analyzed by unpaired t-test with Welch’s correction or 2-way ANOVA with post hoc corrections. In vivo Wnt signaling activity in LRRK2 KO and LRRK2 G2019S KI mice was increased significantly ~ threefold, with a more pronounced effect in males (~ fourfold) than females (~ twofold). NFATc1 signaling was reduced ~ 0.5-fold in LRRK2 G2019S KI mice. Brain tissue analysis showed region-specific expression changes in Wnt and NFAT signaling components. These effects were predominantly observed at the protein level in the striatum and cerebral cortex of LRRK2 KI mice. Primary neuronal cell culture analysis showed significant genotype-dependent alterations in Wnt and NFATc1 signaling under basal and stimulated conditions. Wnt and NFATc1 signaling was primarily dysregulated in cortical and hippocampal neurons respectively. Our study further built on knowledge of LRRK2 as a Wnt and NFAT signaling protein. We identified complex changes in neuronal models of LRRK2 PD, suggesting a role for mutant LRRK2 in the dysregulation of NFAT, and canonical and non-canonical Wnt signaling.

Mutations in leucine-rich repeat kinase 2 gene ( ) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains, including 13 putative armadillo-type repeats at the N-terminus. In this study, we analyzed the functional and molecular consequences of a novel variant, E193K, identified in an Italian family. E193K substitution does not influence LRRK2 kinase activity. Instead it affects LRRK2 biochemical properties, such as phosphorylation at Ser935 and affinity for 14-3-3ε. Primary fibroblasts obtained from an E193K carrier demonstrated increased cellular toxicity and abnormal mitochondrial fission upon 1-methyl-4-phenylpyridinium treatment. We found that E193K alters LRRK2 binding to DRP1, a crucial mediator of mitochondrial fission. Our data support a role for LRRK2 as a scaffolding protein influencing mitochondrial fission.

Leucine-rich repeat kinase 2 (LRRK2) has been associated with a variety of human diseases, including Parkinson’s disease and Crohn’s disease, whereas LRRK2 deficiency leads to accumulation of abnormal lysosomes in aged animals. However, the cellular roles and mechanisms of LRRK2-mediated lysosomal regulation have remained elusive. Here, we reveal a mechanism of stress-induced lysosomal response by LRRK2 and its target Rab GTPases. Lysosomal overload stress induced the recruitment of endogenous LRRK2 onto lysosomal membranes and activated LRRK2. An upstream adaptor Rab7L1 (Rab29) promoted the lysosomal recruitment of LRRK2. Subsequent family-wide screening of Rab GTPases that may act downstream of LRRK2 translocation revealed that Rab8a and Rab10 were specifically accumulated on overloaded lysosomes dependent on their phosphorylation by LRRK2. Rab7L1-mediated lysosomal targeting of LRRK2 attenuated the stress-induced lysosomal enlargement and promoted lysosomal secretion, whereas Rab8 stabilized by LRRK2 on stressed lysosomes suppressed lysosomal enlargement and Rab10 promoted lysosomal secretion, respectively. These effects were mediated by the recruitment of Rab8/10 effectors EHBP1 and EHBP1L1. LRRK2 deficiency augmented the chloroquine-induced lysosomal vacuolation of renal tubules in vivo. These results implicate the stress-responsive machinery composed of Rab7L1, LRRK2, phosphorylated Rab8/10, and their downstream effectors in the maintenance of lysosomal homeostasis.

