Background
Mutations in the
leucine-rich repeat kinase 2 (
LRRK2) gene cause familial Parkinson’s disease (PD) and variations around the
LRRK2 locus increase risk for sporadic PD, indicating that abnormal LRRK2 function contributes to disease pathogenesis [
1,
2]. Various pathogenic LRRK2 mutations have been described which all seem to converge on causing increased phosphorylation of select kinase substrates in intact cells [
3], indicating that LRRK2 kinase activity may represent a therapeutic PD target. However, the downstream event(s) associated with abnormal LRRK2-mediated substrate phosphorylation remain unknown.
LRRK2 has been reported to be involved in a number of intracellular vesicular trafficking events [
4‐
9] and also plays an important role in neurite outgrowth/cell polarity and cell migration [
4,
10‐
14]. In dividing cells, pathogenic LRRK2 is known to impair neuronal precursor cell division in vitro and adult neurogenesis in vivo, deficits which may at least in part contribute to some of the age-dependent non-motor symptoms of PD patients [
15‐
18]. LRRK2 is also highly expressed in various non-neuronal tissues, suggesting that it may play more general cellular role(s) shared amongst distinct cell types. Whilst displaying a broad subcellular distribution, LRRK2 can also partially localize to a centrosomal compartment [
19]. Interestingly, a recent phosphoproteomics study has conclusively identified a subset of Rab proteins including Rab8a as LRRK2 kinase substrates [
3]. Rab8a is a small GTPase localized to various intracellular compartments including Golgi, pericentrosomal recycling endosomes and centrosomes, and is known to regulate centrosome-related events [
20‐
22]. However, the cellular consequences of LRRK2-mediated Rab8a phosphorylation are currently unknown.
Proper centrosome positioning is important for maintenance of cell polarity and directed migration [
23‐
25]. The centrosome also plays an important role during the cell cycle, with centrosome duplication and separation allowing for the formation of a bipolar spindle required for appropriate chromosome segregation [
26]. Finally, the centrosome plays a crucial role for membrane trafficking events to and from the pericentrosomal recycling endosome, and conversely, the pericentrosomal recycling endosome can modulate centrosomal maturation processes [
20,
27], indicating that these two compartments cooperate to regulate various key cellular processes.
In the present study, we report that pathogenic LRRK2 causes alterations in centrosome positioning which are associated with deficits in neurite outgrowth and polarized cell migration. In dividing cells, pathogenic LRRK2 causes centrosomal cohesion deficits which are also observed in two distinct cell types derived from LRRK2-PD patients as compared to healthy controls, and are reverted by distinct LRRK2 kinase inhibitors. Furthermore, centrosomal cohesion and polarity deficits are observed when co-expressing wildtype LRRK2 along with wildtype but not phospho-deficient Rab8a mutant, and are associated with a kinase activity-dependent increase in the centrosomal accumulation of phosphorylated Rab8a. Finally, the centrosomal cohesion defects mediated by pathogenic LRRK2 are largely abolished upon RNAi of Rab8a. Altogether, these data indicate that pathogenic LRRK2 causes centrosomal alterations via Rab8a phosphorylation.
Methods
Cell culture and transfections
SH-SY5Y cells stably expressing GFP, flag-tagged wildtype LRRK2, or flag-tagged G2019S-mutant LRRK2 were cultured as described [
28,
29]. Briefly, cells were grown in full medium (Dulbecco’s modified Eagle’s medium containing high glucose and 15% fetal bovine serum, non-essential amino acids, 50 μg/ml gentamycin (Life Technologies) and 200 μg/ml hygromycin B (Invivogen), and subcultured at a ratio of 1:6 twice a week. Transfection of cells was carried out at 80% confluence with 0.4 μg DNA and 1.5 μl Lipofectamine 2000 (Invitrogen) per well of a 24-well plate in 200 μl OptiMEM. Five h later, cells were changed into full medium, and passaged the following day at a ratio of 1:5 onto coverslips, followed by fixation and staining 72 h after transfection. For differentiation, 10,000 cells were plated onto coverslips in 24-well plates and grown in full medium for 24 h to allow for proper attachment. Cells were then changed to medium containing 3% fetal bovine serum and 10 μM retinoic acid (Sigma), and differentiated during 5 days, with medium changed every 48 h.
HEK293T cells were cultured as described [
5,
8] and transfected at 80% confluence with 2 μg of LRRK2 constructs (and 200 ng of Rab constructs where indicated) and 6 μl of LipoD293 (SignaGen Laboratories) per well of a 6 well plate for 5 h in full medium. Cells were split to 20% confluence the following day, and processed for immunocytochemistry or Western blot analysis 24 h later.
