LRRK2 phosphorylation status and kinase activity regulate (macro)autophagy in a Rab8a/Rab10-dependent manner.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common genetic cause of Parkinson's disease (PD), with growing importance also for Crohn's disease and cancer. LRRK2 is a large and complex protein possessing both GTPase and kinase activity. Moreover, LRRK2 activity and function can be influenced by its phosphorylation status. In this regard, many LRRK2 PD-associated mutants display decreased phosphorylation of the constitutive phosphorylation cluster S910/S935/S955/S973, but the role of these changes in phosphorylation status with respect to LRRK2 physiological functions remains unknown. Here, we propose that the S910/S935/S955/S973 phosphorylation sites act as key regulators of LRRK2-mediated autophagy under both basal and starvation conditions. We show that quadruple LRRK2 phosphomutant cells (4xSA; S910A/S935A/S955A/S973A) have impaired lysosomal functionality and fail to induce and proceed with autophagy during starvation. In contrast, treatment with the specific LRRK2 kinase inhibitors MLi-2 (100 nM) or PF-06447475 (150 nM), which also led to decreased LRRK2 phosphorylation of S910/S935/S955/S973, did not affect autophagy. In explanation, we demonstrate that the autophagy impairment due to the 4xSA LRRK2 phospho-dead mutant is driven by its enhanced LRRK2 kinase activity. We show mechanistically that this involves increased phosphorylation of LRRK2 downstream targets Rab8a and Rab10, as the autophagy impairment in 4xSA LRRK2 cells is counteracted by expression of phosphorylation-deficient mutants T72A Rab8a and T73A Rab10. Similarly, reduced autophagy and decreased LRRK2 phosphorylation at the constitutive sites were observed in cells expressing the pathological R1441C LRRK2 PD mutant, which also displays increased kinase activity. These data underscore the relation between LRRK2 phosphorylation at its constitutive sites and the importance of increased LRRK2 kinase activity in autophagy regulation and PD pathology.

The small GTPase Rit2 modulates LRRK2 kinase activity, is required for lysosomal function and protects against alpha-synuclein neuropathology.

In Parkinson's disease (PD) misfolded alpha-synuclein (aSyn) accumulates in the substantia nigra, where dopaminergic neurons are progressively lost. The mechanisms underlying aSyn pathology are still unclear, but they are hypothesized to involve the autophagy-lysosome pathway (ALP). LRRK2 mutations are a major cause of familial and sporadic PD, and LRRK2 kinase activity has been shown to be involved in pS129-aSyn inclusion modulation. We observed selective downregulation of the novel PD risk factor RIT2 in vitro and in vivo. Rit2 overexpression in G2019S-LRRK2 cells rescued ALP abnormalities and diminished aSyn inclusions. In vivo, viral mediated overexpression of Rit2 operated neuroprotection against AAV-A53T-aSyn. Furthermore, Rit2 overexpression prevented the A53T-aSyn-dependent increase of LRRK2 kinase activity in vivo. On the other hand, reduction of Rit2 levels leads to defects in the ALP, similar to those induced by the G2019S-LRRK2 mutation. Our data indicate that Rit2 is required for correct lysosome function, inhibits overactive LRRK2 to ameliorate ALP impairment, and counteracts aSyn aggregation and related deficits. Targeting Rit2 could represent an effective strategy to combat neuropathology in familial and idiopathic PD.

Increased Levels of the Parkinson's Disease-Associated Gene ITPKB Correlate with Higher Expression Levels of α-Synuclein, Independent of Mutation Status.

