Leucine-rich repeat kinase 2 functionally interacts with microtubules and kinase-dependently modulates cell migration

Neurobiology of Disease 54 (2013) 280–288

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Neurobiology of Disease journal homepage: www.elsevier.com/locate/ynbdi

Leucine-rich repeat kinase 2 functionally interacts with microtubules and kinase-dependently modulates cell migration Mareike Caesar a, Susanne Zach a, Coby B. Carlson b, Kathrin Brockmann c, Thomas Gasser c, Frank Gillardon a,⁎ a b c

Boehringer Ingelheim Pharma GmbH & Co. KG, CNS Diseases Research, 88397 Biberach an der Riss, Germany Primary and Stem Cell Systems, Life Technologies Corporation, Madison, WI 53719, USA Hertie Institut für klinische Hirnforschung, 72076 Tübingen, Germany

a r t i c l e

i n f o

Article history: Received 21 August 2012 Revised 29 November 2012 Accepted 21 December 2012 Available online 11 January 2013 Keywords: LRRK2 Tubulin Fibroblasts Migration Parkinson's disease

a b s t r a c t Recent studies indicate that the Parkinson's disease-linked leucine-rich repeat kinase 2 (LRRK2) modulates cytoskeletal functions by regulating actin and tubulin dynamics, thereby affecting neurite outgrowth. By interactome analysis we demonstrate that the binding of LRRK2 to tubulins is significantly enhanced by pharmacological LRRK2 inhibition in cells. Co-incubation of LRRK2 with microtubules increased the LRRK2 GTPase activity in a cell-free assay. Destabilization of microtubules causes a rapid decrease in cellular LRRK2(S935) phosphorylation indicating a decreased LRRK2 kinase activity. Moreover, both human LRRK2(G2019S) fibroblasts and mouse LRRK2(R1441G) fibroblasts exhibit alterations in cell migration in culture. Treatment of mouse fibroblasts with the selective LRRK2 inhibitor LRRK2-IN1 reduces cell motility. These findings suggest that LRRK2 and microtubules mutually interact both in non-neuronal cells and in neurons, which might contribute to our understanding of its pathogenic effects in Parkinson's disease. © 2013 Elsevier Inc. All rights reserved.

Introduction Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene on average account for 10% of all familial and 4% of all sporadic Parkinson's disease (PD) cases. To date forty different rare variants of LRRK2 have been found, seven of which have been confirmed to be pathogenic (for a review see Lesage and Brice, 2009). These validated PD mutations are clustered in the central catalytic region of the protein, i.e., the ras of complex (ROC) GTPase domain, the kinase domain and the Cterminus of ROC (COR) domain linking the two catalytic domains. LRRK2 is a dimeric protein (Berger et al., 2010; Lu et al., 2010; Sen et al., 2009) and following dimerization the kinase domain phosphorylates multiple sites in the Roc domain and may thus regulate overall LRRK2 function (Gloeckner et al., 2010; Greggio et al., 2008, 2009; Kamikawaji et al., 2009). Despite the information that has been gathered about the LRRK2 structure, little is known about its physiological and pathophysiological functions. The presence of both a Roc-GTPase and a kinase domain suggests a role in intracellular signaling, while the presence of several non-catalytic protein-binding domains points to an additional function as a scaffolding protein. One line of evidence links LRRK2 to

⁎ Corresponding author at: Boehringer Ingelheim Pharma GmbH & Co. KG, CNS Diseases Research, Birkendorfer Strasse 65, 88397 Biberach an der Riss, Germany. Fax: +49 7351 5498928. E-mail address: [email protected] (F. Gillardon). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nbd.2012.12.019

the cellular cytoskeleton and neurite outgrowth (Parisiadou and Cai, 2010). LRRK2 directly interacts with tubulin (Gandhi et al., 2008) and phosphorylates and stabilizes tubulin in the presence of microtubule-associated proteins (Gillardon, 2009b). This effect is further enhanced by the pathogenic G2019S mutation in the kinase domain. LRRK2 also phosphorylates tubulin-associated tau protein in vitro, although at a lower stoichiometry (Kawakami et al., 2012). LRRK2-mediated tau phosphorylation reduces tau binding to tubulin. Drosophila LRRK2 phosphorylates the microtubule-associated protein Futsch, thereby regulating synaptic structure and function (Lee et al., 2010). Several studies have shown that a gene knock-out of LRRK2 or PD-linked mutations in the protein causes changes in neurite outgrowth in embryonic primary neurons (Dächsel et al., 2010; Gillardon, 2009a; MacLeod et al., 2006; Parisiadou et al., 2009; Wang et al., 2008). Changes in neurite sprouting correlate with alterations in phospho-Ezrin/Radixin/Moesin (ERM), putative LRRK2 substrate proteins, and filamentous actin staining in filopodia (Parisiadou et al., 2009). ERM proteins link the actin cytoskeleton to the plasma membrane when phosphorylated (Mangeat et al., 1999). Inhibiting the phosphorylation of ERM or preventing actin polymerization abolishes the effects of the G2019S mutation on neurite outgrowth (Parisiadou et al., 2009). Fibroblasts isolated from PD patients have been previously used to study pathophysiological processes in PD, such as mitochondrial dysfunction due to Parkin (Grunewald et al., 2010) or PINK1 (Hoepken et al., 2007) mutations. Cytoskeleton alterations have been demonstrated in fibroblasts of Friedreich's ataxia patients (Pastore et al., 2003), a