HELA cells were cultured as previously described [
8], Cells were plated in six-well plates at 40% confluence, and transfected the following day with 400 ng of GFP-tagged Rab8a constructs and 5 μl of Lipofectamine 2000 according to manufacturer’s instructions in full medium overnight. The following day, cells were split at a 1:4 ratio, plated onto coverslips, and processed 24 h later.
Primary human skin fibroblasts established from skin biopsies taken from five age- and sex-matched healthy control and five PD patients with the G2019S mutation [
8], with informed consent and ethical approval, were grown in IMDM and 10% fetal bovine serum, with media changed every two days. Cells were subcultured at a ratio of 1:4, and seeded at equal densities on coverslips. All analyses were carried out on passages 3–8, and no passage-dependent differences were observed.
For lymphoblast generation, three healthy control and three PD patients due to the G2019S LRRK2 mutation were recruited at the Movement Disorders Unit of Lille University Medical Center (Lille, France, CPP Nord-Ouest 2008/09), with the two groups matched according to age and gender. Blood samples were collected in BD Vacutainer CPT Cell Preparation Tubes containing sodium heparin (Le Pont-de-Claix, France). The peripheral blood mononuclear cells (PBMCs) were collected and processed according to supplier’s recommendations. Lymphocytes were immortalized by infection with Epstein-Barr virus (EBV) as described [
30]. Briefly, cell lines were established from freshly isolated lymphocytes or from cryopreserved lymphocytes using standard EBV transformation protocols that include cell separation by gradient centrifugation and lymphocyte growth enhancement by the mitogen phytohemagglutinin. Cell lines were maintained in RPMI 1640 medium supplemented with 20% fetal bovine serum, L-glutamine, 20 units/ml penicillin and 20 μg/ml streptomycin in T75 flasks in 5% CO
2 at 37 °C. Cells were maintained at a density of 10
6 cells/ml, with cell density monitored every other day using trypan blue staining.
Where indicated, cells were treated with nocodazole (200 nM, 2 h, SigmaAldrich), brefeldin A (5 μg/ml, 2 h, SigmaAldrich), or the indicated concentrations of MLi2 (MRC PPU, Dundee, UK), LRRK2-IN1 (obtained through the MJFF) or GSK2578215A (Tocris) before fixation.
DNA constructs and site-directed mutagenesis
GFP-tagged human LRRK2 constructs were obtained from Addgene. Where required, mutations were introduced by site-directed mutagenesis (QuikChange, Stratagene). The GFP-G2019S-K1906 M kinase-dead LRRK2 construct was generated by site-directed mutagenesis introducing K1906 M on top of the G2019S construct, and the identity of constructs verified by sequencing of the entire coding region. DNA was prepared from bacterial cultures grown at 28 °C using a midiprep kit (Promega) according to manufacturer’s instructions. Human GFP-tagged Rab8a, Rab8a-Q67L and Rab8a-T22 N, as well as human GFP-tagged Rabin8 were obtained from Addgene. mRFP-tagged or triple-flag (3xFlag)-tagged Rab8a constructs were generated using Gibson Assembly Master Mix (New England Biolabs). Rab8a-T72A, Rab8a-T72D and Rab8a-T72E mutant constructs were generated by site-directed mutagenesis (QuikChange, Stratagene). The siRNA-resistant forms of mRFP-Rab8a and mRFP-Rab8a-T72A were generated by introducing 3 silent mutations into the target sequence of the seed region of the Rab8a siRNA (Silencer Select Rab8a (Ambion, ThermoFisher, ID s8679, nr 4,390,824). Specifically, the original sequence of the mRFP-Rab8a plasmids (5´-GCAAGAGAATTAAACTGCA-3′) was mutated to 5´-GCAAGAGAATTAAGTTACA-3′). Identity of all constructs was verified by sequencing of the entire coding region. HttQ74-GFP was a generous gift from D. Rubinsztein (Cambridge University, UK).
Immunofluorescence and laser confocal imaging
HEK293T cells were fixed using 2% paraformaldehyde (PFA) in PBS for 10 min at room temperature, followed by permeabilization in MeOH for 2 min at − 20 °C. Cells were subsequently incubated in PBS containing 50 mM NH
4Cl for 10 min [
31], and then permeabilized with 0.2% Triton-X100/PBS for 20 min. For determination of the size of pericentrin-positive structures and for phospho-Rab8a staining, the MeOH and NH
4Cl steps were omitted. HELA cells expressing GFP-tagged Rab8a constructs were fixed using 4% PFA in PBS for 15 min at room temperature, permeabilized with 0.5% Triton-X100/PBS for 5 min, rinsed in PBS, and mounted with mounting medium containing DAPI. SH-SY5Y cells were fixed using 2% paraformaldehyde (PFA) in PBS containing 4% sucrose for 20 min at room temperature, followed by permeabilization with 0.2% Triton-X100/PBS for 20 min. Primary fibroblasts were fixed with 2% paraformaldehyde (PFA) in PBS for 10 min at room temperature, followed by permeabilization with 0.2% Triton-X100/PBS for 20 min.