Autosomal dominant mutations in the gene encoding α-synuclein (SNCA) were the first to be linked with hereditary Parkinson's disease (PD). Duplication and triplication of SNCA has been observed in PD patients, together with mutations at the N-terminal of the protein, among which A30P and A53T influence the formation of fibrils. By overexpressing human α-synuclein in the neuronal system of Drosophila, we functionally validated the ability of IP3K2, an ortholog of the GWAS identified risk gene, Inositol-trisphosphate 3-kinase B (ITPKB), to modulate α-synuclein toxicity in vivo. ITPKB mRNA and protein levels were also increased in SK-N-SH cells overexpressing wild-type α-synuclein, A53T or A30P mutants. Kinase overexpression was detected in the cytoplasmatic and in the nuclear compartments in all α-synuclein cell types. By quantifying mRNAs in the cortex of PD patients, we observed higher levels of ITPKB mRNA when SNCA was expressed more (p < 0.05), compared to controls. A positive correlation was also observed between SNCA and ITPKB expression in the cortex of patients, which was not seen in the controls. We replicated this observation in a public dataset. Our data, generated in SK-N-SH cells and in cortex from PD patients, show that the expression of α-synuclein and ITPKB is correlated in pathological situations.

Protein phosphatase 2A holoenzymes regulate leucine-rich repeat kinase 2 phosphorylation and accumulation.

LRRK2 is a highly phosphorylated multidomain protein and mutations in the gene encoding LRRK2 are a major genetic determinant of Parkinson's disease (PD). Dephosphorylation at LRRK2's S910/S935/S955/S973 phosphosite cluster is observed in several conditions including in sporadic PD brain, in several disease mutant forms of LRRK2 and after pharmacological LRRK2 kinase inhibition. However, the mechanism of LRRK2 dephosphorylation is poorly understood. We performed a phosphatome-wide reverse genetics screen to identify phosphatases involved in the dephosphorylation of the LRRK2 phosphosite S935. Candidate phosphatases selected from the primary screen were tested in mammalian cells, Xenopus oocytes and in vitro. Effects of PP2A on endogenous LRRK2 phosphorylation were examined via expression modulation with CRISPR/dCas9. Our screening revealed LRRK2 phosphorylation regulators linked to the PP1 and PP2A holoenzyme complexes as well as CDC25 phosphatases. We showed that dephosphorylation induced by different kinase inhibitor triggered relocalisation of phosphatases PP1 and PP2A in LRRK2 subcellular compartments in HEK-293 T cells. We also demonstrated that LRRK2 is an authentic substrate of PP2A both in vitro and in Xenopus oocytes. We singled out the PP2A holoenzyme PPP2CA:PPP2R2 as a powerful phosphoregulator of pS935-LRRK2. Furthermore, we demonstrated that this specific PP2A holoenzyme induces LRRK2 relocalization and triggers LRRK2 ubiquitination, suggesting its involvement in LRRK2 clearance. The identification of the PPP2CA:PPP2R2 complex regulating LRRK2 S910/S935/S955/S973 phosphorylation paves the way for studies refining PD therapeutic strategies that impact LRRK2 phosphorylation.

ATP13A2 Regulates Cellular α-Synuclein Multimerization, Membrane Association, and Externalization.

ATP13A2, a late endo-/lysosomal polyamine transporter, is implicated in a variety of neurodegenerative diseases, including Parkinson's disease and Kufor-Rakeb syndrome, an early-onset atypical form of parkinsonism. Loss-of-function mutations in ATP13A2 result in lysosomal deficiency as a consequence of impaired lysosomal export of the polyamines spermine/spermidine. Furthermore, accumulating evidence suggests the involvement of ATP13A2 in regulating the fate of α-synuclein, such as cytoplasmic accumulation and external release. However, no consensus has yet been reached on the mechanisms underlying these effects. Here, we aimed to gain more insight into how ATP13A2 is linked to α-synuclein biology in cell models with modified ATP13A2 activity. We found that loss of ATP13A2 impairs lysosomal membrane integrity and induces α-synuclein multimerization at the membrane, which is enhanced in conditions of oxidative stress or exposure to spermine. In contrast, overexpression of ATP13A2 wildtype (WT) had a protective effect on α-synuclein multimerization, which corresponded with reduced αsyn membrane association and stimulation of the ubiquitin-proteasome system. We also found that ATP13A2 promoted the secretion of α-synuclein through nanovesicles. Interestingly, the catalytically inactive ATP13A2 D508N mutant also affected polyubiquitination and externalization of α-synuclein multimers, suggesting a regulatory function independent of the ATPase and transport activity. In conclusion, our study demonstrates the impact of ATP13A2 on α-synuclein multimerization via polyamine transport dependent and independent functions.

