The LRRK2 kinase substrates RAB8a and RAB10 contribute complementary but distinct disease-relevant phenotypes in human neurons.

Mutations in the LRRK2 gene cause familial Parkinson's disease presenting with pleomorphic neuropathology that can involve α-synuclein or tau accumulation. LRRK2 mutations are thought to converge upon a pathogenic increase in LRRK2 kinase activity. A subset of small RAB GTPases has been identified as LRRK2 substrates, with LRRK2-dependent phosphorylation resulting in RAB inactivation. We used CRISPR-Cas9 genome editing to generate a novel series of isogenic iPSC lines deficient in the two most well-validated LRRK2 substrates, RAB8a and RAB10, from deeply phenotyped healthy control lines. Thorough characterization of NGN2-induced neurons revealed opposing effects of RAB8a and RAB10 deficiency on lysosomal pH and Golgi organization, with isolated effects of RAB8a and RAB10 ablation on α-synuclein and tau, respectively. Our data demonstrate largely antagonistic effects of genetic RAB8a or RAB10 inactivation, which provide discrete insight into the pathologic features of their biochemical inactivation by pathogenic LRRK2 mutation in human disease.

Leucine-rich repeat kinase 2 limits dopamine D1 receptor signaling in striatum and biases against heavy persistent alcohol drinking.

The transition from hedonic alcohol drinking to problematic drinking is a hallmark of alcohol use disorder that occurs only in a subset of drinkers. This transition requires long-lasting changes in the synaptic drive and the activity of striatal neurons expressing dopamine D1 receptor (D1R). The molecular mechanisms that generate vulnerability in some individuals to undergo the transition are less understood. Here, we report that the Parkinson's-related protein leucine-rich repeat kinase 2 (LRRK2) modulates striatal D1R function to affect the behavioral response to alcohol and the likelihood that mice transition to heavy, persistent alcohol drinking. Constitutive deletion of the Lrrk2 gene specifically from D1R-expressing neurons potentiated D1R signaling at the cellular and synaptic level and enhanced alcohol-related behaviors and drinking. Mice with cell-specific deletion of Lrrk2 were more prone to heavy alcohol drinking, and consumption was insensitive to punishment. These findings identify a potential novel role for LRRK2 function in the striatum in promoting resilience against heavy and persistent alcohol drinking.

Differential LRRK2 signalling and gene expression in WT-LRRK2 and G2019S-LRRK2 mouse microglia treated with zymosan and MLi2.

Introduction: Mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) gene cause autosomal dominant Parkinson's disease (PD) with the most common causative mutation being the LRRK2 p.G2019S within the kinase domain. LRRK2 protein is highly expressed in the human brain and also in the periphery, and high expression of dominant PD genes in immune cells suggest involvement of microglia and macrophages in inflammation related to PD. LRRK2 is known to respond to extracellular signalling including TLR4 resulting in alterations in gene expression, with the response to TLR2 signalling through zymosan being less known. Methods: Here, we investigated the effects of zymosan, a TLR2 agonist and the potent and specific LRRK2 kinase inhibitor MLi-2 on gene expression in microglia from LRRK2-WT and LRRK2 p.G2019S knock-in mice by RNA-Sequencing analysis. Results: We observed both overlapping and distinct zymosan and MLi-2 mediated gene expression profiles in microglia. At least two candidate Genome-Wide Association (GWAS) hits for PD, CathepsinB (Ctsb) and Glycoprotein-nmb (Gpnmb), were notably downregulated by zymosan treatment. Genes involved in inflammatory response and nervous system development were up and downregulated respectively with zymosan treatment while MLi-2 treatment particularly exhibited upregulated genes for ion transmembrane transport regulation. Furthermore, we observed the top twenty most significantly differentially expressed genes in LRRK2 p.G2019S microglia show enriched biological processes in iron transport and response to oxidative stress. Discussion: Overall, these results suggest that microglial LRRK2 may contribute to PD pathogenesis through altered inflammatory pathways. Our findings should encourage future investigations of these putative avenues in the context of PD pathogenesis.

Perspective on the current state of the LRRK2 field.

