Statement on animal health
All experiments were carried out in accordance with the authorization guidelines of the Swiss federal and cantonal veterinary offices for the care and use of laboratory animals. Studies described in this report were approved by the Swiss cantonal veterinary office and performed according to Novartis animal license numbers 2063, 2382 and 95.
Generation of LRRK2 gene-modified mice
Cloning of the LRRK2-G2019S targeting vector
LRRK2 genomic sequences containing exon 41 were amplified from Balb/c mouse genomic DNA and subcloned. Site-directed mutagenesis was performed to modify codon GGG (Gly) to TCG (Ser), resulting in the desired G2019S mutation in exon 41. A loxP element was inserted into a BglI restriction site 5′ of exon 41. Modified genomic sequences were excised by HindIII and subcloned into the HindIII restriction site of pRAY2 (Accession No. U63120). LRRK2 genomic sequences downstream of exon 41 were amplified as described above and subcloned using ClaI/NotI into pRAY2 containing the cloned modified exon, resulting in the LRRK2 target plasmid.
Cloning of the LRRK2-D1994S targeting vector
LRRK2 genomic sequences flanking exon 41 were amplified from Balb/c mouse genomic DNA and subcloned. Overlapping polymerase chain reaction (PCR) was performed to modify codon GAC (Asp) to TCC (Ser), resulting in the desired D1994S mutation in exon 41. Modified genomic sequences were subcloned into the HpaI/XhoI-digested vector pRAY2-LRRK2 5′ arm. LRRK2 genomic sequences downstream of exon 41 were amplified as described above and subcloned using ClaI/NotI into pRAY2 containing the cloned modified exon, resulting in the plasmid LRRK2-D1994S target. Subcloned sequences were compared with sequences available from the Ensembl database (Ensembl Gene ID ENSMUSG00000036273).
Transfection of mouse embryonic stem cells
Embryonic stem (ES) cells were cultured in 6 cm dishes containing primary X-ray-inactivated embryonic fibroblast cells from DR4 mice (TgN(DR4)1Jae (61
). ES cells were transfected by electroporation of 20 µg of Not
I-digested pLRRK2 target plasmid. Transfected ES cells were selected for neomycin resistance with 0.2 mg/ml geneticin (G418, Invitrogen #10131-019). Ten days after transfection, G418-resistant ES cell clones were isolated and analyzed by PCR (nested PCR: first PCR: sense primer: Neo-4: TCC TCG TGC TTT ACG GTA; antisense primer: LRRK2-rev1: CCC GTC AGT AAG TGA GCT; second PCR: sense primer: Neo-5: CTA TCG CCT TCT TGA CGA A; antisense primer: LRRK2-rev2: CTT TGA CCT TGG GAT GAA) for homologous recombination. An additional PCR was performed to control for the presence of the lox
P element 5′ of LRRK2
exon 41 (sense primer: LRRK2-geno.for: GTG TTA AAG CTC CAG TTG CCT; antisense primer: LRRK2-geno.rev: GCC AGA GAG TAC ACA GGA GGT).
To eliminate the flp recombinase recognition site (FRT)-flanked neomycin cassette in ES cells, cells were co-transfected with the plasmids pEGFP-N1 (Accession No. U55762) and pCAG-FLP at a ratio of 1:4 using Lipofectamine™ 2000 (Invitrogen #11668-027) as transfection reagent. Two days after transfection, enhanced green fluorescent protein-expressing ES cells were collected by fluorescence-activated cell sorting (FACS) (FACSCalibur, Becton Dickinson) and cultured for a further 6 days in normal media. Forty-eight single clone cells were selected and split onto two plates. Cell culture was performed on a 96-well plate with feeder (0.5 × 106 inactivated feeder cells/96-well plate) in the ES medium with or without G418 (0.2 mg/ml G418). PCR to control for Flp-mediated FRT recombination and excision of the neomycin cassette were performed (sense primer: LRRK2-geno.for: GTG TTA AAG CTC CAG TTG CCT; antisense primer: FRT(-neo)rev: TTT CGA ACC CGG GGA AGT TC).