Heterozygous gain-of-kinase function variants in LRRK2 (leucine-rich repeat kinase 2) cause 1–2% of all cases of Parkinson’s disease (PD) albeit with incomplete and age-dependent penetrance. All pathogenic LRRK2 mutations reside within the two catalytic domains of LRRK2—either in its kinase domain (e.g. G2019S) with modest effect or its ROC-COR GTPase domain (e.g. R1441G/H) with large effect on LRRK2 kinase activity. We have previously reported assays to interrogate LRRK2 kinase pathway activity in human bio-samples measuring phosphorylation of its endogenous substrate Rab10, that mirrors LRRK2 kinase activation status. Here, we isolated neutrophils from fresh peripheral blood from 101 participants including 42 LRRK2 mutation carriers (21 with the G2019S and 21 with the R1441G mutations), 27 patients with idiopathic PD, and 32 controls. Using a dual approach, LRRK2 dependent Rab10 phosphorylation at Threonine 73 (pRab10Thr73) was measured by quantitative multiplexed immunoblotting for pRab10Thr73/total Rab10 as well as targeted mass-spectrometry for absolute pRab10Thr73 occupancy. We found a significant over fourfold increase in pRab10Thr73 phosphorylation in carriers of the LRRK2 R1441G mutation irrespective of clinical disease status. The effect of the LRRK2 G2019S mutation did not reach statistical significance. Furthermore, we show that LRRK2 phosphorylation at Serine 935 is not a marker for LRRK2 kinase activity in human neutrophils. When analysing pRab10Thr73 phosphorylation in post-mortem brain samples, we observed overall high variability irrespective of clinical and LRRK2 mutation status and attributed this mainly to the adverse effect of the peri- and post-mortem period on the stability of posttranslational modifications such as protein phosphorylation. Overall, in vivo LRRK2 dependent pRab10Thr73 phosphorylation in human peripheral blood neutrophils is a specific, robust and promising biomarker for significant LRRK2 kinase hyperactivation, as with the LRRK2 R1441G mutation. Additional readouts and/or assays may be needed to increase sensitivity to detect modest LRRK2 kinase activation, as with the LRRK2 G2019S mutation. Our assays could be useful for patient stratification and target engagement studies for LRRK2 kinase inhibitors.

IntroductionMutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant Parkinson’s disease (PD). LRRK2 encodes a multi-domain protein containing a Roc GTPase domain and a serine/threonine-directed protein kinase domain, with PD-linked mutations known to enhance LRRK2 kinase activity and neuronal toxicity. Our previous studies identified the Golgi protein, ADP-Ribosylation Factor GTPase-Activating Protein 1 (ArfGAP1), as a novel modifier of LRRK2-induced cellular toxicity, where it can serve as a GAP-like protein and a robust kinase substrate of LRRK2.MethodsHere, we further explore the phosphorylation of ArfGAP1 by LRRK2 and its functional consequences.ResultsLRRK2 mediates the robust phosphorylation of ArfGAP1 in vitro within its lipid-sensing ALPS2 motif at residues Ser284, Thr291, and Thr292. We mutated these three candidate phosphorylation sites, either alone or combined, to create hydrophobic phospho-null or charged phospho-mimicking versions of ArfGAP1. We find that modulating ArfGAP1 phosphorylation at these sites impairs its normal capacity to induce Golgi fragmentation upon overexpression in neural cells. Blocking phosphorylation impairs ArfGAP1-induced neurite outgrowth inhibition in primary neurons and protects against the pathogenic effects of PD-linked G2019S LRRK2. ArfGAP1 interactome analysis in neural cells identifies 114 putative interacting proteins with a proportion of these localized to mitochondria, including the outer membrane proteins Voltage-Dependent Anion Channel (VDAC) 1–3. An ArfGAP1 triple phospho-mimic mutant displays an increased interaction with mitochondrial VDACs owing to the redistribution of ArfGAP1 from the cis-Golgi to the cytoplasm. Mimicking ArfGAP1 phosphorylation also blocks the formation of Golgi-derived vesicles following mild ER stress.DiscussionOur data provides evidence for a complex functional interaction between LRRK2 and ArfGAP1 that serves to regulate ArfGAP1 subcellular localization, protein interactions, activity and neuronal integrity via LRRK2-mediated phosphorylation of its membrane-binding ALPS2 motif. Our findings support additional validation of ArfGAP1 as a putative therapeutic target for modulating LRRK2-linked PD.

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.

We previously reported that Parkinson’s disease (PD) kinase LRRK2 phosphorylates a subset of Rab GTPases on a conserved residue in their switch-II domains (Steger et al., 2016) (PMID: 26824392). Here, we systematically analyzed the Rab protein family and found 14 of them (Rab3A/B/C/D, Rab5A/B/C, Rab8A/B, Rab10, Rab12, Rab29, Rab35 and Rab43) to be specifically phosphorylated by LRRK2, with evidence for endogenous phosphorylation for ten of them (Rab3A/B/C/D, Rab8A/B, Rab10, Rab12, Rab35 and Rab43). Affinity enrichment mass spectrometry revealed that the primary ciliogenesis regulator, RILPL1 specifically interacts with the LRRK2-phosphorylated forms of Rab8A and Rab10, whereas RILPL2 binds to phosphorylated Rab8A, Rab10, and Rab12. Induction of primary cilia formation by serum starvation led to a two-fold reduction in ciliogenesis in fibroblasts derived from pathogenic LRRK2-R1441G knock-in mice. These results implicate LRRK2 in primary ciliogenesis and suggest that Rab-mediated protein transport and/or signaling defects at cilia may contribute to LRRK2-dependent pathologies.