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neurodegenerative disease affecting nerve fibers. Actin cytoskeleton changes were also detected in epithelial cell lines expressing mutant alpha-synuclein (Sousa et al., 2009). In the present study, we analyzed the functional interdependency between LRRK2 and the microtubule cytoskeleton in more detail. PD-linked LRRK2 mutations and LRRK2 kinase inhibitors were assessed to get more insight into the (patho)physiological function of LRRK2. Although the sequence of events remains to be defined, our findings indicate that tubulins influence the LRRK2 structural organization and enzymatic function and vice versa. Material and methods Ethics statement The isolation of skin biopsies from human subjects was approved by the ethics committee of the University of Tuebingen and all participants gave a written informed consent. PD patients carrying a LRRK2(G2019S) mutation were neurologically largely indistinguishable from late-onset sporadic PD patients (Brockmann et al., 2011). As reported by others (Wider et al., 2010), LRRK2(G2019S) PD brains display neuronal loss and Lewy bodies in the substantia nigra. In some autopsied cases tau-positive neurofibrillary tangles were observed. The isolation of primary fibroblasts from mouse skin was approved by the appropriate institutional governmental agency (Regierungspraesidium Tuebingen, Germany) and performed in accordance with the European Convention for Animal Care and Use of Laboratory Animals. All efforts were made to minimize animal suffering, and to reduce the number of animals for ex-vivo experiments. Swiss 3T3 fibroblast cell culture and SILAC labeling The Swiss 3T3 fibroblast cell line was cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). For stable isotope labeling of amino acids in culture (SILAC), the SILAC Protein Quantitation Kit (Pierce, Rockford, USA) was used according to the manufacturer's instructions. Actively growing 3T3 cells were metabolically labeled by culturing four passages in SILAC DMEM containing ‘heavy’ 13C6 L-lysine and 13C6 15N4 L-arginine or the corresponding ‘light’ amino acids. Thereafter, cells were lysed in a buffer supplemented with 1% Triton X-100. Heavy and light labeled sample pairs were pooled and subjected to immunoprecipitation by using a polyclonal sheep anti-LRRK2 (amino acids 100–500) antibody (Dzamko et al., 2010). Protein identification by quantitative tandem mass spectrometry was performed by Kinaxo (Munich, Germany) as described elsewhere (www.kinaxo.com). Fluorescent resonance energy transfer-based LRRK2–tubulin interaction assay Flp-In™ T-REx™ HEK293 cells inducibly expressing GFP-tagged LRRK2 (Dzamko et al., 2010) were plated on black, clear-bottomed, poly-D-lysine-coated 96-well plates at a density of 30,000 cells per well in complete growth medium (DMEM plus 10% FCS) in the presence of 0.1 μg/ml doxycycline to induce expression of GFP-LRRK2. After 24 h induction, the medium was replaced by fresh growth medium supplemented again with doxycycline and either CellLight® Tubulin-RFP or MAP4-RFP BacMam 2.0 reagents (Invitrogen, Madison, USA). Cells were transduced in triplicates with BacMam virus at a concentration of 25 particles per cell in a volume of 100 μl per well. After overnight transduction, cells were treated with LRRK2 Inhibitor-1 (IN1, 3 μM) for 90 min. The medium was then removed from each well and replaced with PBS. The fluorescent resonance energy transfer (FRET) signal was measured immediately on a Tecan Safire2 by exciting

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GFP at 488 nm and acquiring the emission of RFP at 590 nm. Additionally, total levels of GFP and RFP were also measured. Fluorescent resonance energy transfer-based LRRK2(S935) phosphorylation assay To examine the effect of tubulin destabilizing agents on LRRK2(S935) phosphorylation, a cell-based assay using LanthaScreen® technology (Carlson et al., 2009) was established. In brief, Flp-In™ T-REx™ HEK293 cells inducibly expressing GFP-tagged LRRK2 (Dzamko et al., 2010) were cultured in DMEM (high Glucose; Gibco, Darmstadt, Germany) supplemented with 10% FCS, 1% penicillin/ streptomycin, 15 μg/ml blasticidin (Invitrogen, Darmstadt, Germany) and 100 μg/ml hygromycin B (Invitrogen, Darmstadt, Germany). Cells were treated with 0.1 μg/ml doxycycline (Clonetech, SaintGermain-en-Laye, France) for 24 h to induce LRRK2 expression. Cells were then harvested, resuspended in Opti-MEM-I assay medium (Gibco, Darmstadt, Germany) containing 0.5% FCS, 0.1 μg/ml doxycycline and seeded into 384-well, flat bottom assay plates (Corning, Amsterdam, Netherlands) at a density of 20,000 cells/well. The following day colchicine, vinblastine, taxol (Sigma, Munich, Germany) or Latrunculin A (Invitrogen, Darmstadt, Germany), or vehicle were added to the wells at concentrations ranging from 0.6 nM to 20 μM, and cells were incubated at 37 °C for 90 min. Cells were then lysed in a LanthaScreen cellular lysis buffer (Invitrogen, Darmstadt, Germany) supplemented with phosphatase and protease inhibitor cocktails (Sigma, Munich, Germany). A Terbium-conjugated (Carlson et al., 2009) polyclonal phospho-LRRK2(Ser935) antibody (Dzamko et al., 2010) was added, and assay plates were incubated in the dark at 20 °C for 2 h. Thereafter, the time-resolved fluorescent resonance energy transfer signal between Terbium and GFP was measured by using an EnVision 2102 fluorescence plate reader (Perkin Elmer, Waltham, USA). Fluorescence emission ratios of GFP (520 nm) to Terbium (495 nm) were calculated by using the Wallac EnVision Manager software (Perkin Elmer, Waltham, USA). IC50 values were determined by using XLfit. LRRK2 GTPase assay GTP hydrolysis assays based on thin-layer chromatography were performed as described (Binns et al., 1999; Gillardon, 2009b). Briefly, microtubules assembled from purified tubulin (Cytoskeleton, Denver, USA) (2.0 μM in terms of tubulin concentration) and recombinant full-length LRRK2 (0.2 μM) were pre-incubated with 50 nM [α- 33P] GTP (3000 Ci/mmol) in 20 M Tris, pH 8.0, 1 mM EDTA, 2 mM dithiothreitol, for 5 min at room temperature. The reaction was started by adding MgCl2 to a final concentration of 5 mM and incubating at 37 °C. After 20 min the reaction was stopped by mixing with a solution containing 0.2% (v/v) sodiumdodecylsulfate, 2 mM EDTA, 2 mM dithiothreitol, 0.5 mM GTP, 0.5 mM GDP. The mixture was vortexed and heated to 65 °C for 20 min. 2 μl aliquots were spotted onto thin-layer plates (Merck, Darmstadt, Germany) and separated in a 1 M KH2PO4, pH 3.5, buffer. The plates were exposed to phosphor screens, the screens were imaged on a Typhoon 9400 laser scanner (GE Healthcare, Freiburg, Germany), and the radiolabeled spots were quantified by using the Quantity One software (Bio-Rad, Munich, Germany). Immunocytochemical staining Flp-In™ T-REx™ HEK293 cells inducibly expressing GFP-tagged LRRK2 were seeded into 8 well Permanox Lab Tek Chamber slides (Thermo Scientific, Langenselbold, Germany) in growth medium consisting of DMEM, 10% FCS and 0.1 μg/ml doxycycline. Cells were left to adhere at 37 °C, 5% CO2 overnight. The following day medium was exchanged for fresh medium containing 1 μM LRRK2