LRRK2 Ablation Attenuates Αlpha-Synuclein-Induced Neuroinflammation Without Affecting Neurodegeneration or Neuropathology In Vivo.

The development of disease-modifying therapies for Parkinson's disease is a major challenge which would be facilitated by a better understanding of the pathogenesis. Leucine-rich repeat kinase 2 (LRRK2) and α-synuclein are key players in Parkinson's disease, but their relationship remains incompletely resolved. Previous studies investigating the effect of LRRK2 on α-synuclein-induced neurotoxicity and neuroinflammation in preclinical Parkinson's disease models have reported conflicting results. Here, we aimed to further explore the functional interaction between α-synuclein and LRRK2 and to evaluate the therapeutic potential of targeting physiological LRRK2 levels. We studied the effects of total LRRK2 protein loss as well as pharmacological LRRK2 kinase inhibition in viral vector-mediated α-synuclein-based Parkinson's disease models developing early- and late-stage neurodegeneration. Surprisingly, total LRRK2 ablation or in-diet treatment with the LRRK2 kinase inhibitor MLi-2 did not significantly modify α-synuclein-induced motor deficits, dopaminergic cell loss, or α-synuclein pathology. Interestingly, we found a significant effect on α-synuclein-induced neuroinflammatory changes in the absence of LRRK2, with a reduced microglial activation and CD4+ and CD8+ T cell infiltration. This observed lack of protection against α-synuclein-induced toxicity should be well considered in light of the ongoing therapeutic development of LRRK2 kinase inhibitors for idiopathic Parkinson's disease. Future studies will be crucial to understand the link between these neuroinflammatory processes and disease progression as well as the role of α-synuclein and LRRK2 in these pathological events.

Inflammatory bowel disease and Parkinson's disease: common pathophysiological links.

Inflammatory bowel disease and Parkinson's disease are chronic progressive disorders that mainly affect different organs: the gut and brain, respectively. Accumulating evidence has suggested a bidirectional link between gastrointestinal inflammation and neurodegeneration, in accordance with the concept of the 'gut-brain axis'. Moreover, recent population-based studies have shown that inflammatory bowel disease might increase the risk of Parkinson's disease. Although the precise mechanisms underlying gut-brain interactions remain elusive, some of the latest findings have begun to explain the link. Several genetic loci are shared between both disorders with a similar direction of effect on the risk of both diseases. The most interesting example is LRRK2 (leucine-rich repeat kinase 2), initially identified as a causal gene in Parkinson's disease, and recently also implicated in Crohn's disease. In this review, we highlight recent findings on the link between these seemingly unrelated diseases with shared genetic susceptibility. We discuss supporting and conflicting data obtained from epidemiological and genetic studies along with remaining questions and concerns. In addition, we discuss possible biological links including the gut-brain axis, microbiota, autoimmunity, mitochondrial function and autophagy.

Divergent Effects of G2019S and R1441C LRRK2 Mutations on LRRK2 and Rab10 Phosphorylations in Mouse Tissues.