For lymphoblast immunocytochemistry, 13 mm diameter coverslips were placed into 24-well plates and coated with Cell-Tak Cell and Tissue Adhesive solution (Corning) following manufacturer’s protocols. After 20 min at room temperature, the solution was removed, coverslips were rinsed twice with distilled water and air-dried. Lymphoblast cells (200′000 per well) were added to dry coated coverslips, and cells attached by slight centrifugation at 690 g for 10 min at room temperature (without brake). Lymphoblast cells were fixed using 2% PFA in PBS for 20 min at room temperature, followed by permeabilization with 0.2% Triton-X100/PBS for 20 min at room temperature.
After fixation and permeabilization, coverslips were blocked for 1 h with 0.5% (
w/
v) BSA in 0.2% Triton-X100/PBS (blocking solution), followed by incubation with primary antibodies in blocking solution overnight at 4°C. Primary antibodies included rabbit polyclonal anti-pericentrin (Abcam, ab4448, 1:1000), mouse monoclonal anti-pericentrin (Abcam, ab28144, 1:1000), mouse monoclonal anti-γ-tubulin (Abcam, ab11316, 1:1000), mouse monoclonal anti-c-Myc (Sigma, clone 9E10, M4439 1:500), mouse monoclonal anti-flag (Sigma, clone M2, 1:500), rabbit polyclonal anti-Rab8a (Millipore, ABC423, 1:1000), mouse anti-Golgin97 (Molecular Probes, A-21270, 1:100), rabbit polyclonal anti-β-COP (Invitrogen, PA1–061, 1:750), and mouse monoclonal p230/Golgin-245 (Becton Dickinson, 611,280, 1:400). The sheep anti-Rab8a and anti-T72-phospho-Rab8a antibodies have been previously described (MRC PPU, S969D and S874D, respectively) [
3]. For the sheep anti-Rab8a antibody, the NH
4Cl step was omitted, and for the sheep anti-T72-phospho-Rab8a antibody, both the MeOH and NH
4Cl steps were omitted. Sheep antibodies were used at a 1:50 dilution, and the anti-T72-phospho-Rab8a antibody was used in the presence of a 10-fold molar excess of dephospho-peptide, or of phospho-peptide where indicated. Importantly, all double- and triple-immunocytochemistry involving sheep antibodies were performed sequentially, with the sheep antibodies employed first.
Secondary antibodies included Alexa 405-conjugated goat anti-mouse or goat anti-rabbit, Alexa 488-conjugated goat anti-mouse or goat anti-rabbit, Alexa 594-conjugated goat anti-mouse or goat anti-rabbit, Alexa 633-conjugated goat anti-mouse or goat anti-rabbit (Invitrogen, 1:1000), Alexa 488-conjugated donkey anti-sheep (Invitrogen, 1:1000) or Alexa 594-conjugated donkey anti-sheep (Abcam, 1:1000). As indicated, cells were either mounted using mounting medium containing DAPI (Vector Laboratories), or incubated with TO-PRO-3 Iodide (642/661) (Invitrogen, 1:1000) for 3 min, followed by washes in PBS before mounting in ProLong Gold Antifade mounting medium (Invitrogen).
Images were acquired on a Leica TCS-SP5 confocal microscope using a 63X 1.4 NA oil UV objective (HCX PLAPO CS). Images were collected using single excitation for each wavelength separately and dependent on secondary antibodies (405 nm UV diode and a 415–455 nm emission band pass; 488 nm Argon Laser line and a 510–540 nm emission band pass; 543 HeNe Laser line and a 600–630 nm emission band pass; 633 HeNe Laser line and a 640–670 nm emission band pass). GFP-tagged proteins were excited with 488 nm Argon Laser line and a 500–530 nm emission band pass, and RFP-tagged proteins with 543 nm HeNe Laser line and a 560–590 nm emission band pass, respectively. DAPI was excited with the 405 nm UV diode and a 430–480 nm emission band pass, and TO-PRO with 633 nm HeNe Laser line and a 650–675 nm emission band pass, respectively.
10–15 image sections of selected areas were acquired with a step size of 0.5 μm, and z-stack images analyzed and processed using Leica Applied Systems (LAS AF6000) image acquisition software. The same laser intensity settings and exposure times were used for image acquisition of individual experiments to be quantified. For quantification of centrosome size, a circle was drawn around individual centrosomes and area quantified using image acquisition software as described above, where mature centrosomes were around 1 μm2, and immature centrosomes around 0.5 μm2. Centrosomes were scored as being separated when the distance between their centers was > 1.5 μm (for HEK293T and SH-SY5Y cells) as analyzed by ImageJ software. For fibroblasts, the mean distance between separated centrosomes in control cells was 2.25 ± 0.2 μm (mean ± s.e.m., n = 10 cells), and centrosomes scored as being separated when the distance was > 2.5 μm. Using equivalent criteria for lymphoblasts, centrosomes were scored as being separated when the distance was > 1.3 μm. In all cases, mitotic cells were excluded from this analysis.
