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.

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.

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.

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.

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.

Unbiased screen for interactors of leucine-rich repeat kinase 2 supports a common pathway for sporadic and familial Parkinson disease.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson disease (PD), and common variants around LRRK2 are a risk factor for sporadic PD. Using protein-protein interaction arrays, we identified BCL2-associated athanogene 5, Rab7L1 (RAB7, member RAS oncogene family-like 1), and Cyclin-G-associated kinase as binding partners of LRRK2. The latter two genes are candidate genes for risk for sporadic PD identified by genome-wide association studies. These proteins form a complex that promotes clearance of Golgi-derived vesicles through the autophagy-lysosome system both in vitro and in vivo. We propose that three different genes for PD have a common biological function. More generally, data integration from multiple unbiased screens can provide insight into human disease mechanisms.

Chemical genetic approach identifies microtubule affinity-regulating kinase 1 as a leucine-rich repeat kinase 2 substrate.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of autosomal-dominant forms of Parkinson's disease. LRRK2 is a modular, multidomain protein containing 2 enzymatic domains, including a kinase domain, as well as several protein-protein interaction domains, pointing to a role in cellular signaling. Although enormous efforts have been made, the exact pathophysiologic mechanisms of LRRK2 are still not completely known. In this study, we used a chemical genetics approach to identify LRRK2 substrates from mouse brain. This approach allows the identification of substrates of 1 particular kinase in a complex cellular environment. Several of the identified peptides are involved in the regulation of microtubule (MT) dynamics, including microtubule-associating protein (MAP)/microtubule affinity-regulating kinase 1 (MARK1). MARK1 is a serine/threonine kinase known to phosphorylate MT-binding proteins such as Tau, MAP2, and MAP4 at KXGS motifs leading to MT destabilization. In vitro kinase assays and metabolic-labeling experiments in living cells confirmed MARK1 as an LRRK2 substrate. Moreover, we also showed that LRRK2 and MARK1 are interacting in eukaryotic cells. Our findings contribute to the identification of physiologic LRRK2 substrates and point to a potential mechanism explaining the reported effects of LRRK2 on neurite morphology.

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.

MicroRNA-205 regulates the expression of Parkinson's disease-related leucine-rich repeat kinase 2 protein.

Recent genome-wide association studies indicate that a simple alteration of Leucine-rich repeat kinase 2 (LRRK2) gene expression may contribute to the etiology of sporadic Parkinson's disease (PD). However, the expression and regulation of LRRK2 protein in the sporadic PD brains remain to be determined. Here, we found that the expression of LRRK2 protein was enhanced in the sporadic PD patients using the frontal cortex tissue from a set of 16 PD patients and 7 control samples. In contrast, no significant difference was detected in the level of LRRK2 mRNA expression between the control and PD cases, suggesting a potential post-transcriptional modification of the LRRK2 protein expression in the sporadic PD brains. Indeed, it was identified that microRNA-205 (miR-205) suppressed the expression of LRRK2 protein through a conserved-binding site at the 3'-untranslated region (UTR) of LRRK2 gene. Interestingly, miR-205 expression was significantly downregulated in the brains of patients with sporadic PD, showing the enhanced LRRK2 protein levels. Also, in vitro studies in the cell lines and primary neuron cultures further established the role of miR-205 in modulating the expression of LRRK2 protein. In addition, introduction of miR-205 prevented the neurite outgrowth defects in the neurons expressing a PD-related LRRK2 R1441G mutant. Together, these findings suggest that downregulation of miR-205 may contribute to the potential pathogenic elevation of LRRK2 protein in the brains of patients with sporadic PD, while overexpression of miR-205 may provide an applicable therapeutic strategy to suppress the abnormal upregulation of LRRK2 protein in PD.

Differential protein-protein interactions of LRRK1 and LRRK2 indicate roles in distinct cellular signaling pathways.

Genetic studies show that LRRK2, and not its closest paralogue LRRK1, is linked to Parkinson's disease. To gain insight into the molecular and cellular basis of this discrepancy, we searched for LRRK1- and LRRK2-specific cellular processes by identifying their distinct interacting proteins. A protein microarray-based interaction screen was performed with recombinant 3xFlag-LRRK1 and 3xFlag-LRRK2 and, in parallel, co-immunoprecipitation followed by mass spectrometry was performed from SH-SY5Y neuroblastoma cell lines stably expressing 3xFlag-LRRK1 or 3xFlag-LRRK2. We identified a set of LRRK1- and LRRK2-specific as well as common interactors. One of our most prominent findings was that both screens pointed to epidermal growth factor receptor (EGF-R) as a LRRK1-specific interactor, while 14-3-3 proteins were LRRK2-specific. This is consistent with phosphosite mapping of LRRK1, revealing phosphosites outside of 14-3-3 consensus binding motifs. To assess the functional relevance of these interactions, SH-SY5Y-LRRK1 and -LRRK2 cell lines were treated with LRRK2 kinase inhibitors that disrupt 14-3-3 binding, or with EGF, an EGF-R agonist. Redistribution of LRRK2, not LRRK1, from diffuse cytoplasmic to filamentous aggregates was observed after inhibitor treatment. Similarly, EGF induced translocation of LRRK1, but not of LRRK2, to endosomes. Our study confirms that LRRK1 and LRRK2 can carry out distinct functions by interacting with different cellular proteins. LRRK1 and LRRK2 (leucine-rich repeat kinase) interaction partners were identified by two different protein-protein interaction screens. These confirmed epidermal growth factor receptor (EGR-R) as a LRRK1-specific interactor, while 14-3-3 proteins were LRRK2-specific. Functional analysis of these interactions and the pathways they mediate shows that LRRK1 and LRRK2 signaling do not intersect, reflective of the differential role of both LRRKs in Parkinson's disease.