Mutations in LRRK2 cause familial Parkinson's disease and common variants increase disease risk. LRRK2 kinase activity and cellular localization are tightly regulated by phosphorylation of key residues, primarily Ser1292 and Ser935, which impacts downstream phosphorylation of its substrates, among which Rab10. A comprehensive characterization of LRRK2 activity and phosphorylation in brain as a function of age and mutations is missing. Here, we monitored Ser935 and Ser1292 phosphorylation in midbrain, striatum, and cortex of 1, 6, and 12 months-old mice carrying G2019S and R1441C mutations or murine bacterial artificial chromosome (BAC)-Lrrk2-G2019S. We observed that G2019S and, at a greater extent, R1441C brains display decreased phospho-Ser935, while Ser1292 autophosphorylation increased in G2019S but not in R1441C brain, lung, and kidney compared to wild-type. Further, Rab10 phosphorylation, is elevated in R1441C carrying mice, indicating that the effect of LRRK2 mutations on substrate phosphorylation is not generalizable. In BAC-Lrrk2-G2019S striatum and midbrain, Rab10 phosphorylation, but not Ser1292 autophosphorylation, decreases at 12-months, pointing to autophosphorylation and substrate phosphorylation as uncoupled events. Taken together, our study provides novel evidence that LRRK2 phosphorylation in mouse brain is differentially impacted by mutations, brain area, and age, with important implications as diagnostic markers of disease progression and stratification.

Pathogenic LRRK2 requires secondary factors to induce cellular toxicity.

Pathogenic mutations in the leucine-rich repeat kinase 2 (LRRK2) gene belong to the most common genetic causes of inherited Parkinson's disease (PD) and variations in its locus increase the risk to develop sporadic PD. Extensive research efforts aimed at understanding how changes in the LRRK2 function result in molecular alterations that ultimately lead to PD. Cellular LRRK2-based models revealed several potential pathophysiological mechanisms including apoptotic cell death, LRRK2 protein accumulation and deficits in neurite outgrowth. However, highly variable outcomes between different cellular models have been reported. Here, we have investigated the effect of different experimental conditions, such as the use of different tags and gene transfer methods, in various cellular LRRK2 models. Readouts included cell death, sensitivity to oxidative stress, LRRK2 relocalization, α-synuclein aggregation and neurite outgrowth in cell culture, as well as neurite maintenance in vivo. We show that overexpression levels and/or the tag fused to LRRK2 affect the relocalization of LRRK2 to filamentous and skein-like structures. We found that overexpression of LRRK2 per se is not sufficient to induce cellular toxicity or to affect α-synuclein-induced toxicity and aggregate formation. Finally, neurite outgrowth/retraction experiments in cell lines and in vivo revealed that secondary, yet unknown, factors are required for the pathogenic LRRK2 effects on neurite length. Our findings stress the importance of technical and biological factors in LRRK2-induced cellular phenotypes and hence imply that conclusions based on these types of LRRK2-based assays should be interpreted with caution.

Kinase inhibition of G2019S-LRRK2 enhances autolysosome formation and function to reduce endogenous alpha-synuclein intracellular inclusions.

The Parkinson's disease (PD)-associated kinase Leucine-Rich Repeat Kinase 2 (LRRK2) is a crucial modulator of the autophagy-lysosome pathway, but unclarity exists on the precise mechanics of its role and the direction of this modulation. In particular, LRRK2 is involved in the degradation of pathological alpha-synuclein, with pathogenic mutations precipitating neuropathology in cellular and animal models of PD, and a significant proportion of LRRK2 patients presenting Lewy neuropathology. Defects in autophagic processing and lysosomal degradation of alpha-synuclein have been postulated to underlie its accumulation and onset of neuropathology. Thus, it is critical to obtain a comprehensive knowledge on LRRK2-associated pathology. Here, we investigated a G2019S-LRRK2 recombinant cell line exhibiting accumulation of endogenous, phosphorylated alpha-synuclein. We found that G2019S-LRRK2 leads to accumulation of LC3 and abnormalities in lysosome morphology and proteolytic activity in a kinase-dependent fashion, but independent from constitutively active Rab10. Notably, LRRK2 inhibition was ineffective upon upstream blockade of autophagosome-lysosome fusion events, highlighting this step as critical for alpha-synuclein clearance.

Multiple-Hit Hypothesis in Parkinson's Disease: LRRK2 and Inflammation.