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Inhibitor-1, 1 μM Colchicine or solvent. After 2 h of incubation cells were fixed for 10 min in 4% PFA and prepared for microscopic inspection.

then imaged at 10 × magnification and the cell-free space was determined by using the ImageJ software (NIH, Rockville, USA). Analysis of cell adhesion and cell morphology

Culture of primary human fibroblasts Human fibroblasts isolated from skin biopsies of PD patients and age/gender matched healthy donors were cultured in RPMI medium 1640 plus GlutaMax™ (Gibco, Darmstadt, Germany) containing 20% FCS (PAA Laboratories, Coelbe, Germany), 1% Penicillin/Streptomycin (Gibco, Darmstadt, Germany) and 0.3% Fungizone (Sigma, Munich, Germany) at 37 °C and 5% CO2. Fibroblast cultures were split 1:2 whenever cells became confluent. Before the medium change, cells were washed with 1% Fungizone in PBS. Fibroblast migration assay Primary human fibroblasts were suspended in culture medium at 200,000 cells/ml and 70 μl cell suspensions were pipetted into each chamber of the cell culture insert (ibidi, Munich, Germany; www. ibidi.de). The cell culture insert was removed after 8 h leaving a defined cell-free gap of 500 μm. Fourteen hours later cells were fixed and stained with HCS CellMask™ Orange (Invitrogen, Darmstadt, Germany) according to the manufacturer's protocol. Images were taken at 10 × magnification and the cell-free space was determined by using the ImageJ software (NIH, Rockville, USA). Isolation and culture of primary mouse fibroblasts Heterozygous LRRK2(R1441G) transgenic mice on an FVB background were acquired from the Jackson Laboratory (Bar Harbor, USA) (Li et al., 2009). LRRK2(R1441G) transgenic mice exhibit hyperphosphorylated tau protein in brain lysates and phospho-tau immunopositive axonal swellings and dystrophic neurites in the brain sections (Li et al., 2009). LRRK2 knockout mice on a C57BL/6 background were generated as described previously (Gillardon, 2009b). Primary mouse skin fibroblasts were obtained from the male mutant mice and the respective male wildtype controls (all 12 months of age) as described by others (Leiser and Miller, 2010) with some modifications. Earlaps were briefly washed in ethanol, diced, and digested for 6 h in 1.5 ml DMEM (Gibco, Darmstadt, Germany) supplemented with collagenase type II (400 U/ml; Gibco, Darmstadt, Germany) at 37 °C. Tissue was then triturated and transferred to 25 cm2 cell culture flasks containing 5 ml of culture medium (DMEM, 10% heat-inactivated FCS, 1% Penicillin/Streptomycin, 0.3% Fungizone). Cells were left to adhere for at least 12 h before the medium containing remaining tissue fragments was exchanged for fresh culture medium. From this point, the medium was exchanged twice a week and cells were split 1:2 when confluent. Primary mouse fibroblasts were used for the functional studies from passages 3 to 7. Wound healing assay Primary mouse fibroblasts were plated into 12 well plates in a culture medium. Eight hours after seeding the confluent cell layers were wounded by a linear scratch by using a sterile 200 μl pipette tip (Francis et al., 2011; Larson et al., 2010). Thereafter, the culture medium was aspirated and replaced with fresh medium. For LRRK2 inhibition experiments, 3 μM of the selective LRRK2 inhibitor LRRK2-IN1 (Deng et al., 2011) or vehicle was added to the fresh medium. Fourteen hours after scratching, the cells were fixed in 4% paraformaldehyde (PFA) in PBS (Gibco, Darmstadt, Germany), permeabilized by using 0.1% Triton X-100 (Sigma, Munich, Germany) and stained with HCS CellMask™ Orange (Invitrogen, Darmstadt, Germany) according to the manufacturer's protocol. The scratched area was