Almost 2 decades after linking LRRK2 to Parkinson's disease, a vibrant research field has developed around the study of this gene and its protein product. Recent studies have begun to elucidate molecular structures of LRRK2 and its complexes, and our understanding of LRRK2 has continued to grow, affirming decisions made years ago to therapeutically target this enzyme for PD. Markers of LRRK2 activity, with potential to monitor disease progression or treatment efficacy, are also under development. Interestingly, there is a growing understanding of the role of LRRK2 outside of the central nervous system in peripheral tissues such as gut and immune cells that may also contribute to LRRK2 mediated pathology. In this perspective, our goal is to take stock of LRRK2 research by discussing the current state of knowledge and critical open questions in the field.

The LRRK2 kinase substrates Rab8a and Rab10 contribute complementary but distinct disease-relevant phenotypes in human neurons.

Mutations in the LRRK2 gene cause familial Parkinson's disease presenting with pleomorphic neuropathology that can involve α-synuclein or tau accumulation. LRRK2 mutations are thought to converge toward a pathogenic increase in LRRK2 kinase activity. A subset of small Rab GTPases have been identified as LRRK2 substrates, with LRRK2-dependent phosphorylation resulting in Rab inactivation. We used CRISPR/Cas9 genome editing to generate a novel series of isogenic iPSC lines deficient in the two most well validated LRRK2 substrates, Rab8a and Rab10, from two independent, deeply phenotyped healthy control lines. Thorough characterization of NGN2-induced neurons revealed divergent effects of Rab8a and Rab10 deficiency on lysosomal pH, LAMP1 association with Golgi, α-synuclein insolubility and tau phosphorylation, while parallel effects on lysosomal numbers and Golgi clustering were observed. Our data demonstrate largely antagonistic effects of genetic Rab8a or Rab10 inactivation which provide discrete insight into the pathologic features of their biochemical inactivation by pathogenic LRRK2 mutation.

Disease mechanisms as subtypes: Lysosomal dysfunction in the endolysosomal Parkinson's disease subtype.

Parkinson's disease (PD) remains one of the most prevalent neurodegenerative disorders. It has become increasingly recognized that PD is not one disease but a constellation of many, with distinct cellular mechanisms driving pathology and neuronal loss in each given subtype. Endolysosomal trafficking and lysosomal degradation are crucial to maintain neuronal homeostasis and vesicular trafficking. It is clear that deficits in endolysosomal signaling data support the existence of an endolysosomal PD subtype. This chapter describes how cellular pathways involved in endolysosomal vesicular trafficking and lysosomal degradation in neurons and immune cells can contribute to PD. Last, as inflammatory processes including phagocytosis and cytokine release are central in glia-neuron interactions, a spotlight on the role of neuroinflammation plays in the pathogenesis of this PD subtype is also explored.

Differential LRRK2 Signalling and Gene Expression in WT-LRRK2 and G2019S-LRRK2 Mouse Microglia Treated with Zymosan and MLi2.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause autosomal dominant Parkinson's disease (PD), with the most common causative mutation being the LRRK2 p.G2019S within the kinase domain. LRRK2 protein is highly expressed in the human brain and also in the periphery, and high expression of dominant PD genes in immune cells suggests involvement of microglia and macrophages in inflammation related to PD. LRRK2 is known to respond to extracellular signalling including TLR4, resulting in alterations in gene expression, with the response to TLR2 signalling through zymosan being less known. Here, we investigated the effects of zymosan, a TLR2 agonist and the potent and specific LRRK2 kinase inhibitor MLi-2 on gene expression in microglia from LRRK2-WT and LRRK2 p.G2019S knock-in mice by RNA-sequencing analysis. We observed both overlapping and distinct zymosan and MLi-2 mediated gene expression profiles in microglia. At least two candidate genome-wide association (GWAS) hits for PD, CathepsinB (Ctsb) and Glycoprotein-nmb (Gpnmb), were notably downregulated by zymosan treatment. Genes involved in inflammatory response and nervous system development were up and downregulated, respectively, with zymosan treatment, while MLi-2 treatment particularly exhibited upregulated genes for ion transmembrane transport regulation. Furthermore, we observed that the top twenty most significantly differentially expressed genes in LRRK2 p.G2019S microglia show enriched biological processes in iron transport and response to oxidative stress. Overall, these results suggest that microglial LRRK2 may contribute to PD pathogenesis through altered inflammatory pathways. Our findings should encourage future investigations of these putative avenues in the context of PD pathogenesis.

Correction: Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia.

[This corrects the article DOI: 10.1371/journal.pbio.3001480.].

Convergence of signalling pathways in innate immune responses and genetic forms of Parkinson's disease.

