Viral construction and production
Adeno-associated viral vectors (AAVs). AAV6 viral particles were obtained by encapsidation of AAV2 recombinant genomes into serotype 6 AAV capsids as described previously [36]. Briefly, viral particles were produced by co-transfection of HEK-293T cells with (1) an adenovirus helper plasmid (pXX6-80), (2) an AAV packaging plasmid carrying the rep2 and cap6 genes, and (3) a plasmid encoding a recombinant AAV2 genome containing the transgene expression cassette. Seventy-two hours following transfection, viral particles were purified and concentrated from cell lysates and supernatants by ultracentrifugation on an iodixaniol density gradient followed by dialysis against PBSMK (0.5 mM MgCl2 and 1.25 mM KCl in PBS). The concentration of vector stocks was estimated by real-time-PCR following the method described by Aurnhammer et al. [37] and expressed as viral genomes per ml of concentrated stocks (Vg/ml). AAVs coding for human ∆LRRK2 (WT, G2019S, and G2019S plus D1994A mutation, i.e. "kinase dead"), α-synA53T, and GFP under the PGK1 (mouse phosphoglycerate kinase) promoter were produced.
Lentivirus. DNA sequences encoding GFP and the C-terminal part of human LRRK2 (kinase,K; ROC-COR-kinase, RCK; RCK plus the WD40 domain, called hereafter “DLRRK2”) were synthesised and inserted into the self-inactivated vector (SIN) backbone containing the WPRE element (W) and the murine PGK promoter. We generated lentivirus vectors LV-GFP, and LV-DLRRK2 coding for the WT form or G2019S forms of the fragments. Viral particles were produced as described elsewhere [38]. All the SIN vectors were pseudotyped with VSV glycoprotein G. Briefly, the viral particles were produced in HEK-293T cells by a four plasmid transient transfection system [39]. The supernatant was collected 48 hours later and filtered. High-titre stocks were obtained by ultracentrifugation. The pellet was re-suspended in 1% BSA in PBS, frozen and stored at −80°C. Particle content of the viral batches was determined by ELISA for the p24 antigen (Gentaur, Paris, France). LV-DLRRK2 vectors were used at a concentration of 100 ng/μl p24.
Stereotaxic injection
Experiments in rats. Adult Sprague-Dawley rats (Charles River Laboratories), weighing ∼250 g (Charles River, Saint Germain sur l’Arbresle, France), were housed under a 12-h light/dark cycle with ad libitum access to food and water, in accordance with European Community (Directive 2010-63/EEC) and French (Code Rural R214/87-130) regulations. Experimental procedures were approved by the local ethics committee and registered with the French Research Ministry (committee #44, approval #12-100, and APAFIS#1372-2015080415269690v2). For stereotaxic injections, the animals were deeply anaesthetized with 4% isoflurane, followed by a mixture of ketamine (75 mg/kg) and xylazine (5 mg/kg), and placed in a stereotaxic frame. Recombinant AAVs were injected unilaterally into the SNpc, at the following stereotaxic coordinates: +3.4 mm anterior to the interaural zero and ±2.0 mm lateral to bregma, at a depth of -7.8 mm relative to the skull, with the tooth bar set at -3.3 mm. We injected 4 µl of virus at a concentration of 2.5x1010 Vg per site for single injections and 2.5x1010 Vg of each vector for co-injections for a total of 5x1010 Vg per site, with a 34-gauge blunt-tipped needle linked to a 10-μl Hamilton syringe by a polyethylene catheter at a rate of 0.25 μl/min using an automatic pump (CMA-4004). The needle was left in place for five minutes and then slowly withdrawn.
Experiments in mice. Adult male C57BL/6J mice (each weighing 25 g; Charles River, Saint Germain sur l’Arbresle, France) were used for lentiviral infections. Mice were housed (five/cage in enriched environment) in a controlled-temperature room maintained on a 12 h-hour light/dark cycle. Food and water were available ad libitum. All animal studies were conducted in accordance with French regulations (EU Directive 86/609 – French Act Rural Code R 214-87 to 131). The animal facility was approved by veterinary inspectors (authorization no. A 92-032-02) and complies with the Standards for Humane Care and Use of Laboratory Animals of the Office of Laboratory Animal Welfare (OLAW – n°#A5826-01). All procedures were approved by the ethics committee and the Research Ministry [no. 2015060417243726vl (APAFIS#770)].
LV-Htt171-82Q was used at a concentration of 150 ng/µl of p24. LVs coding for LV-DLRRK2 forms (WT, G2019S or the dead kinase G2019S/D1994A were used at a concentration of 100 ng/µl of p24.