Adhesion assays using fibroblasts were performed as described by others (Guo et al., 2006) with minor modifications. In brief, 10,000 cells per well were plated in culture medium on a 96-well BD Imager plate (Becton Dickinson Biosciences, Heidelberg, Germany) and incubated at 37 °C. At the indicated time points the medium was removed, cells were washed with prewarmed PBS and adherent cells were fixed with 4% PFA in PBS for 10 min. Nuclei were stained with 1 μM Hoechst 33342 and quantified by using the Becton Dickinson Pathway 435 imager and AttoVision 1.6.2 software (Becton Dickinson Biosciences, Heidelberg, Germany). To analyze the cell size, cell shape, and cell spreading, fibroblasts were plated in culture medium on a 96-well plate at a density of 1000 cells per well. After 1, 4, or 24 h cells were fixed in 4% PFA in PBS and stained with CellMask Orange as described above. Microscopic images were taken at a 5-fold magnification by using the Becton Dickinson Pathway 435 imager. The area, perimeter, circularity (area × 4π/perimeter 2), and aspect ratio (major axis/minor axis) of 10 cells per well from a total of 6 wells per fibroblast cell line were measured by using the ImageJ software. Fibroblast spreading was calculated based on the ratio of not phase-bright cells with processes versus phase-bright round cells 4 h after seeding (Larson et al., 2010). Immunoprecipitation Samples were lysed in ice-cold lysis buffer (50 mM Tris pH 7.5, 0.27 M sucrose, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 5 mM sodium pyrophosphate, 50 mM sodium fluoride, 1% Triton X-100, 0.1% mercaptoethanol, 1 mM benzamidine, 1 mM PMSF) in a Teflon-glass douncer. Lysates were centrifuged at 16,000 ×g for 15 min at 4 °C. The aliquots of the supernatants were taken for protein determination by using the Bio-Rad protein assay (Biorad, Munich, Germany). Three mg of total proteins was used per reaction. Lysate volume was adjusted to 700 μl with lysis buffer. LRRK2 was immunoprecipitated by using a rat monoclonal antibody against LRRK2 (clone 1E11, GSF, Munich, Germany). A pre-immune serum was used as a negative control. Fifteen μl protein G-agarose beads (Protein G Immunoprecipitation Kit, Sigma, SaintLouis, USA) were incubated with five μg antibody or pre-immune serum for 4 h at 4 °C under constant agitation. The beads were pelleted by centrifugation at 16,000 ×g for 1 min at 4 °C. The supernatants were removed, the beads were resuspended in 1 ml of lysis buffer, and washed twice. Thereafter, the protein extracts were added and incubated overnight at 4 °C under constant agitation. Beads were pelleted and washed sequentially with lysis buffer plus 0.5 M NaCl, lysis buffer alone, and PBS. After a final centrifugation step beads were resuspended in 20 μl Laemmli buffer (1% sodium dodecyl sulfate, 100 mM dithiothreitol, 50 mM Tris, pH 7.5) and heated to 55 °C for 15 min. The supernatants were separated from the beads by centrifugation in spin columns (Protein G immunoprecipitation kit, Sigma, Saint-Louis, USA), heated to 75 °C for 10 min and subjected to gel electrophoresis followed by immunoblotting. Gel electrophoresis and immunoblotting For immunoblot analysis, samples were lysed in lysis buffer, vortexed and centrifuged at 16,000 ×g for 7 min. The supernatant was collected and stored at − 80 °C. Proteins were resolved by electrophoresis on 4–12% NuPAGE Bis–Tris gradient gels according to the manufacturer's protocol by using the NuPAGE MOPS running buffer (Invitrogen, Darmstadt, Germany). After their transfer to nitrocellulose membranes (Invitrogen, Darmstadt, Germany), the

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membranes were blocked for 1 h at 20 °C in 5% skimmed milk powder in Tris-buffered saline and 0.1% Tween-20. Membranes were then incubated overnight at 4 °C with rabbit monoclonal antibodies against LRRK2 (MJFF c41-2, MJFF c81-8, Epitomics, Burlingame, USA), a polyclonal sheep antibody against phospho-LRRK2(S935), a monoclonal rabbit antibody against phospho-LRRK2(S910) (Dzamko et al., 2010), a polyclonal rabbit antibody against ERK1/2, or a polyclonal rabbit antibody against phospho-ERK1/2 (Cell Signaling, Frankfurt am Main, Germany). Horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence reagents (ECL kit, GE Healthcare, Freiburg, Germany) were used for detection. All membranes were checked for protein load and protein transfer by using total protein staining (MemCode, Pierce, Rockford, USA) (Aldridge et al., 2008) and beta-actin immunoblotting (1:5000, Sigma, Munich, Germany), respectively. Densitometric analysis of immunoblots was performed by using the Quantity One software (Biorad, Munich, Germany). Statistical analysis For all statistical tests, variances between the groups were compared and the appropriate two tailed t-test was used. Statistical analysis was performed by using the GraphPad Prism 4 (GraphPad Software, La Jolla, USA). Mean values ± SEM are indicated. Results