In recent years progress in molecular biology and genetics have advanced our understanding of neurological disorders and highlighted synergistic relationships with inflammatory and age-related processes. Parkinson's disease (PD) is a common neurodegenerative disorder that is characterized by loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Increasing extensive evidence supports the contribution of genetic risk variants and inflammation in the pathobiology of this disease. Functional and genetic studies demonstrate an overlap between genes linked to increased risk for PD and autoimmune diseases. Variants identified in loci adjacent to LRRK2, GBA, and HLA establish a crosstalk between the pathobiologies of the two disease spectra. Furthermore, common signalling pathways associated with the pathogenesis of genetic PD are also relevant to inflammatory signaling include MAPK, NF-κB, Wnt and inflammasome signaling. Importantly, post-mortem analyses of brain and cerebrospinal fluid from PD patients show the accumulation of proinflammatory cytokines. In this review we will focus on the principal mechanisms of genetic, inflammatory and age-related risk that intersect in the pathogenesis of PD.

Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal dominant Parkinson disease (PD), while polymorphic LRRK2 variants are associated with sporadic PD. PD-linked mutations increase LRRK2 kinase activity and induce neurotoxicity in vitro and in vivo. The small GTPase Rab8a is a LRRK2 kinase substrate and is involved in receptor-mediated recycling and endocytic trafficking of transferrin, but the effect of PD-linked LRRK2 mutations on the function of Rab8a is poorly understood. Here, we show that gain-of-function mutations in LRRK2 induce sequestration of endogenous Rab8a to lysosomes in overexpression cell models, while pharmacological inhibition of LRRK2 kinase activity reverses this phenotype. Furthermore, we show that LRRK2 mutations drive association of endocytosed transferrin with Rab8a-positive lysosomes. LRRK2 has been nominated as an integral part of cellular responses downstream of proinflammatory signals and is activated in microglia in postmortem PD tissue. Here, we show that iPSC-derived microglia from patients carrying the most common LRRK2 mutation, G2019S, mistraffic transferrin to lysosomes proximal to the nucleus in proinflammatory conditions. Furthermore, G2019S knock-in mice show a significant increase in iron deposition in microglia following intrastriatal LPS injection compared to wild-type mice, accompanied by striatal accumulation of ferritin. Our data support a role of LRRK2 in modulating iron uptake and storage in response to proinflammatory stimuli in microglia.

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

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.

LRRK2 mediates tubulation and vesicle sorting from lysosomes.

Genetic variation around the LRRK2 gene affects risk of both familial and sporadic Parkinson's disease (PD). However, the biological functions of LRRK2 remain incompletely understood. Here, we report that LRRK2 is recruited to lysosomes after exposure of cells to the lysosome membrane-rupturing agent LLOME. Using an unbiased proteomic screen, we identified the motor adaptor protein JIP4 as an LRRK2 partner at the lysosomal membrane. LRRK2 can recruit JIP4 to lysosomes in a kinase-dependent manner via the phosphorylation of RAB35 and RAB10. Using super-resolution live-cell imaging microscopy and FIB-SEM, we demonstrate that JIP4 promotes the formation of LAMP1-negative tubules that release membranous content from lysosomes. Thus, we describe a new process orchestrated by LRRK2, which we name LYTL (LYsosomal Tubulation/sorting driven by LRRK2), by which lysosomal tubulation is used to release vesicles from lysosomes. Given the central role of the lysosome in PD, LYTL is likely to be disease relevant.

LRRK2 mediates microglial neurotoxicity via NFATc2 in rodent models of synucleinopathies.

Synucleinopathies are neurodegenerative disorders characterized by abnormal α-synuclein deposition that include Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. The pathology of these conditions also includes neuronal loss and neuroinflammation. Neuron-released α-synuclein has been shown to induce neurotoxic, proinflammatory microglial responses through Toll-like receptor 2, but the molecular mechanisms involved are poorly understood. Here, we show that leucine-rich repeat kinase 2 (LRRK2) plays a critical role in the activation of microglia by extracellular α-synuclein. Exposure to α-synuclein was found to enhance LRRK2 phosphorylation and activity in mouse primary microglia. Furthermore, genetic and pharmacological inhibition of LRRK2 markedly diminished α-synuclein-mediated microglial neurotoxicity via lowering of tumor necrosis factor-α and interleukin-6 expression in mouse cultures. We determined that LRRK2 promoted a neuroinflammatory cascade by selectively phosphorylating and inducing nuclear translocation of the immune transcription factor nuclear factor of activated T cells, cytoplasmic 2 (NFATc2). NFATc2 activation was seen in patients with synucleinopathies and in a mouse model of synucleinopathy, where administration of an LRRK2 pharmacological inhibitor restored motor behavioral deficits. Our results suggest that modulation of LRRK2 and its downstream signaling mediator NFATc2 might be therapeutic targets for treating synucleinopathies.