For intracerebral infections, animal were anesthetized (100 mg/kg ketamine and 10 mg/kg xylazine). Local analgesia included subcutaneous lidocaine (5 mg/kg). A total volume of 2 µl of LV or AAV suspension was injected into the mouse striatum, as described [40], at the following stereotaxic coordinates: 1.0 mm anterior and 2.0 mm lateral to bregma, at a depth of 2.7 mm from the dura, with the tooth bar set at 0.0 mm. The mice were then left for one to two hours in a heated (30°C) ventilated box, until they had woken up and recovered fully from anesthesia. Post-surgery analgesia included acetaminophen (Doliprane) in drinking water for 48 h (1.6 mg/ml).
Tissue processing
For all procedures, rats were first deeply anesthetized by isoflurane inhalation, followed by the intraperitoneal injection of a lethal dose of sodium pentobarbital.
Rats were transcardially perfused with 300 ml 4% paraformaldehyde (4% PFA) in phosphate buffer saline (PBS - 0.1 M phosphate buffer, 9 g/L NaCl) at a rate of 30 ml/min. After perfusion, the brain of each rat was quickly removed and immersed in ice-cold 4% PFA/PBS for at least 24 h, before transfer to 15% sucrose in PBS for 24 h and then 30% sucrose in PBS the next day, for cryoprotection. The brains were then cut into 40-μm sections on a freezing microtome (SM2400, Leica, Germany). Serial sections of the striatum and midbrain were stored in antifreeze solution (30% glycerol/30% ethylene glycol in PBS) and stored at -20°C until use.
Mice were deeply anesthetized by the intraperitoneal injection of sodium pentobarbital solution (50 µg per gram body weight). They were then transcardially perfused with 100 ml 4% PFA in PBS at a flow rate of 8 ml/min. The brains of the animals were removed, post-fixed overnight in the same solution, then cryoprotected by immersion in 30% sucrose in PBS for 36 hours. Free-floating 30-µm serial coronal sections from throughout the striatum were collected with a freezing sliding microtome. Brain slices were placed in a storage solution (30% glycerol, 30% ethylene glycol in PBS) and stored at -20°C before use.
Immunohistological analysis and quantification
Immunohistochemistry
Sections were removed from the antifreeze solution and washed in PBS. Endogenous peroxidase activity was quenched by transferring them to 1% H2O2 and incubation for 30 min at room temperature (RT) and washing them three times with PBS for 10 min each. The sections were then blocked by incubation with 4.5% normal goat serum for 30 min in PBS-T (0.2% Triton X-100 in PBS) and then incubated overnight with primary antibody in 3% normal goat serum in PBS-T at 4°C with gentle shaking.
For histological evaluation using rat brain sections, the following primary antibodies were used for the present study: anti-tyrosine hydroxylase (TH) antibody: MAB318 clone LNC1, Merk-Millipore, 1:3000; anti-hemagglutinin tag (HA), Covance clone 11, 1:1000; anti-human α-synuclein, syn 211, 1:1000; anti-phospho-α-synS129, ab51253, Abcam, 1:5000]. The next day, the sections were removed from the primary antibody solution, washed three times, and incubated for 1 h at RT with the appropriate biotinylated secondary antibody in PBS-T (Vector Laboratories, Burlingame, CA, USA, 1:1000). The sections were then washed and incubated with ABC complex solution in PBS-T (1:250, reagents A and B combined in a 1:1 ratio, Vector Laboratories) for 1 h.
For histological evaluation using mouse brain sections, we used the anti-NeuN (1:2000, mouse, Millipore, Molsheim, France), anti-HA (1:500, mouse, clone 11 Covance, Princeton, NJ) and the EM48 (Chemicon, MAB5374, Temecula, CA, USA; 1 : 1000) antibodies. Sections were rinsed three times in PBS and then incubated with the appropriate anti-IgG biotinylated antibody (Vector Laboratories, Burlingame, CA) at a dilution of 1:5000, for one hour. Staining was visualized by adding avidin-biotinylated peroxidase (Vector Laboratories, Burlingame, CA) for one minute. For NeuN immunostaining, we used the MOM immunodetection kit (Vector Laboratories, Burlingame, CA).
The rat and mouse sections were then incubated with DAB for 30 s to 1 min and after dehydration mounted on slides in Eukitt mounting medium.
Cell counting
Optical fractionator sampling was carried out on a Zeiss AxioPlan microscope. Midbrain dopaminergic neurons were outlined on the basis of TH immunolabelling with reference to a coronal atlas of the rat brain (Paxinos and Watson, 6th edition). TH-positive cells were counted by unbiased stereology in the entire SNpc and the number of positive neurons per section was calculated using the Mercator Software (Explora Nova, France). We placed 100 × 100 μm grids in a systematically random manner, 80 × 80 μm apart, with a 3-µm offset from the surface of the section. Quantification was performed on 12 serial sections spaced by 200 µm, corresponding to the entire SNpc.
The phosphorylation of α-syn on S129 (p-synS129) was evaluated by counting the number of p-synS129-positive neurons in the SNpc using stereology methods. The SNpc was delimited by Nissl staining and the grids (250 x 250 µm) placed, with a space of 100 x 100 µm. Quantification was performed on six serial sections spaced by 400 µm, corresponding to the entire SNpc. In the striatum, a threshold was applied to select only the p-synS129-positive neurons by immunostaining and quantification performed on three slices, corresponding to the beginning, middle, and end of the striatum.