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In order to confirm the kinase activity-dependent LRRK2-tubulin interaction within living cells, we transiently expressed either RFP-tagged Tubulin or RFP-tagged MAP4 by using BacMam gene delivery in HEK293 cells overexpressing GFP-tagged LRRK2. As shown in Fig. 1, the GFP-to-RFP FRET signal from both microtubuleassociated fusion proteins increased significantly (p b 0.05) when cells were treated with LRRK2-IN1 (3 μM, 90 min). Similar results were obtained by using the structurally-different, less selective LRRK2 inhibitor sunitinib (not shown) (Nichols et al., 2009). The 2to 3-fold assay windows observed here were obtained in an unoptimized assay format where cell-background-specific effects and the role of endogenous tubulin (which is abundant and nonRFP-tagged) could influence the FRET signal. Previous studies demonstrated that the inhibition of GFP-tagged LRRK2 using the nonselective LRRK2 inhibitor H-1152 induces the accumulation of LRRK2 in cytoplasmic fibrillar aggregates that partially colocalized with tubulin immunoreactivity (Dzamko et al., 2010). In the present study, heterogeneous expression of GFP-tagged LRRK2 in stably transfected HEK293 cells was detected and the aggregates of GFP-LRRK2 were already visible in control cells expressing high levels of LRRK2 (Supplementary Fig. 2). After treatment with LRRK2-IN1 cytoplasmic GFP-LRRK2 accumulated into fibrillar structures in some cells as described by others (Deng et al., 2011). After colchicine treatment GFP-LRRK2 accumulated in the cell periphery (Supplementary Fig. 2). Overall, the expression and localization of GFP-tagged LRRK2 in overexpressing HEK293 cells were too heterogeneous for quantitative digital image analysis.

LRRK2 interactome analysis in Swiss 3T3 fibroblasts The endogenous expression of active LRRK2 in the Swiss 3T3 fibroblast cell line has been shown by others (Nichols et al., 2009). For quantitative interactome analysis, we immunoprecipitated endogenous LRRK2 from SILAC-labeled 3T3 fibroblasts and identified interacting proteins by liquid chromatography followed by tandem mass spectrometry (n= 4 biological replicates per group). Both lysine- and arginine-containing peptides were isotopically labeled with an efficiency of approximately 92%. Importantly, short-term treatment of the heavy-labeled 3T3 cells with the highly selective LRRK2 inhibitor LRRK2-IN1 (3 μM, 90 min) (Deng et al., 2011) before immunoprecipitation caused a significant, 18-fold increase in LRRK2 binding to tubulin-beta-2 (normalized Heavy to Light Ratio: 17.9± 1.6; p b 0.02). The pharmacological inhibition of LRRK2 kinase activity in 3T3 cells was confirmed by a decline in LRRK2(S935) phosphorylation (Dzamko et al., 2010) detectable both by mass spectrometry and immunoblotting (Supplementary Fig. 1).

LRRK2(S935) phosphorylation is decreased by microtubule destabilizing drugs Since the pharmacological inhibition of LRRK2 kinase activity promotes its binding to tubulin in cells (shown above), we tested whether the microtubule cytoskeleton might influence LRRK2 kinase activity as shown for other kinases (Archambault et al., 2008; Toya et al., 2011). For medium-throughput compound testing we established a cellular assay for LRRK2(S935) phosphorylation based on LanthaScreen timeresolved FRET technology. Two distinct microtubule destabilizing agents, colchicine and vinblastine (Jordan and Wilson, 2004), were tested in this assay format and a rapid, dose-dependent signal decrease by approximately 40% was observed (Figs. 2A, B), whereas complete dephosphorylation was detected after LRRK2-IN-1 treatment (Fig. 2C). This effect was reproduced in mouse primary fibroblasts treated with 1 μM colchicine for 90 min and immunoblotted for endogenous phospho-LRRK2(S935) (decrease by 42.0% ± 11.1%, n = 6, p b 0.05)

Fig. 1. Effect of LRRK2 inhibition on LRRK2-tubulin protein interaction in living cells. FRET-based signal between GFP-tagged LRRK2 and RFP-tagged tubulin or RFP-tagged MAP4 in HEK293 cells. Cells were treated with the LRRK2 inhibitor LRRK2-IN1 (3 μM, 90 min) or vehicle control (Ctrl, DMSO) following doxycycline-induced expression of GFP-LRRK2 and BacMam-mediated delivery of RFP-fusion proteins. The FRET signal from non-transduced cells (i.e., cells expressing only GFP-LRRK2) was subtracted from all samples. Total fluorescence measurements of GFP and RFP were also acquired to confirm consistent expression across the 96-well plates (data not shown). Bars depict mean ± SEM, * p b 0.05, *** p b 0.001.

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(Fig. 2G). By contrast, neither the microtubule stabilizing drug taxol nor the actin depolymerizing agent Latrunculin A (Spector et al., 1989) affected LRRK2(S935) phosphorylation when tested in a broad concentration range in our cellular FRET assay (Figs. 2D, E). By microscopical inspection shortly before the cell lysis, the colchicine-treated fibroblasts appeared indistinguishable from the vehicle-treated control cultures. Moreover, total LRRK2 immunoreactive bands, phospho-ERK1/2 immunoreactive bands, and MemCode-stained protein bands did not decline on immunoblots of the colchicine-treated cells speaking against an unspecific cytotoxic effect (Fig. 2G). By testing cantharidin

and cantharidic acid in our FRET assay, we identified protein phosphatase 2A as a potent LRRK2(S935) phosphatase (Fig. 2F). Recombinant LRRK2 GTPase activity is increased by co-incubation with microtubules LRRK2 and dynamin are members of a class of G proteins that are regulated by homodimerization (Gasper et al., 2009). Since the GTPase activity of dynamin is stimulated to high levels by binding to microtubules in vitro (Binns et al., 1999; Shpetner and Vallee, 1992), we