Quantification of the phospho-Rab8a signal in SH-SY5Y cells was done over non-processed and non-saturated images acquired during the same day with the same laser intensities. Quantification was performed with Leica Applied Systems (LAS AF6000) image acquisition software. Circular ROIs of 2.2 μm width and 2.2 μm height were set on top of the centrosomal signal, and mean intensity from the phospho-Rab8a signal obtained from at least 50 cells per condition and experiment. Background signal was subtracted in all cases by placing the ROI in a different aleatory place within the same cell. Similarly, to quantify phospho-Rab8a signals in lymphoblasts, a circle of 3 μm diameter was drawn around individual centrosomes as assessed by pericentrin staining, and the phospho-Rab8a fluorescence intensity from around 30–50 individual cells quantified from maximal intensity projections using Leica Applied Systems (LAS AF6000) image acquisition software.
Most experiments were quantified by two independent observers, and some experiments were quantified by a third observer blind to condition, with similar results obtained in all cases.
Knockdown of Rab8a by RNA interference
HEK293T cells were seeded in 6-well plates at 30–40% confluence one day prior to transfection such that they were at a confluence of 70–80% the following day. They were transfected with 2 μg of GFP-LRRK2 DNA and 25 nM siRNA using 4 μl of jetPRIME Transfection Reagent (Polyplus-Transfection SA, no 114–15) in 200 μl jetPRIME buffer. The mix was incubated for 15 min at room temperature and added to 2 ml of full medium per well of a 6-well plate overnight. For knockdown experiments in the presence of both GFP-LRRK2 and mRFP-Rab8a or mRFP-Rab8a-T72A (sensitive or resistant to siRNA, respectively), cells were transfected with 50 nM of the indicated siRNA using 4 μl of jetPRIME Transfection Reagent as described above. Four hours later, media was replaced and cells transfected with 2 μg of the indicated LRRK2 constructs and 200 ng of the indicated Rab8a constructs and 6 μl of LipoD293 (SignaGen Laboratories) per well of a 6-well plate overnight in full medium. In all cases, cells were passaged 24 h later and processed for Western blot analyis or immunocytochemistry 48 h after transfection. RNAi reagents included Silencer Select Negative Control no. 1 siRNA (Ambion, ThermoFisher, cat. nr 4,390,843) and Silencer Select Rab8a (Ambion, ThermoFisher, ID s8679, cat. nr 4,390,824).
For determination of Rab8a levels, cells were washed in PBS and resuspended in cell lysis buffer (1% SDS in PBS containing 1 mM PMSF, 1 mM Na3VO4, 5 mM NaF). Extracts were sonicated, boiled and centrifuged at 13,500 rpm for 10 min at 4 °C. Protein concentration of supernatants was estimated using the BCA assay (Pierce), and 30 μg of extracts resolved by SDS-PAGE and analyzed by Western blotting.
Wound healing and cell migration assays
For wound-healing assays, 40,000 SH-SY5Y cells were seeded on each side of 35 mm insert-containing dishes (IBIDI) and grown to confluence in full medium. The wound (500 μm diameter) was generated by removing the insert, cells were gently washed three times in full medium, and phase contrast images acquired every 10 min for 15 h on a Leica TCS-SP5 confocal microscope using a 10X objective (C-PLAN 10.0 × 0.22 POL) and 1.3X zoom. Overall wound healing migration speed was calculated as average speed of the cell front using ImageJ software analysis. For single cell tracking, individual cells in the first row facing the wound were tracked (ImageJ manual tracking plugin) until reaching the middle of the wound [
32], and individual cell migration speed, directionality (D) and forward migration index in Y (FMI Y) calculated from at least 30 cells per condition and experiment using the Chemotaxis and Migration Tool (IBIDI).
For determination of cell polarity, cells were processed for immunocytochemistry 4 h after generating the wound using anti-pericentrin and anti-golgin97 antibodies as described above. The first row of cells facing the wound was analyzed, and cells were scored as polarized when located in a 90° sector emerging from the center of the nucleus and facing the wound edge [
33]. Basal levels of expected random orientation of 25% were confirmed by analyzing centrosome orientation immediately after generating the wound.