Immunofluorescence
The procedure used was similar to that for immunohistochemistry, but without the incubation in 1% H2O2. The primary antibodies used for the immunofluorescence procedure were the same as previously described (IBA1, Wako, 1:1000). Sections were first incubated with the primary antibody overnight at 4°C. The next day, they were incubated with a fluorescent secondary antibody (Alexa Fluor 594-labeled goat anti-rabbit IgG or Alexa Fluor 488-labeled goat anti-rabbit IgG (1:1000, Life Technologies)) for 1h at RT. Sections were then washed and incubated overnight at 4°C with another primary antibody. Finally, they were incubated with a second fluorescent secondary antibody (Alexa Fluor 488-labeled goat anti-mouse IgG or 594-labeled goat anti-mouse IgG (1:1000, Life Technologies)) for 1h at RT. The sections were stained with DAPI, washed, and mounted in a fluorescence mounting medium. Images were acquired with a laser confocal microscope (SP8, Leica, Germany) or an epifluorescence microscope (DM6000, Leica, Germany).
Thioflavin-S staining
A double-staining protocol was used to verify that accumulation of positive p-synS19 inside cells could colocalize with aggregated form of a-syn. The immunostaining procedure for p-synS19 and DAPI staining was performed on floating sections before the Thioflavin-S (Thio-S) staining. Floating sections were washed in PBS and mounted on Superfrost Plus slides. Slides were place in holders and dive into 70% EtOH and 80% EtOH, for 1 min each. Then, Slides were incubated in Thio-S diluted at 1% in distilled water for 7 min. The Thio-S solution must be protected from light, filtrated before use, and should be stored at 4°C. Then, slides were washed in 80% EtOH, 70% EtOH and distilled water for 1 min each before being coverslipped with the fluorescence-mounting medium.
Colocalization
The percentage of co-localization between ΔLRRK2 and α-syn was determined by counting the number of cells co-expressing both ΔLRRK2 and α-syn proteins divided by the number of cells expressing α-syn alone. Images were acquired with a laser confocal microscope (SP8, Leica, Germany). On the same acquisitions, the levels of ΔLRRK2 and α-syn proteins were evaluated on three coronal sections in the SNpc. Twenty cells co-expressing both ΔLRRK2 and α-syn proteins were delineated per animal using image J software and the mean fluorescence intensity in the red and in green channels (corresponding to ΔLRRK2 and α-syn proteins, respectively) was measured in each cell.
Fluorescence intensity measurement
Striatal dopaminergic innervation at 15 weeks was quantified by measuring the fluorescence intensity of TH-immunoreactive terminals on three coronal striatal sections. The sections were observed by epifluorescence microscopy at a magnification of 63X and the fluorescence intensity determined using MorphoStrider software (Explora Nova, France).
Microglia area measurement
The area occupied by microglia was evaluated by confocal microscopy at a magnification of 20X in the dorso-medial part of the striatum and in the SN pars reticulata. A threshold was applied and the area of 20 microglia cells measured per acquisition. Three acquisitions per animal were used.
Image analysis of lesion area.
Observation of sections and calculation of the surface of lesion were performed using a Leica
DM6000 equipped with a motorized stage and an automated image acquisition and analysis system (Mercator software, Explora Nova, La Rochelle, France). The area covered by striatal lesions resulting from LV-Htt171-82Q infection was delineated manually using a 10x objective by identifying the border of the lesion on each coronal brain section and the corresponding area was calculated. The volume of the striatal lesion was determined by the Cavalieri method and the number of EM48-positive inclusions was determined as previously described with an inter-section distance of 210 µm (i.e. we used one in every seven sections) [40–42]. Automatic detection of EM48-positive objects was performed using the Mercator software to count the number of aggregates on the entire cross-sectional area of the striatum and to measure the size of all the detected EM48-positive objects. Objects with an apparent cross-sectional area exceeding 5 µm2 were reliably detected with this method.
Statistical analysis
Normality of data distribution was tested using the Shapiro-Wilk test and homogeneity of variance was tested with Levene’s test using a commercially available software (Statistica, 13.0; Statsoft Inc., Tulsa, Oklahoma, USA). When normality and homogeneity of variance were met, unpaired Student’s t-test was used for pairwise comparisons between groups. For comparisons of more than two groups, one-way ANOVA for multiple comparisons was carried out, with Fisher's post hoc PLSD test. In the cases where assumption of normality and/or homogeneity of variance were not met, non-parametric tests where applied: Mann-Whitney and Kruskall-Wallis for comparison of 2 and more groups, respectively. The annotations used to indicate the level of significance are as follows: *p < 0.05, **p < 0.01, ***p < 0.001.