The multiple hit hypothesis for Parkinson's disease (PD) suggests that an interaction between multiple (genetic and/or environmental) risk factors is needed to trigger the pathology. Leucine-Rich Repeat Kinase 2 (LRRK2) is an interesting protein to study in this context and is the focus of this review. More than 15 years of intensive research have identified several cellular pathways in which LRRK2 is involved, yet its exact physiological role or contribution to PD is not completely understood. Pathogenic mutations in LRRK2 are the most common genetic cause of PD but most likely require additional triggers to develop PD, as suggested by the reduced penetrance of the LRRK2 G2019S mutation. LRRK2 expression is high in immune cells such as monocytes, neutrophils, or dendritic cells, compared to neurons or glial cells and evidence for a role of LRRK2 in the immune system is emerging. This has led to the hypothesis that an inflammatory trigger is needed for pathogenic LRRK2 mutations to induce a PD phenotype. In this review, we will discuss the link between LRRK2 and inflammation and how this could play an active role in PD etiology.

RAB8, RAB10 and RILPL1 contribute to both LRRK2 kinase-mediated centrosomal cohesion and ciliogenesis deficits.

Mutations in the LRRK2 kinase are the most common cause of familial Parkinson's disease, and variants increase risk for the sporadic form of the disease. LRRK2 phosphorylates multiple RAB GTPases including RAB8A and RAB10. Phosphorylated RAB10 is recruited to centrosome-localized RILPL1, which may interfere with ciliogenesis in a disease-relevant context. Our previous studies indicate that the centrosomal accumulation of phosphorylated RAB8A causes centrosomal cohesion deficits in dividing cells, including in peripheral patient-derived cells. Here, we show that both RAB8 and RAB10 contribute to the centrosomal cohesion deficits. Pathogenic LRRK2 causes the centrosomal accumulation not only of phosho-RAB8 but also of phospho-RAB10, and the effects on centrosomal cohesion are dependent on RAB8, RAB10 and RILPL1. Conversely, the pathogenic LRRK2-mediated ciliogenesis defects correlate with the centrosomal accumulation of both phospho-RAB8 and phospho-RAB10. LRRK2-mediated centrosomal cohesion and ciliogenesis alterations are observed in patient-derived peripheral cells, as well as in primary astrocytes from mutant LRRK2 mice, and are reverted upon LRRK2 kinase inhibition. These data suggest that the LRRK2-mediated centrosomal cohesion and ciliogenesis defects are distinct cellular readouts of the same underlying phospho-RAB8/RAB10/RILPL1 nexus and highlight the possibility that either centrosomal cohesion and/or ciliogenesis alterations may serve as cellular biomarkers for LRRK2-related PD.

Rab32 interacts with SNX6 and affects retromer-dependent Golgi trafficking.

The Rab family of small GTPases regulate various aspects of cellular dynamics in eukaryotic cells. Membrane trafficking has emerged as central to the functions of leucine-rich repeat kinase 2 (LRRK2), which is associated with inherited and sporadic forms of Parkinson's disease (PD). Rabs act as both regulators of the catalytic activity and targets for serine/threonine phosphorylation by LRRK2. Rab32, Rab38 and Rab29 have been shown to regulate LRRK2 sub-cellular localization through direct interactions. Recently, Rab29 was shown to escort LRRK2 to the Golgi apparatus and activate the phosphorylation of Rab8 and Rab10. Rab32 is linked to multiple cellular functions including endosomal trafficking, mitochondrial dynamics, and melanosome biogenesis. A missense mutation in Rab32 has also recently been linked to PD. Here, we demonstrate that Rab32 directly interacts with sorting nexin 6 (SNX6). SNX6 is a transient subunit of the retromer, an endosome-Golgi retrieval complex whose Vps35 subunit is strongly associated with PD. We could further show that localization of cation-independent mannose-6-phosphate receptors, which are recycled to the trans-Golgi network (TGN) by the retromer, was affected by both Rab32 and SNX6. These data imply that Rab32 is linked to SNX6/retromer trafficking at the Golgi, and also suggests a possible connection between the retromer and Rab32 in the trafficking and biological functions of LRRK2.

RAB7L1-Mediated Relocalization of LRRK2 to the Golgi Complex Causes Centrosomal Deficits via RAB8A.