Cell extracts and western blotting
HEK293T cells were collected 48 h after transfection, washed in PBS and resuspended in cell lysis buffer (1% SDS in PBS containing 1 mM PMSF, 1 mM Na3VO4, 5 mM NaF). Extracts were sonicated, boiled and centrifuged at 13,500 rpm for 10 min at 4 °C. Protein concentration of supernatants was estimated using the BCA assay (Pierce), and 40 μg of extracts resolved by SDS-PAGE and analyzed by Western blot, using a rabbit polyclonal anti-GFP antibody (ab6556, 1:3000, Abcam), phospho-S935-LRRK2 antibody (ab133450, Abcam, 1:500), a mouse monoclonal anti-myc antibody (clone 9E10, 1:1000, Sigma), a sheep polyclonal anti-Rab8a (MRC PPU, S969D, 1:200), a sheep polyclonal anti-phospho-Rab8a (MRC PPU, S874D, 1:200), a mouse monoclonal anti-flag antibody (clone M2, 1:2000, Sigma) and a mouse monoclonal anti-tubulin antibody (clone DM1A, 1:10,000, Sigma) as loading control.
For lymphoblast cell extracts, 106 cells were centrifuged at 1030 g for 10 min at 4 °C. The pellet was resuspended in 100 μl of lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Triton-X100) containing protease and phosphatase inhibitor cocktails (SigmaAldrich), and incubated for 30 min on a rotary wheel at 4 °C. Cell extracts were centrifuged at 13,500 rpm for 5 min at 4 °C, and supernatants quick-frozen in liquid N2 and stored at − 80 °C. Protein concentration of supernatants was estimated using the BCA assay (Pierce), and 20 μg of extracts resolved by SDS-PAGE and analyzed by Western blot, using a rabbit polyclonal phospho-S935-LRRK2 antibody (ab133450, Abcam, 1:500), a mouse monoclonal anti-LRRK2 antibody (UC Davies/NIH NeuroMab clone 75–253, 1:1000), a sheep polyclonal anti-Rab8a (MRC PPU, S969D, 1:200) and a mouse monoclonal anti-tubulin antibody (clone DM1A, 1:10,000, Sigma) as loading control. Some Westerns were developed with ECL reagents (Roche), and a series of timed exposures to ensure that densitometric analyses were performed at exposures within the linear range. Quantification was performed using QuantityOne software (BioRad). Western blotting of phospho-S935 and total LRRK2 in extracts from lymphoblasts was performed with ECL Prime Western Blotting Detection Reagent (GEHealthcare), and analysis in Amersham Imager 600 (GEHealthcare). The majority of Western blotting was performed according to the protocol described by LI-COR for Near-Infrared Western Blot Detection. In all cases, incubation with primary antibodies was performed overnight at 4 °C, and secondary antibodies were incubated for 1 h at RT. For analysis of Rab8a or phospho-Rab8a levels using this technology, a rabbit monoclonal anti-Rab8a antibody (ab188574, Abcam, 1:1000), and a rabbit polyclonal phospho-Rab8a antibody were employed. Blots were imaged using an Odyssey CLx system, and quantification was done using the instrument’s Image Studio software.
Immunoprecipitation of GFP-Rabin8
HEK293T cells were cultured as described and co-transfected at 80% confluence with 600 ng of GFP-Rabin8 and 200 ng of flag-tagged Rab8a constructs using 6 μl of LipoD293 (SignaGen Laboratories) per well of a 6 well plate overnight in full medium. The following day, cells were split into 100 mm tissue culture plates, and were collected 48 h after transfection. Cells were washed in PBS, followed by resuspension in 1 ml of lysis buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 2 mM MgCl2, 1% Triton-X100, 1 mM DTT, protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail 3 (Sigma)), and incubated on a rotary wheel for 1 h at 4 °C. Lysates were subsequently spun at 13′000 rpm for 10 min at 4 °C, and protein concentration of supernatants estimated by BCA assay (Sigma), with 1 mg of total protein subjected to immunoprecipitation with a rabbit polyclonal anti-GFP antibody (Abcam, Ab 6556, 1 μg per sample). Lysates were incubated with antibody for 2 h at 4 °C, followed by addition of protein G Sepharose Fast Flow (Amersham) and incubation overnight at 4 °C. The next day, beads were washed three times with lysis buffer, and bound proteins eluted with Laemmli sample buffer and heating at 95 °C for 4 min prior to separation by SDS-PAGE and Western blotting as indicated above, using a mouse monoclonal anti-GFP antibody (Roche, 1:1000) or a mouse monoclonal anti-flag antibody (clone M2, 1:1000, Sigma), respectively.
Cell transfection, protein purification and in vitro kinase assays
HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum, penicillin (100 units/ml) and streptomycin (100 μg/ml) (Life Technologies) at 37 °C and 5% CO2. 3xFlag-LRRK2 (wildtype and G2019S mutant) and 3xFlag-Rab8a (wildtype and T72A mutant) were transiently transfected using linear polyethylenimine (PEI, Polyscience) with a 1:2 DNA:PEI ratio. The transfection mixes were prepared by dissolving 40 μg of DNA in 1 ml of OPTI-MEM with 80 μl of PEI (40 μM final). Mixes were incubated at room temperature for 20 min and subsequently added to HEK293T cells previously plated on a 15 cm2 Petri dish.