Sequential screening nominates the Parkinson's disease associated kinase LRRK2 as a regulator of Clathrin-mediated endocytosis.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are an established cause of inherited Parkinson's disease (PD). LRRK2 is expressed in both neurons and glia in the central nervous system, but its physiological function(s) in each of these cell types is uncertain. Through sequential screens, we report a functional interaction between LRRK2 and Clathrin adaptor protein complex 2 (AP2). Analysis of LRRK2 KO tissue revealed a significant dysregulation of AP2 complex components, suggesting LRRK2 may act upstream of AP2. In line with this hypothesis, expression of LRRK2 was found to modify recruitment and phosphorylation of AP2. Furthermore, expression of LRRK2 containing the R1441C pathogenic mutation resulted in impaired clathrin-mediated endocytosis (CME). A decrease in activity-dependent synaptic vesicle endocytosis was also observed in neurons harboring an endogenous R1441C LRRK2 mutation. Alongside LRRK2, several PD-associated genes intersect with membrane-trafficking pathways. To investigate the genetic association between Clathrin-trafficking and PD, we used polygenetic risk profiling from IPDGC genome wide association studies (GWAS) datasets. Clathrin-dependent endocytosis genes were found to be associated with PD across multiple cohorts, suggesting common variants at these loci represent a cumulative risk factor for disease. Taken together, these findings suggest CME is a LRRK2-mediated, PD relevant pathway.

The Parkinson's Disease Protein LRRK2 Interacts with the GARP Complex to Promote Retrograde Transport to the trans-Golgi Network.

Mutations in Leucine-rich repeat kinase 2 (LRRK2) cause Parkinson's disease (PD). However, the precise function of LRRK2 remains unclear. We report an interaction between LRRK2 and VPS52, a subunit of the Golgi-associated retrograde protein (GARP) complex that identifies a function of LRRK2 in regulating membrane fusion at the trans-Golgi network (TGN). At the TGN, LRRK2 further interacts with the Golgi SNAREs VAMP4 and Syntaxin-6 and acts as a scaffolding platform that stabilizes the GARP-SNAREs complex formation. Therefore, LRRK2 influences both retrograde and post-Golgi trafficking pathways in a manner dependent on its GTP binding and kinase activity. This action is exaggerated by mutations associated with Parkinson's disease and can be blocked by kinase inhibitors. Disruption of GARP sensitizes dopamine neurons to mutant LRRK2 toxicity in C. elegans, showing that these pathways are interlinked in vivo and suggesting a link in PD.

Transcriptome analysis of LRRK2 knock-out microglia cells reveals alterations of inflammatory- and oxidative stress-related pathways upon treatment with α-synuclein fibrils.

Several previous studies have linked the Parkinson's disease (PD) gene LRRK2 to the biology of microglia cells. However, the precise ways in which LRRK2 affects microglial function have not been fully resolved. Here, we used the RNA-Sequencing to obtain transcriptomic profiles of LRRK2 wild-type (WT) and knock-out (KO) microglia cells treated with α-synuclein pre-formed fibrils (PFFs) or lipopolysaccharide (LPS) as a general inflammatory insult. We observed that, although α-synuclein PFFs and LPS mediate overlapping gene expression profiles in microglia, there are also distinct responses to each stimulus. α-Synuclein PFFs trigger alterations of oxidative stress-related pathways with the mitochondrial dismutase Sod2 as a strongly differentially regulated gene. We validated SOD2 at mRNA and protein levels. Furthermore, we found that LRRK2 KO microglia cells reported attenuated induction of mitochondrial SOD2 in response to α-synuclein PFFs, indicating a potential contribution of LRRK2 to oxidative stress-related pathways. We validate several genes in vivo using single-cell RNA-Seq from acutely isolated microglia after striatal injection of LPS into the mouse brain. Overall, these results suggest that microglial LRRK2 may contribute to the pathogenesis of PD via altered oxidative stress signaling.

LRRK2 links genetic and sporadic Parkinson's disease.