Fig. 2. Effect of microtubules on LRRK2 enzymatic activity. Dose-dependent decrease of LRRK2(S935) phosphorylation after incubation of HEK293 cells with tubulin depolymerizing compounds colchicine (A) and vinblastine (B) for 90 min compared to cells incubated with vehicle control (DMSO) only. (C) Complete dephosphorylation of LRRK2 was achieved by incubation with the small molecule LRRK2 inhibitor LRRK2-IN1. (G) Western blot analysis of colchicine-treated (lane 2) mouse fibroblasts also showed a significant decrease in phospho-LRRK2(S935) compared to DMSO-treated (lane 1) controls. Levels of total LRRK2, total ERK1/2, and phospho-ERK1/2 remain unchanged. (D) Stabilization of microtubules by incubation with Taxol or (E) depolymerization of the actin cytoskeleton by Latrunculin A treatment had no effect on LRRK2(S935) phosphorylation. (F) Treatment with the protein phosphatase 2A inhibitor cantharidine resulted in a 4-fold increase LRRK2(S935) phosphorylation. LRRK2(S935) phosphorylation was quantified by using the FRET-based signal between GFP-tagged LRRK2 and a Terbium-conjugated phospho-specific antibody. (H) Effect of microtubules on LRRK2 GTPase activity. Recombinant LRRK2 was co-incubated with microtubule-depleted assembly buffer (lanes 1, 2), microtubules stabilized by taxol (lanes 3, 4), or microtubules incubated at 4 °C to induce depolymerization (lanes 5, 6). Hydrolysis of [α-33P]GTP was analyzed by thin layer chromatography. Representative autoradiogram depicts a 2-fold increase in 33P-GDP levels in samples containing LRRK2 and microtubules compared to LRRK2 alone. Only the background levels of 33P-GDP are detectable in microtubule-depleted assembly buffer (lanes 7, 8), taxol-stabilized microtubules (lanes 9, 10), and cold-treated microtubules (lanes 11, 12) in the absence of LRRK2.

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performed similar GTPase assays by using recombinant full-length LRRK2 (Gillardon, 2009a). LRRK2 was co-incubated with taxolstabilized microtubules, cold-treated microtubules, or microtubuledepleted assembly buffer. The hydrolysis of [α-33P]GTP was analyzed by thin layer chromatography. Microtubule preparations alone did not exhibit significant GTPase activity under these assay conditions. In two independent experiments however, LRRK2 co-incubation with microtubules lead to a significant (pb 0.05, t-test), 2-fold increase in GTPase activity compared to LRRK2 only (Fig. 2H). Collectively, these data provide strong evidence for LRRK2-tubulin interaction and support the hypothesis that microtubules may act as a scaffold for LRRK2 structural organization and emanating enzymatic activity. Cell migration is affected in LRRK2(G2019S) human fibroblasts The microtubule cytoskeleton plays an essential role in cell migration, cell division, and cell shape (Gardel et al., 2010; Parisiadou and Cai, 2010). To assess a potential influence of LRRK2 on cell migration, human primary fibroblasts were confluently plated in cell culture inserts with a defined cell-free space of 500 μm. Fourteen hours after the removal of the culture insert, the cell-free space that had been invaded by migrating fibroblasts was measured. LRRK2(G2019S) mutant fibroblasts showed a trend (p > 0.05) towards an enhanced migration (Fig. 3) (remaining cell-free space: G2019S 38.4% ± 4.5%; Control 47.0% ± 4.7%). Three different LRRK2(G2019S) and four different healthy donor fibroblast lines were used. Four biological replicates per cell line were analyzed. Additionally, cell size, cell shape (perimeter, circularity, aspect ratio), and cell spreading did not significantly differ between mutant and control fibroblasts. Similar LRRK2

Fig. 3. Migration of human LRRK2(G2019S) fibroblasts in culture. (A) Histogram showing that human LRRK2(G2019S) fibroblasts exhibit a trend towards faster invasion of the cell-free space created by a cell culture insert compared to corresponding wildtype control cultures. Immunoblot analysis demonstrating that fibroblasts isolated from Parkinson's disease patients carrying the LRRK2(G2019S) mutation (right immunoreactive band) and from healthy control donors (left band), respectively, express LRRK2 protein at comparable levels. MemCode total protein stain and beta-actin immunoblotting were used as loading control and for normalization, respectively. (B) Representative images of LRRK2(G2019S) mutant fibroblasts (right panel) and wild-type control cultures (left panel) are shown 14 h after the removal of the cell culture insert. Bars depict mean±SEM.