Seventy-two hours after transfection, cells were resuspended in 1 ml of lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 2.5 mM Na4P2O7, 1 mM beta-glycerophosphate, 1 mM Na3VO4, supplemented with protease inhibitor mixture (Sigma-Aldrich) and 1% (v/v) Tween-20). Samples were left on ice for 30 min, and cell lysates collected after centrifugation at 18000×g for 35 min at 4 °C. Supernatants were collected and incubated overnight with 40 μl of Anti-Flag M2 Affinity Gel beads (Sigma-Aldrich) at 4 °C with mild agitation. Beads were pelleted and washed with 1 ml of five different wash buffers (WB): WB1 (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 1% (v/v) Tween-20) twice, WB2 (20 mM Tris-HCl pH 7.5, 350 mM NaCl, 1% (v/v) Tween-20) twice, WB3 (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% (v/v) Tween-20) twice, WB4 (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% (v/v) Tween-20) twice and WB5 (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.02% (v/v) Tween-20) once. All 3xFlag-tagged proteins were eluted in elution buffer (25 mM Tris-HCl pH 7.5, 5 mM beta-glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, 10 mM MgCl2 supplemented with 0.007% Tween-20, 150 ng/μl 3xFlag peptide (Sigma-Aldrich) for subsequent in vitro kinase assays.
For experiments in which Rab8a was loaded with specific guanine nucleotides, affinity resin bound-protein was washed as above, rinsed in loading buffer (20 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.02% (
v/v) Tween-20) and incubated with an excess (200 μM) GDP or GTPγS for 30 min at 30 °C with slight agitation. Nucleotide exchange was stopped and excess nucleotides removed by rinsing beads three times in kinase buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl
2, 2 mM dithiothreitol (DTT), 0.02% (v/v) Tween-20, 5 mM beta-glycerophosphate, 0.1 mM Na
3VO
4) [
34] followed by elution with kinase buffer supplemented with 150 ng/μl 3xFlag peptide.
For in vitro kinase assays, purified wildtype or T72A-mutant 3xFlag-Rab8a proteins eluted in kinase buffer, with and without nucleotide loading, were incubated with wildtype or G2019S mutant 3xFlag-LRRK2 at a ratio of 50:1 (Rab8a:LRRK2). Reactions were kept at 30 °C for 1 h in the presence of ATP-γ33P (1 μCi/reaction) and 2.5 μM cold ATP and then stopped by adding Laemmli buffer (100 mM Tris-HCl pH 6.8, 4% (w/v) SDS, 200 mM DTT, 20% (v/v) glycerol and Bromophenol Blue). Incorporated 33P was detected by autoradiography with a Phospho-Imager system (Cyclone, Perkin-Elmer). The same membranes were stained with Coomassie Blue or probed with an anti-Flag antibody for total protein loading, and quantification performed using ImageJ software.
Rab8a GTP binding and GTP retention assays
HEK293T cells were transfected with 3xFlag-Rab8a wildtype or mutant plasmids as indicated and 24 h later cells were lysed in buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol and protease inhibitor cocktail (Roche) for 30 min on ice. Lysates were centrifuged (10 min, 20,000×g), supernatants were precleared with Easy view Protein G agarose (Sigma-Aldrich) for 30 min at 4 °C, followed by incubation with anti-Flag M2 agarose beads (Sigma-Aldrich) for 1 h at 4 °C with mild agitation. Proteins bound to beads were washed 4 times with 25 mM Tris-HCl pH 7.5, 400 mM NaCl, and 1% Triton X-100.
For GTP or GDP binding assays, equal amounts of wildtype or mutant 3xFlag-Rab8a fusion proteins bound to anti-Flag M2 agarose beads (Sigma-Aldrich) were washed twice with Buffer A (20 mM Tris–HCl pH 7.5, 100 mM NaCl, 5 mM MgCl2, 1 mM NaH2PO4, 2 mM DTT) and incubated overnight on ice in Buffer A containing 0.1 μM 3H–GTP or 3H–GDP. Beads were then washed twice in Buffer A to remove unbound nucleotides, added to Bio-safe II (RPI) scintillation cocktail, and binding quantified using scintillation counting for H3 (Tri-Carb 2810TR scintillation counter, Perkin Elmer).