Mutations in the LRRK2 gene cause autosomal-dominant Parkinson's disease (PD), and both LRRK2 as well as RAB7L1 have been implicated in increased susceptibility to idiopathic PD. RAB7L1 has been shown to increase membrane-association and kinase activity of LRRK2, and both seem to be mechanistically implicated in the same pathway. Another RAB protein, RAB8A, has been identified as a prominent LRRK2 kinase substrate, and our recent work demonstrates that aberrant LRRK2-mediated phosphorylation of RAB8A leads to centrosomal alterations. Here, we show that RAB7L1 recruits LRRK2 to the Golgi complex, which causes accumulation of phosphorylated RAB8A in a pericentrosomal/centrosomal location as well as centrosomal deficits identical to those observed with pathogenic LRRK2. The centrosomal alterations induced by wildtype LRRK2 in the presence of RAB7L1 depend on Golgi integrity. This is in contrast to pathogenic LRRK2 mutants, which cause centrosomal deficits independent of Golgi integrity or largely independent on RAB7L1 expression. Furthermore, centrosomal alterations in the presence of wildtype LRRK2 and RAB7L1 are at least in part mediated by aberrant LRRK2-mediated RAB8A phosphorylation, as abolished by kinase inhibitors and reduced upon knockdown of RAB8A. These results indicate that pathogenic LRRK2, as well as increased levels of RAB7L1, cause centrosomal deficits in a manner dependent on aberrant RAB8A phosphorylation and centrosomal/pericentrosomal accumulation, suggesting that centrosomal cohesion deficits may comprise a useful cellular readout for a broader spectrum of the disease.

LRRK2 Phosphorylation: Behind the Scenes.

Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are known today as the most common genetic cause of Parkinson's disease (PD). LRRK2 is a large protein that is hypothesized to regulate other proteins as a scaffold in downstream signaling pathways. This is supported by the multiple domain composition of LRRK2 with several protein-protein interaction domains combined with kinase and GTPase activity. LRRK2 is highly phosphorylated at sites that are strictly controlled by upstream regulators, including its own kinase domain. In cultured cells, most pathogenic mutants display increased autophosphorylation at S1292, but decreased phosphorylation at sites controlled by other kinases. We only begin to understand how LRRK2 phosphorylation is regulated and how this impacts its physiological and pathological function. Intriguingly, LRRK2 kinase inhibition, currently one of the most prevailing disease-modifying therapeutic strategies for PD, induces LRRK2 dephosphorylation at sites that are also dephosphorylated in pathogenic variants. In addition, LRRK2 kinase inhibition can induce LRRK2 protein degradation, which might be related to the observed inhibitor-induced adverse effects on the lung in rodents and non-human primates, as it resembles the lung pathology in LRRK2 knock-out animals. In this review, we will provide an overview of how LRRK2 phosphorylation is regulated and how this complex regulation relates to several molecular and cellular features of LRRK2.

Parkinson disease-associated mutations in LRRK2 cause centrosomal defects via Rab8a phosphorylation.

BACKGROUND: Mutations in LRRK2 are a common genetic cause of Parkinson's disease (PD). LRRK2 interacts with and phosphorylates a subset of Rab proteins including Rab8a, a protein which has been implicated in various centrosome-related events. However, the cellular consequences of such phosphorylation remain elusive. METHODS: Human neuroblastoma SH-SY5Y cells stably expressing wildtype or pathogenic LRRK2 were used to test for polarity defects in the context of centrosomal positioning. Centrosomal cohesion deficits were analyzed from transiently transfected HEK293T cells, as well as from two distinct peripheral cell types derived from LRRK2-PD patients. Kinase assays, coimmunoprecipitation and GTP binding/retention assays were used to address Rab8a phosphorylation by LRRK2 and its effects in vitro. Transient transfections and siRNA experiments were performed to probe for the implication of Rab8a and its phosphorylated form in the centrosomal deficits caused by pathogenic LRRK2. RESULTS: Here, we show that pathogenic LRRK2 causes deficits in centrosomal positioning with effects on neurite outgrowth, cell polarization and directed migration. Pathogenic LRRK2 also causes deficits in centrosome cohesion which can be detected in peripheral cells derived from LRRK2-PD patients as compared to healthy controls, and which are reversed upon LRRK2 kinase inhibition. The centrosomal cohesion and polarity deficits can be mimicked when co-expressing wildtype LRRK2 with wildtype but not phospho-deficient Rab8a. The centrosomal defects induced by pathogenic LRRK2 are associated with a kinase activity-dependent increase in the centrosomal localization of phosphorylated Rab8a, and are prominently reduced upon RNAi of Rab8a. CONCLUSIONS: Our findings reveal a new function of LRRK2 mediated by Rab8a phosphorylation and related to various centrosomal defects.