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protein levels were detected in mutant and wildtype human fibroblasts by immunoblotting (Fig. 3). Lesioning-induced cell migration is enhanced in LRRK2(R1441G) mouse fibroblasts Next we investigated the influence of LRRK2 on the migration of primary fibroblasts when the cell layer is damaged by a mechanical scratch, a well established cell culture model of wound healing (Larson et al., 2010). To assess a more homogenous cell population, we performed the scratch assay by using dermal fibroblasts isolated from LRRK2(R1441G) transgenic mice (Li et al., 2009) and their wildtype littermates. Consistent with our findings in human fibroblast cultures, mouse fibroblasts carrying the PD-linked LRRK2(R1441G) mutation showed a significantly (pb 0.005) enhanced invasion of the cell-free space (Fig. 4) (remaining cell-free space: Wildtype 21.5% ± 2.4%; R1441G 10.4%± 1.9%). In contrast, the lesion-induced migration of fibroblasts from LRRK2 knockout mice was significantly (pb 0.05) slower compared to the corresponding wildtype mice (remaining cell-free space: wildtype 24.4% ± 4.1%; knockout 37.0% ± 4.1%). In all experiments, the fibroblasts were isolated from 3 individual mice per genotype, and at least 3 biological replicates each were tested. The effect size of LRRK2 mutation/deficiency on fibroblast migration in the scratch assay is comparable to other gene mutations that are known to affect the microtubule cytoskeleton (Francis et al., 2011; Larson et al., 2010). Treatment of mouse fibroblasts with the highly selective LRRK2 inhibitor LRRK2-IN1 slowed down cell migration in LRRK2 expressing fibroblasts, but not in LRRK2-deficient fibroblasts clearly indicating that LRRK2 influences fibroblast motility (Fig. 4E). LRRK2IN1 inhibits MAPK7 and DCLK1 at higher concentrations (Deng et al., 2011) which might contribute to its effects on cell migration. However, our gene chip database indicates that neither MAPK7 nor DCLK1 is expressed in the skin samples or fibroblast cultures further supporting a role of LRRK2 in fibroblast migration. LRRK2IN1 completely blocked the phosphorylation of LRRK2 at Ser-910 and Ser-935 (Fig. 4F) indicating the pharmacological inhibition of LRRK2 kinase activity (Dzamko et al., 2010). Total LRRK2 protein levels in dermal fibroblasts of LRRK2(R1441G) transgenic mice did not significantly differ from the wildtype controls (111% ± 8%, n = 7, p = 0.2) as detected by immunoblotting by using a monoclonal antibody (MJFF c41-2) that recognizes both human and endogenous mouse LRRK2 (Fig. 4F). Immunoblotting using a monoclonal antibody (MJFF c81-8) that preferentially recognizes human LRRK2 confirmed the expression of human LRRK2(R1441G) in primary fibroblasts from transgenic mice. Since cell adhesion is an essential factor influencing cell migration, a standard cell adhesion assay was performed (Guo et al., 2006). The number of fibroblasts that were adherent at different time points after seeding and washing did not differ between the genotypes demonstrating that LRRK2 does not influence cell adhesion (Supplementary Fig. 3). Similarly, cell size, cell shape (perimeter, circularity, aspect ratio), and cell spreading did not significantly differ between the genotypes. Finally, an Alamar blue assay was performed to assure that differences in fibroblast migration are not due to alterations in cell viability or proliferation rate. Mutant and wildtype fibroblasts did not show any differences in this assay over 24 h (data not shown). Discussion We and others have shown that LRRK2 interacts with tubulin and promotes tubulin polymerization which is enhanced by the G2019S mutation (Gandhi et al., 2008; Gillardon, 2009a). Consistently, mutations in LRRK2 impair neurite outgrowth, a process that is highly dependent on a dynamic cytoskeleton (Dächsel et al., 2010; MacLeod et al., 2006; Melrose et al., 2010; Parisiadou et al., 2009; Wang et al., 2008). LRRK2 also interacts with actin and actin-related proteins

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Fig. 4. Lesioning-induced migration of cultured mouse LRRK2(R1441G) fibroblasts. (A) Primary dermal fibroblasts isolated from LRRK2(R1441G) transgenic mice exhibit a significantly faster closure of the cell-free space created by scratching the confluent cell layer compared to wildtype (WT) control cultures. (B) Fibroblasts from LRRK2 gene knockout (KO) fibroblasts show a delayed closure compared to wildtype controls. Representative cell culture images are shown in C and D. (E, F) Treatment with the highly selective LRRK2 inhibitor LRRK2-IN1 (3 μM) completely blocks LRRK2(S935) phosphorylation (F) and significantly slows the migration of wildtype fibroblasts and LRRK2(R1441G) fibroblasts compared to the corresponding vehicle-treated controls. Migration of LRRK2 knockout fibroblasts is not significantly impaired by LRRK2-IN1 (E). Bars depict mean ± SEM, * p b 0.05, ** p b 0.01. MemCode total protein stain and beta-actin immunoblotting were used as loading control and for normalization, respectively.

(Meixner et al., 2011). Although Parkinson's disease is a disorder of the central nervous system, LRRK2 protein expression and kinase activity have been shown in various non-neuronal cells, e.g., Swiss 3T3 fibroblasts and RAW macrophages (Dzamko et al., 2010). Here, we demonstrate that LRRK2 protein is also expressed and active in primary fibroblasts making them a useful model to investigate alterations in the cytoskeleton of fibroblasts isolated from PD patients carrying LRRK2 mutations. Reorganization of microtubules is a key event in directed cell movement (Gundersen and Bulinski, 1988), and LRRK2 binding to microtubules (Gandhi et al., 2008; Gillardon, 2009a, 2009b) might contribute to the alterations in cell migration presented here. We observed that a small molecule LRRK2 inhibitor significantly increases LRRK2–tubulin interaction in cells, and that microtubule destabilization by vinblastine or colchicine leads to a rapid decrease in LRRK2(S935) phosphorylation pointing to a decline in LRRK2 kinase activity (Dzamko et al., 2010). An interaction with microtubules requires the LRRK2 ROC/GTPase domain which is autophosphorylated on several residues (Gandhi et al., 2008; Greggio et al., 2009; Kamikawaji et al., 2009). The inhibition of LRRK2 kinase activity may cause decreased autophosphorylation of the ROC domain residues and increased binding to microtubules. It should be mentioned however, that overexpression of kinase-inactive GFP-tagged LRRK2 in cell culture reduces the formation of fluorescent filamentous structures suggesting reduced LRRK2-microtubule association (Kett et al., 2012). Differences in experimental design (e.g., overexpression of an artificial kinase-dead LRRK2 variant versus pharmacological