To assay GTP or GDP retention, agarose-bound Rab8a proteins were incubated in Buffer A containing 0.1 μM 3H–GTP or 3H–GDP overnight on ice and washed twice with Buffer A to remove unbound nucleotide. Subsequently, proteins were incubated in Buffer A containing a 100-fold excess of unlabeled GTP or GDP for 0, 15, 30 or 60 min, and shaking at 37 °C. After each time point, Rab8a proteins were washed twice with Buffer A, and retained 3H–GTP or 3H–GDP bound to proteins was quantified using scintillation counting. The amount of 3H–GTP or 3H–GDP bound at 15, 30 and 60 min for each sample was calculated as a fraction of initial binding.
Co-immunoprecipitation
HEK293FT cells transfected with 3xflag-Rab8a plasmids were lysed in IP buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.3% Triton X-100, 10% Glycerol, and protease inhibitor cocktail (Roche)) for 30 min on ice. Lysates were centrifuged at 4 °C for 10 min at 20,000 g and supernatants were further cleared by incubation with Easy view Protein G agarose beads (Sigma) for 30 min at 4 °C. After agarose bead removal by centrifugation, lysates were incubated with anti-flag M2 agarose beads (Sigma) for 1 h at 4 °C with mild agitation. Beads were washed three times with IP wash buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, 10% Glycerol) and eluted in buffer containing 20 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl2 and 150 ng/μl of 3xflag peptide by shaking for 15 min at 4 °C. Each co-immunoprecipitation was repeated in 3 independent experiments, and samples analyzed by Western blotting using an anti-GDI1/2 antibody (1:2000, Life Technologies, 710,300) or an anti-flag antibody (1:500, Sigma, F3165).
Mass spectrometry
Purified wildtype and mutant Rab8a proteins were separated on 4–20% SDS-PAGE, stained with Coomassie brilliant blue staining (Thermo Scientific) and a band corresponding to a ~ 50 kDa protein was excised. Protein identification was performed using MASCOT.
Statistical analysis
All data are expressed as means ± s.e.m. Unless otherwise noted, data were analyzed by one-way ANOVA with Tukey’s post-hoc test, and p < 0.05 was considered significant. Statistical details to all experiments can be found in the figure legends. ***p < 0.005; **p < 0.01; *p < 0.05.
Discussion
Here, we provide evidence that mutant LRRK2 causes centrosomal alterations in both dividing and non-dividing cells. Centrosomal deficits were observed with distinct pathogenic LRRK2 mutants and in distinct cell lines, as well as in two different patient-derived cell types. The effects were reverted by various specific and structurally distinct LRRK2 kinase inhibitors, suggesting that LRRK2 causes centrosomal alterations in a kinase activity-dependent manner. Together, the data indicate that endogenous pathogenic LRRK2 protein impacts upon the same cellular pathway, and highlight the potential applicability of such cellular readout as possible pharmacodynamic assay in clinical studies employing LRRK2 kinase inhibitors.
Our data confirm that Rab8a is robustly phosphorylated on T72 by LRRK2 in vitro, and further indicate that phosphorylation is largely independent on nucleotide-bound status. As compared to wildtype or phosphorylation-deficient Rab8a, phospho-mimetic Rab8a mutants did not display altered nucleotide binding/dissociation, but were unable to interact with GDI1/2 or with Rabin8. However, when transiently expressed in cells, the phospho-mimetic Rab8a mutants were not able to mimick the localization or action of phosphorylated Rab8a in intact cells. Both the negative charge and the size of the ionic shell produced by aspartate or glutamate substitutions are different from those of a phosphorylated residue at physiological pH. In addition, if the phosphorylation site serves as a recognition signal for a binding partner, phospho-mimetic mutants cannot bind as not fitting into the binding pocket [
54,
55]. Therefore, and as observed here, phospho-mimetic mutations often fail to reproduce the changes to a protein caused by its phosphorylation.
Whilst clearly a LRRK2 kinase substrate, previous studies have shown that the stoichiometry of Rab8a phosphorylation in intact cells is low, with the phospho-state-specific Rab8a antibody only detecting phosphorylated protein by Western blotting techniques upon co-transfection with exogenous Rab8a [
3]. By immunocytochemistry, we could detect accumulation of endogenous phosphorylated Rab8a in HEK293T cells expressing high levels of exogenous pathogenic LRRK2, or in SH-SY5Y cells stably expressing LRRK2 upon co-transfection with exogenous Rab8a. In lymphoblasts which express both high levels of endogenous LRRK2 and of Rab8a, a pericentrosomal/centrosomal phospho-Rab8a signal was detectable, but there were no statistically significant differences between G2019S LRRK2 mutant and control lymphoblasts. These data are consistent with the interpretation that the centrosomal effects mediated by pathogenic LRRK2 are, at least in part, mediated by Rab8a and Rab8a phosphorylation, even though currently available tools do not allow us to detect endogenous phospho-Rab8a in G2019S mutant lymphoblasts.