The past two decades in research has revealed the importance of leucine-rich repeat kinase 2 (LRRK2) in both monogenic and sporadic forms of Parkinson's disease (PD). In families, mutations in LRRK2 can cause PD with age-dependent but variable penetrance and genome-wide association studies have found variants of the gene that are risk factors for sporadic PD. Functional studies have suggested that the common mechanism that links all disease-associated variants is that they increase LRRK2 kinase activity, albeit in different ways. Here, we will discuss the roles of LRRK2 in areas of inflammation and vesicular trafficking in the context of monogenic and sporadic PD. We will also provide a hypothetical model that links inflammation and vesicular trafficking together in an effort to outline how these pathways might interact and eventually lead to neuronal cell death. We will also highlight the translational potential of LRRK2-specific kinase inhibitors for the treatment of PD.

Differences in Stability, Activity and Mutation Effects Between Human and Mouse Leucine-Rich Repeat Kinase 2.

Mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene have been implicated in the pathogenesis of Parkinson's disease (PD). Identification of PD-associated LRRK2 mutations has led to the development of novel animal models, primarily in mice. However, the characteristics of human LRRK2 and mouse Lrrk2 protein have not previously been directly compared. Here we show that proteins from different species have different biochemical properties, with the mouse protein being more stable but having significantly lower kinase activity compared to the human orthologue. In examining the effects of PD-associated mutations and risk factors on protein function, we found that conserved substitutions such as G2019S affect human and mouse LRRK2 proteins similarly, but variation around position 2385, which is not fully conserved between humans and mice, induces divergent in vitro behavior. Overall our results indicate that structural differences between human and mouse LRRK2 are likely responsible for the different properties we have observed for these two species of LRRK2 protein. These results have implications for disease modelling of LRRK2 mutations in mice and on the testing of pharmacological therapies in animals.

Genetic analysis of neurodegenerative diseases in a pathology cohort.

Molecular genetic research provides unprecedented opportunities to examine genotype-phenotype correlations underlying complex syndromes. To investigate pathogenic mutations and genotype-phenotype relationships in diverse neurodegenerative conditions, we performed a rare variant analysis of damaging mutations in autopsy-confirmed neurodegenerative cases from the Johns Hopkins Brain Resource Center (n = 1243 patients). We used NeuroChip genotyping and C9orf72 hexanucleotide repeat analysis to rapidly screen our cohort for disease-causing mutations. In total, we identified 42 individuals who carried a pathogenic mutation in LRRK2, GBA, APP, PSEN1, MAPT, GRN, C9orf72, SETX, SPAST, or CSF1R, and we provide a comprehensive description of the diverse clinicopathological features of these well-characterized cases. Our study highlights the utility of high-throughput genetic screening arrays to establish a molecular diagnosis in individuals with complex neurodegenerative syndromes, to broaden disease phenotypes and to provide insights into unexpected disease associations.

Analysis of macroautophagy related proteins in G2019S LRRK2 Parkinson's disease brains with Lewy body pathology.

LRRK2, the gene encoding the multidomain kinase Leucine-Rich Repeat Kinase 2 (LRRK2), has been linked to familial and sporadic forms of Parkinson's disease (PD), as well as cancer, leprosy and Crohn's disease, establishing it as a target for discovery therapeutics. LRRK2 has been associated with a range of cellular processes, however its physiological and pathological functions remain unclear. The most prevalent LRRK2 mutations in PD have been shown to affect macroautophagy in various cellular models while a role in autophagy signalling has been recapitulated in vivo. Dysregulation of autophagy has been implicated in PD pathology, and this raises the possibility that differential autophagic activity is relevant to disease progression in PD patients carrying LRRK2 mutations. To examine the relevance of LRRK2 to the regulation of macroautophagy in a disease setting we examined the levels of autophagic markers in the basal ganglia of G2019S LRRK2 PD post-mortem tissue, in comparison to pathology-matched idiopathic PD (iPD), using immunoblotting (IB). Significantly lower levels of p62 and LAMP1 were observed in G2019S LRRK2 PD compared to iPD cases. Similarly, an increase in ULK1 was observed in iPD but was not reflected in G2019S LRRK2 PD cases. Furthermore, examination of p62 by immunohistochemistry (IH) recapitulated a distinct signature for G2019S PD. IH of LAMP1, LC3 and ULK1 broadly correlated with the IB results. Our data from a small but pathologically well-characterized cases highlights a divergence of G2019S PD carriers in terms of autophagic response in alpha-synuclein pathology affected brain regions compared to iPD.