inhibition of endogenous LRRK2 kinase activity) make a direct comparison difficult. Additionally, kinase-inactive LRRK2 variants are prone to misfolding and degeneration (Rudenko et al., 2012). Interestingly, the regulation of Polo kinase activity by sequestration to microtubules was shown in proliferating cells (Archambault et al., 2008), and differences in the localization and function of kinaseactive versus kinase-inactive Aurora A on microtubules were recently reported (Toya et al., 2011). It may thus be hypothesized that similar mechanisms contribute to the spatial and temporal regulations of LRRK2. Moreover, our in vitro data indicate that the binding of the LRRK2 ROC/GTPase domain to microtubules increases GTP hydrolysis. Similar findings have been reported following the co-incubation of dynamin GTPase and microtubules (Binns et al., 1999; Shpetner and Vallee, 1992). Microtubules provide a scaffold for dynamin oligomerization which regulates dynamin GTPase activity. Dynamin and LRRK2 are members of a class of G proteins that are regulated by homodimerization (Gasper et al., 2009), and microtubules may provide a scaffold for self-assembly for both GTPases. Unexpectedly, cold-treated microtubules also increased LRRK2 GTPase activity suggesting that microtubules were not completely depolymerized or that binding to tubulin monomers may be sufficient to enhance LRRK2 GTPase activity. In the present study, cultured primary fibroblasts from LRRK2(R1441G) transgenic mice migrated more rapidly, whereas fibroblasts from LRRK2 knockout mice migrated more slowly compared to the corresponding wildtype control cultures. One out of

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three human LRRK2(G2019S) fibroblast cell lines migrated as slow as the control fibroblasts, while the other two LRRK2(G2019S) cell lines migrated significantly faster. The slowly migrating fibroblast line was isolated from an LRRK2 mutation carrier that had a long-term history of cigarette smoking. The effects of nicotine and other constituents of cigarette smoke on cell migration are well described (Fang and Svoboda, 2005; Peña et al., 2011; Wong et al., 2004). Thus, it may be speculated that long-term nicotine exposure may underlie the LRRK2-independent changes in the migration of this fibroblast line. Basically, cell migration represents a four step process: advance of the leading edge, formation of focal adhesions, generation of tractile forces, and disassembly of focal adhesions at the rear of the cell. While the actin cytoskeleton is considered to be the key player at the leading edge of a migrating cell, dynamic microtubules regulate the retraction of the rear end (WehrleHaller and Imhof, 2003). To advance the leading edge, polymerization of actin is required, and disassembly of focal adhesions is achieved by repeated targeting by polymerizing microtubules (Kaverina et al., 1999). LRRK2 binds to both actin and tubulin and modulates their polymerization in vitro (Gillardon, 2009b; Meixner et al., 2011). Moreover, binding to tubulin and modulation of polymerization are influenced by LRRK2 kinase activity (Gillardon, 2009b; present study) further substantiating the role of LRRK2 in cytoskeletal dynamics and cell migration. This physiological role is detectable both in neuronal cells (neurite outgrowth) and in non-neuronal cells (fibroblast motility) in culture indicating that LRRK2 acts within a conserved pathway in different cell types. Moreover, the LRRK2-related kinase Roco2 promotes pseudopod extension and cell motility in the slime mold Dictyostelium discoideum (Kicka et al., 2011). Unexpectedly, mutant LRRK2 decreases neurite outgrowth (Dächsel et al., 2010; MacLeod et al., 2006), whereas the motility of fibroblasts is increased (present study) suggesting that different effector proteins are involved. Wound healing following isolation of skin biopsies has not been monitored in the present study, but biologically relevant differences in LRRK2 mutation carriers are unlikely. In vivo, postmitotic neurons with their long processes may be more susceptible to cytoskeletal alterations caused by LRRK2 mutations favoring a neurodegenerative phenotype. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.nbd.2012.12.019. Acknowledgments We thank Dario Alessi, Jeremy Nichols, and Nicolas Dzamko for providing LRRK2 overexpressing HEK293 cells and antibodies against LRRK2. References Aldridge, G.M., Podrebarac, D.M., Greenough, W.T., Weiler, I.J., 2008. The use of total protein stains as loading controls: an alternative to high-abundance singleprotein controls in semi-quantitative immunoblotting. J. Neurosci. Methods 172, 250–254. Archambault, V., D'Avino, P.P., Deery, M.J., Lilley, K.S., Glover, D.M., 2008. Sequestration of Polo kinase to microtubules by phosphopriming-independent binding to Map205 is relieved by phosphorylation at a CDK site in mitosis. Genes Dev. 22, 2707–2720. Berger, Z., Smith, K.A., LaVoie, M.J., 2010. Membrane localization of LRRK2 is associated with increased formation of the highly active LRRK2 dimer and changes in its phosphorylation. Biochemistry 49, 5511–5523. Binns, D.D., Barylko, B., Grichine, N., Atkinson, M.A.L., Helms, M.K., Jameson, D.M., et al., 1999. Correlation between self-association modes and GTPase activation of dynamin. J. Protein Chem. 18, 277–290. Brockmann, K., Groger, A., Di, S.A., Liepelt, I., Schulte, C., Klose, U., et al., 2011. Clinical and brain imaging characteristics in leucine-rich repeat kinase 2-associated PD and asymptomatic mutation carriers. Mov. Disord. 26, 2335–2342. Carlson, C.B., Robers, M.B., Vogel, K.W., Machleidt, T., 2009. Development of LanthaScreen ™ cellular assays for key components within the PI3K/AKT/mTOR pathway. J. Biomol. Screen. 14, 121–132. Dächsel, J.C., Behrouz, B., Yue, M., Beevers, J.E., Melrose, H.L., Farrer, M.J., 2010. A comparative study of Lrrk2 function in primary neuronal cultures. Parkinsonism Relat. Disord. 16, 650–655.

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