The LRRK2-mediated phosphorylation of Rab8a does not seem to cause a mere loss-of-function phenotype, as knockdown of Rab8a in control cells did not cause centrosomal cohesion deficits. Conversely, pathogenic LRRK2 expression caused centrosomal defects accompanied by an accumulation of endogenous phosphorylated Rab8a in a pericentrosomal/centrosomal compartment, and increasing the amount of phosphorylated Rab8a by coexpression of wildtype LRRK2 with wildtype but not phospho-deficient Rab8a caused centrosomal cohesion deficits and centrosomal polarity defects in a kinase activity-mediated manner as well. Finally, RNAi of Rab8a in pathogenic LRRK2-expressing cells caused a significant reversal of centrosomal cohesion deficits. Whilst future studies will be required to address whether the centrosomal cohesion deficits remaining upon Rab8a knockdown are mediated by remnant Rab8a, by other functionally redundant Rab protein LRRK2 kinase substrates such as Rab8b or Rab10 [
3], or indeed by other, non-Rab-related LRRK2 substrates, these results indicate that a significant part of the phenotype is dependent on the presence of Rab8a. Altogether, our data are consistent with a model whereby pathogenic LRRK2 kinase activity causes an abnormal accumulation of phosphorylated Rab8a in a pericentrosomal/centrosomal compartment with various downstream effects on centrosome functioning as described here.
Centrosomal deficits were also observed in non-dividing cells. Differentiated SH-SY5Y cells expressing pathogenic LRRK2 displayed a deficit in cell polarity as evidenced by a significant increase in the amount of cells with abnormal positioning of the centrosome with respect to the longest neurite and a decrease in overall differentiation capability, consistent with previous reports that pathogenic LRRK2 interferes with neurite outgrowth [
4,
6,
11,
15]. Mutant LRRK2 also caused deficits in cell polarity associated with an impairment in directional cell migration. Whilst both positive and negative effects of mutant LRRK2 on cell migration have been previously described [
12‐
14], this may relate to cell type-specific differences in the position of the centrosome with respect to the leading edge of migratory cells [
56]. In addition, impaired adult neurogenesis in mutant LRRK2-expressing cells seems to be accompanied by a reduction in the number of newly generated neurons migrating to the olfactory bulb [
15]. As deficits in these processes may contribute to early clinical signs of PD such as anosmia, it will be interesting to determine whether these migrational deficits are due to a lack of proper centrosome positioning and cell polarization. Since Rab8a has also been implicated in neurite formation and polarized membrane transport [
57,
58], and since polarity deficits were also observed when co-expressing wildtype but not phospho-deficient Rab8a with wildtype LRRK2, it is tempting to speculate that the effects on neurite outgrowth and directional migration may also involve altered Rab8a-mediated processes. Apart from its abnormal phosphorylation by LRRK2 [
3], Rab8a has also been shown to be phosphorylated by a PINK1-mediated mechanism [
59], and mutations in PINK1 are known to cause autosomal-recessive PD. In addition, Rab8a has been shown to modulate α-synuclein-mediated aggregation and toxicity in cellular and animal models of PD [
60]. Thus, whilst the precise mechanism(s) remain to be further determined, there is increasing evidence linking abnormal Rab8a function with PD pathogenesis.
The link between centrosomal alterations as described here and its relevance to PD remains unclear. Deficits in adult neurogenesis have been reported to contribute to the age-dependent non-motor symptoms of PD patients [
17,
18], and it will be interesting to determine whether alterations in centrosomal cohesion parallel the deficits in cell growth of neuronal precursor cells derived from LRRK2 PD patients in vitro, or the impairment of adult neurogenesis in mutant LRRK2-transgenic mice in vivo [
15,
16]. In addition, as centrosomal alterations are frequently associated with cancer, the changes reported here may further contribute to the reported increased cancer risk in LRRK2 PD patients [
61]. Importantly, centrosomes are the major microtubule-nucleating centers within a cell, and proper centrosome functioning and orientation ensure appropriate microtubule-mediated vesicular trafficking. Interestingly, pathogenic LRRK2 has been linked to alterations in microtubule stability [
62] and to intracellular vesicular trafficking steps including the autophagy, endolysosomal and retromer-mediated trafficking pathways [
4‐
9,
19,
63]. Therefore, the centrosomal alterations described here may contribute to the observed alterations in microtubule-mediated membrane trafficking pathways which have been directly related to the pathobiology of PD [
64,
65].
In summary, we here provide evidence that pathogenic LRRK2 causes deficits in centrosome positioning and cohesion in a manner dependent on kinase activity and Rab8a phosphorylation. Our findings hopefully provide a useful framework for future studies aimed at determining the relative contribution of centrosomal deficits to the various cellular alterations described to be relevant in the context of PD pathogenesis and amenable to LRRK2 kinase inhibitor-mediated strategies.