Mutations in Park8, encoding for the multidomain Leucine-rich repeat kinase 2 (LRRK2) protein, comprise the predominant genetic cause of Parkinson's disease (PD). G2019S, the most common amino acid substitution activates the kinase two- to threefold. This has motivated the development of LRRK2 kinase inhibitors; however, poor consensus on physiological LRRK2 substrates has hampered clinical development of such therapeutics. We employ a combination of phosphoproteomics, genetics, and pharmacology to unambiguously identify a subset of Rab GTPases as key LRRK2 substrates. LRRK2 directly phosphorylates these both in vivo and in vitro on an evolutionary conserved residue in the switch II domain. Pathogenic LRRK2 variants mapping to different functional domains increase phosphorylation of Rabs and this strongly decreases their affinity to regulatory proteins including Rab GDP dissociation inhibitors (GDIs). Our findings uncover a key class of bona-fide LRRK2 substrates and a novel regulatory mechanism of Rabs that connects them to PD. Parkinson’s disease is a degenerative disorder of the nervous system that affects approximately 1% of the elderly population. Mutations in the gene that encodes an enzyme known as LRRK2 are the most common causes of the inherited form of the disease. Such mutations generally increase the activity of LRRK2 and so drug companies have developed drugs that inhibit LRRK2 to prevent or delay the progression of Parkinson’s disease. However, it was not known what role LRRK2 plays in cells, and why its over-activation is harmful. Steger et al. used a 'proteomics' approach to find other proteins that are regulated by LRRK2. The experiments tested a set of newly developed LRRK2 inhibitors in cells and brain tissue from mice. The mice had mutations in the gene encoding LRRK2 that are often found in human patients with Parkinson’s disease. The experiments show that LRRK2 targets some proteins belonging to the Rab GTPase family, which are involved in transporting molecules and other 'cargoes' around cells. Several Rab GTPases are less active in the mutant mice, which interferes with the ability of these proteins to correctly direct the movement of cargo around the cell. Steger et al.’s findings will help to advance the development of new therapies for Parkinson’s disease. The next challenges are to identify how altering the activity of Rab GTPases leads to degeneration of the nervous system and how LRRK2 inhibitors may slow down these processes.

It has been 15 years since the ( ) gene was identified as the most common genetic cause for Parkinson's disease (PD). The two most common mutations are the LRRK2-G2019S, located in the kinase domain, and the LRRK2-R1441C, located in the ROC-COR domain. While the LRRK2-G2019S mutation is associated with increased kinase activity, the LRRK2-R1441C exhibits a decreased GTPase activity and altered kinase activity. Multiple lines of evidence have linked the LRRK2 protein with a role in the autophagy pathway and with lysosomal activity in neurons. Neurons rely heavily on autophagy to recycle proteins and process cellular waste due to their post-mitotic state. Additionally, lysosomal activity decreases with age which can potentiate the accumulation of α-synuclein, the pathological hallmark of PD, and subsequently lead to the build-up of Lewy bodies (LBs) observed in this disorder. This review provides an up to date summary of the LRRK2 field to understand its physiological role in the autophagy pathway in neurons and related cells. Careful assessment of how LRRK2 participates in the regulation of phagophore and autophagosome formation, autophagosome and lysosome fusion, lysosomal maturation, maintenance of lysosomal pH and calcium levels, and lysosomal protein degradation are addressed. The autophagy pathway is a complex cellular process and due to the variety of LRRK2 models studied in the field, associated phenotypes have been reported to be seemingly conflicting. This review provides an in-depth discussion of different models to assess the normal and disease-associated role of the LRRK2 protein on autophagic function. Given the importance of the autophagy pathway in Parkinson's pathogenesis it is particularly relevant to focus on the role of LRRK2 to discover novel therapeutic approaches that restore lysosomal protein degradation homeostasis.