PAK6 Phosphorylates 14-3-3γ to Regulate Steady State Phosphorylation of LRRK2.

Mutations in Leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease (PD) and, as such, LRRK2 is considered a promising therapeutic target for age-related neurodegeneration. Although the cellular functions of LRRK2 in health and disease are incompletely understood, robust evidence indicates that PD-associated mutations alter LRRK2 kinase and GTPase activities with consequent deregulation of the downstream signaling pathways. We have previously demonstrated that one LRRK2 binding partner is P21 (RAC1) Activated Kinase 6 (PAK6). Here, we interrogate the PAK6 interactome and find that PAK6 binds a subset of 14-3-3 proteins in a kinase dependent manner. Furthermore, PAK6 efficiently phosphorylates 14-3-3γ at Ser59 and this phosphorylation serves as a switch to dissociate the chaperone from client proteins including LRRK2, a well-established 14-3-3 binding partner. We found that 14-3-3γ phosphorylated by PAK6 is no longer competent to bind LRRK2 at phospho-Ser935, causing LRRK2 dephosphorylation. To address whether these interactions are relevant in a neuronal context, we demonstrate that a constitutively active form of PAK6 rescues the G2019S LRRK2-associated neurite shortening through phosphorylation of 14-3-3γ. Our results identify PAK6 as the kinase for 14-3-3γ and reveal a novel regulatory mechanism of 14-3-3/LRRK2 complex in the brain.

Leucine-rich repeat kinase 2 interacts with p21-activated kinase 6 to control neurite complexity in mammalian brain.

Leucine-rich repeat kinase 2 (LRRK2) is a causative gene for Parkinson's disease, but the physiological function and the mechanism(s) by which the cellular activity of LRRK2 is regulated are poorly understood. Here, we identified p21-activated kinase 6 (PAK6) as a novel interactor of the GTPase/ROC domain of LRRK2. p21-activated kinases are serine-threonine kinases that serve as targets for the small GTP binding proteins Cdc42 and Rac1 and have been implicated in different morphogenetic processes through remodeling of the actin cytoskeleton such as synapse formation and neuritogenesis. Using an in vivo neuromorphology assay, we show that PAK6 is a positive regulator of neurite outgrowth and that LRRK2 is required for this function. Analyses of post-mortem brain tissue from idiopathic and LRRK2 G2019S carriers reveal an increase in PAK6 activation state, whereas knock-out LRRK2 mice display reduced PAK6 activation and phosphorylation of PAK6 substrates. Taken together, these results support a critical role of LRRK2 GTPase domain in cytoskeletal dynamics in vivo through the novel interactor PAK6, and provide a valuable platform to unravel the mechanism underlying LRRK2-mediated pathophysiology. We propose p21-activated kinase 6 (PAK6) as a novel interactor of leucine-rich repeat kinase 2 (LRRK2), a kinase involved in Parkinson's disease (PD). In health, PAK6 regulates neurite complexity in the brain and LRRK2 is required for its function, (a) whereas PAK6 is aberrantly activated in LRRK2-linked PD brain (b) suggesting that LRRK2 toxicity is mediated by PAK6.