Identification of protein phosphatase 1 as a regulator of the LRRK2 phosphorylation cycle.

A cluster of phosphorylation sites in LRRK2 (leucine-rich repeat kinase 2), including Ser910, Ser935, Ser955 and Ser973, is important for PD (Parkinson's disease) pathogenesis as several PD-linked LRRK2 mutants are dephosphorylated at these sites. LRRK2 is also dephosphorylated in cells after pharmacological inhibition of its kinase activity, which is currently proposed as a strategy for disease-modifying PD therapy. Despite this importance of LRRK2 dephosphorylation in mutant LRRK2 pathological mechanism(s) and in LRRK2's response to inhibition, the mechanism by which this occurs is unknown. Therefore we aimed to identify the phosphatase for LRRK2. Using a panel of recombinant phosphatases, we found that PP1 (protein phosphatase 1) efficiently dephosphorylates LRRK2 in vitro. PP1 activity on LRRK2 dephosphorylation was confirmed in cells using PP1 inhibition to reverse LRRK2 dephosphorylation induced by the potent LRRK2 kinase inhibitor LRRK2-IN1 as well as in R1441G mutant LRRK2. We also found that PP1 and LRRK2 can form a complex in cells. Furthermore, we observed that PP1 inhibition modulates LRRK2's cellular phenotype by reducing skein-like LRRK2-positive structures associated with dephosphorylation. In conclusion, the present study reveals PP1 as the physiological LRRK2 phosphatase, responsible for LRRK2 dephosphorylation observed in PD mutant LRRK2 and after LRRK2 kinase inhibition.

Expression, purification and preliminary biochemical and structural characterization of the leucine rich repeat namesake domain of leucine rich repeat kinase 2.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease. Much research effort has been directed towards the catalytic core region of LRRK2 composed of GTPase (ROC, Ras of complex proteins) and kinase domains and a connecting COR (C-terminus of ROC) domain. In contrast, the precise functions of the protein-protein interaction domains, such as the leucine-rich repeat (LRR) domain, are not known. In the present study, we modeled the LRRK2 LRR domain (LRR(LRRK2)) using a template assembly approach, revealing the presence of 14 LRRs. Next, we focused on the expression and purification of LRR(LRRK2) in Escherichia coli. Buffer optimization revealed that the protein requires the presence of a zwitterionic detergent, namely Empigen BB, during solubilization and the subsequent purification and characterization steps. This indicates that the detergent captures the hydrophobic surface patches of LRR(LRRK2) thereby suppressing its aggregation. Circular dichroism (CD) spectroscopy measured 18% α-helices and 21% ß-sheets, consistent with predictions from the homology model. Size exclusion chromatography (SEC) and dynamic light scattering measurements showed the presence of a single species, with a Stokes radius corresponding to the model dimensions of a protein monomer. Furthermore, no obvious LRR(LRRK2) multimerization was detected via cross-linking studies. Finally, the LRR(LRRK2) clinical mutations did not influence LRR(LRRK2) secondary, tertiary or quaternary structure as determined via SEC and CD spectroscopy. We therefore conclude that these mutations are likely to affect putative LRR(LRRK2) inter- and intramolecular interactions.

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.

Phosphorylation of LRRK2: from kinase to substrate.

The PD (Parkinson's disease) protein LRRK2 (leucine-rich repeat kinase 2) occurs in cells as a highly phosphorylated protein, with the majority of phosphosites clustering in the region between the ankyrin repeat and leucine-rich repeat domains. The observation that several pathogenic variants of LRRK2 display strongly reduced cellular phosphorylation suggests that phosphorylation of LRRK2 is involved in the PD pathological process. Furthermore, treatment of cells with inhibitors of LRRK2 kinase activity, which are currently considered as potential disease-modifying therapeutics for PD, leads to a rapid decrease in the phosphorylation levels of LRRK2. For these reasons, understanding the cellular role and regulation of LRRK2 as a kinase and as a substrate has become the focus of intense investigation. In the present review, we discuss what is currently known about the cellular phosphorylation of LRRK2 and how this relates to its function and dysfunction.