Emerging evidence shows that α-synuclein seed amplification assays (SAAs) have the potential to differentiate people with Parkinson's disease from healthy controls. We used the well characterised, multicentre Parkinson's Progression Markers Initiative (PPMI) cohort to further assess the diagnostic performance of the α-synuclein SAA and to examine whether the assay identifies heterogeneity among patients and enables the early identification of at-risk groups. This cross-sectional analysis is based on assessments done at enrolment for PPMI participants (including people with sporadic Parkinson's disease from LRRK2 and GBA variants, healthy controls, prodromal individuals with either rapid eye movement sleep behaviour disorder (RBD) or hyposmia, and non-manifesting carriers of LRRK2 and GBA variants) from 33 participating academic neurology outpatient practices worldwide (in Austria, Canada, France, Germany, Greece, Israel, Italy, the Netherlands, Norway, Spain, the UK, and the USA). α-synuclein SAA analysis of CSF was performed using previously described methods. We assessed the sensitivity and specificity of the α-synuclein SAA in participants with Parkinson's disease and healthy controls, including subgroups based on genetic and clinical features. We established the frequency of positive α-synuclein SAA results in prodromal participants (RBD and hyposmia) and non-manifesting carriers of genetic variants associated with Parkinson's disease, and compared α-synuclein SAA to clinical measures and other biomarkers. We used odds ratio estimates with 95% CIs to measure the association between α-synuclein SAA status and categorical measures, and two-sample 95% CIs from the resampling method to assess differences in medians between α-synuclein SAA positive and negative participants for continuous measures. A linear regression model was used to control for potential confounders such as age and sex. This analysis included 1123 participants who were enrolled between July 7, 2010, and July 4, 2019. Of these, 545 had Parkinson's disease, 163 were healthy controls, 54 were participants with scans without evidence of dopaminergic deficit, 51 were prodromal participants, and 310 were non-manifesting carriers. Sensitivity for Parkinson's disease was 87·7% (95% CI 84·9–90·5), and specificity for healthy controls was 96·3% (93·4–99·2). The sensitivity of the α-synuclein SAA in sporadic Parkinson's disease with the typical olfactory deficit was 98·6% (96·4–99·4). The proportion of positive α-synuclein SAA was lower than this figure in subgroups including LRRK2 Parkinson's disease (67·5% [59·2–75·8]) and participants with sporadic Parkinson's disease without olfactory deficit (78·3% [69·8–86·7]). Participants with LRRK2 variant and normal olfaction had an even lower α-synuclein SAA positivity rate (34·7% [21·4–48·0]). Among prodromal and at-risk groups, 44 (86%) of 51 of participants with RBD or hyposmia had positive α-synuclein SAA (16 of 18 with hyposmia, and 28 of 33 with RBD). 25 (8%) of 310 non-manifesting carriers (14 of 159 [9%] LRRK2 and 11 of 151 [7%] GBA) were positive. This study represents the largest analysis so far of the α-synuclein SAA for the biochemical diagnosis of Parkinson's disease. Our results show that the assay classifies people with Parkinson's disease with high sensitivity and specificity, provides information about molecular heterogeneity, and detects prodromal individuals before diagnosis. These findings suggest a crucial role for the α-synuclein SAA in therapeutic development, both to identify pathologically defined subgroups of people with Parkinson's disease and to establish biomarker-defined at-risk cohorts. PPMI is funded by the Michael J Fox Foundation for Parkinson's Research and funding partners, including: Abbvie, AcureX, Aligning Science Across Parkinson's, Amathus Therapeutics, Avid Radiopharmaceuticals, Bial Biotech, Biohaven, Biogen, BioLegend, Bristol-Myers Squibb, Calico Labs, Celgene, Cerevel, Coave, DaCapo Brainscience, 4D Pharma, Denali, Edmond J Safra Foundation, Eli Lilly, GE Healthcare, Genentech, GlaxoSmithKline, Golub Capital, Insitro, Janssen Neuroscience, Lundbeck, Merck, Meso Scale Discovery, Neurocrine Biosciences, Prevail Therapeutics, Roche, Sanofi Genzyme, Servier, Takeda, Teva, UCB, VanquaBio, Verily, Voyager Therapeutics, and Yumanity.