Insight into the mode of action of the LRRK2 Y1699C pathogenic mutant.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most prevalent known cause of autosomal dominant Parkinson's disease. The LRRK2 gene encodes a Roco protein featuring a Ras of complex proteins (ROC) GTPase and a kinase domain linked by the C-terminal of ROC (COR) domain. Here, we explored the effects of the Y1699C pathogenic LRRK2 mutation in the COR domain on GTPase activity and interactions within the catalytic core of LRRK2. We observed a decrease in GTPase activity for LRRK2 Y1699C comparable to the decrease observed for the R1441C pathogenic mutant and the T1348N dysfunctional mutant. To study the underlying mechanism, we explored the dimerization in the catalytic core of LRRK2. ROC-COR dimerization was significantly weakened by the Y1699C or R1441C/G mutation. Using a competition assay, we demonstrated that the intra-molecular ROC : COR interaction is favoured over ROC : ROC dimerization. Interestingly, the intra-molecular ROC : COR interaction was strengthened by the Y1699C mutation. This is supported by a 3D homology model of the ROC-COR tandem of LRRK2, showing that Y1699 is positioned at the intra-molecular ROC : COR interface. In conclusion, our data provides mechanistic insight into the mode of action of the Y1699C LRRK2 mutant: the Y1699C substitution, situated at the intra-molecular ROC : COR interface, strengthens the intra-molecular ROC : COR interaction, thereby locally weakening the dimerization of LRRK2 at the ROC-COR tandem domain resulting in decreased GTPase activity.

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.

Phosphorylation of ezrin/radixin/moesin proteins by LRRK2 promotes the rearrangement of actin cytoskeleton in neuronal morphogenesis.

Leucine-rich repeat kinase 2 (LRRK2) functions as a putative protein kinase of ezrin, radixin, and moesin (ERM) family proteins. A Parkinson's disease-related G2019S substitution in the kinase domain of LRRK2 further enhances the phosphorylation of ERM proteins. The phosphorylated ERM (pERM) proteins are restricted to the filopodia of growing neurites in which they tether filamentous actin (F-actin) to the cytoplasmic membrane and regulate the dynamics of filopodia protrusion. Here, we show that, in cultured neurons derived from LRRK2 G2019S transgenic mice, the number of pERM-positive and F-actin-enriched filopodia was significantly increased, and this correlates with the retardation of neurite outgrowth. Conversely, deletion of LRRK2, which lowered the pERM and F-actin contents in filopodia, promoted neurite outgrowth. Furthermore, inhibition of ERM phosphorylation or actin polymerization rescued the G2019S-dependent neuronal growth defects. These data support a model in which the G2019S mutation of LRRK2 causes a gain-of-function effect that perturbs the homeostasis of pERM and F-actin in sprouting neurites critical for neuronal morphogenesis.

Immunohistochemical detection of transgene expression in the brain using small epitope tags.

BACKGROUND: In vivo overexpression of proteins is a powerful approach to study their biological function, generate disease models or evaluate gene therapy approaches. In order to investigate an exogenously expressed protein, specific and sensitive detection is essential. Unfortunately, antibodies that allow histological detection of the protein of interest are not always readily available. The use of an epitope tag fused to the protein can circumvent this problem as well as provide the possibility to discriminate endogenous from overexpressed proteins. In order to minimize impact on the bioactivity and biodistribution of the overexpressed protein, preference is given to small tags. RESULTS: In the present study, we evaluated several small epitope tags together with corresponding anti-tag antibodies for the detection of overexpressed proteins in rat brain, using eGFP as a reference. We generated several lentiviral vectors encoding eGFP with different N-terminally fused small epitope tags (AU1, flag, 3flag, HA, myc and V5). After confirmation of their functionality in cell culture, we injected these lentiviral vectors stereotactically into the striatum of rats and prepared paraformaldehyde fixed floating sections for immunohistochemical analysis. Using multiple antibodies and antibody dilutions per epitope tag, we extensively assessed the efficiency of several anti-tag antibodies for chromogenic immunohistochemical detection of the epitope tagged eGFPs by determining the proportion of immunoreactivity detected by anti-tag antibodies compared to anti-GFP antibody. Using fluorescence immunohistochemistry and confocal microscopy, we also quantified the proportion of eGFP-positive cells detected by anti-tag antibodies. Our results show that all the examined small epitope tags could be detected by anti-tag antibodies both in cell extracts as well as in vivo, although to varying degrees depending on the tag and antibody used. Using the presented protocol, V5/anti-V5 and HA/HA11 tag/antibody combinations provided the most sensitive detection in brain tissue. We confirmed the applicability of these optimized in vivo tag detection conditions for a difficult to detect protein, firefly luciferase (fLuc), using lentiviral vector constructs expressing V5 tagged and 3flag tagged fLuc protein. CONCLUSIONS: We show here that several small epitope tags are useful for immunohistochemical detection of exogenous proteins in vivo. Our study also provides a generic methodology which is broadly applicable for the detection of overexpressed transgenes in mammalian brain tissue.

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.