Southern blot analysis
Homologous recombination of the targeting plasmid into the LRRK2 locus was verified by southern hybridization. For this purpose, genomic DNA from ES cell colonies was digested with the restriction enzyme EcoRV, blotted and hybridized with a labeled neomycin probe. In brief, 5 μg of genomic DNA were digested with 30 units of the restriction enzyme and separated on a 0.9% agarose gel. After denaturation, the DNA was blotted on a Hybond N+ membrane (Amersham #RPN203B) followed by UV crosslinking. Hybridization with the 32P-labeled DNA probe (Rediprime II Random prime labeling kit, Amersham #RPN1633) was performed in Perfect Plus Hybridization buffer (Sigma #H7033) at 65°C overnight. After washing of the hybridized membrane, image analysis was performed using a phosphoimager.
Blastocyst injection and generation of gene-modified animals
Targeted Balb/c ES cells were injected into C57Bl/6J host blastocysts, which were then transferred into pseudopregnant B6CF1 foster mothers. Chimeric offspring were identified by coat pigmentation [white (Balb/c) on a black (C57Bl/6J) background]. White offspring indicated the germline transmission of the targeted ES cells and were further analyzed for their correct genotype.
We generated LRRK2 KO mice by crossing chimeric LRRK2 G2019S KI males with either BALB/c or C57Bl/6J Cre-deleter females (B6-TgN(CMV-Cre)#Cgn (62
) that express the Cre recombinase in the fertilized oocyte, resulting in deletion of the floxed fragment in all cells of the organism (KO). Offsprings of the crossing were analyzed by PCR to identify mice in which Cre recombination has taken place. Cre recombination should take place in offspring containing both the targeted LRRK2 allele as well as the gene for Cre recombinase. This event should result in a PCR product of 150 bp instead of 816bp from the non-recombined allele (sense primer: LRRK2-geno.for: GTG TTA AAG CTC CAG TTG CCT; antisense primer: FRT(-neo)rev: TTT CGA ACC CGG GGA AGT TC).
Mice were kept either in BALB/c background or were backcrossed into C57Bl/6J. For backcrossing of LRRK2 KO and KI lines into C57Bl/6J we applied speed congenics. For the experiments, we used mice that were backcrossed either at least six generations or at least two generations (LRRK2 KD and aged LRRK2 KI) into C57Bl/6J.
Non-transgenic control mice were either littermates (from a heterozygous breeding) or from a WT breeding of the respective line from the same generation to reduce animal numbers.
Gene-modified mice were selected by standard Taqman PCR analysis of tail DNA with primers and fluorescently labeled probes: KD: LRRK2-2lox.for TA (5′-CGA AAA CAG CAA CAA CGA CAA C-3′), LRRK2-2lox.rev TA (5′-ATA GGA ACT TCT TGG CTG GAC-3′), LRRK2.KD-probe (5′-FAM-TCC GAG CCA AAA ACT CTC GAG GAA TT-TAMRA-3′); KI: LRRK2-2lox.for TA (5′-CGA AAA CAG CAA CAA CGA CAA C-3′), LRRK2-2lox.rev TA (5′-ATA GGA ACT TCT TGG CTG GAC-3′), LRRK2.2lox-probe (5′-FAM-TCC GAG CCA AAA ACT AAG CTT CTC GA-TAMRA-3′); KO: LRRK2.for (5′-TGT ATC CCA ATG CTG CCA TC-3′), LRRK2.rev (5′-CTA TAT CTC CTA GAC CCA CAC-3′), LRRK2.Ex41.probe (5′-YYE-TGG GAA TAA AGA CAT CAG AGG GCA C-TAMRA-3′). As endogenous control PCR: αSN-5′ (5′-GCT GGA AAG ACA AAA GAG GG-3′), αSN-3′ (5′-ATT CTC TCA CCT CCA CAC AG-3′), αSN-probe (5′-FAM or YYE-TGG CTG GTG TGT GGT GTC TGA TT-TAMRA-3′). Rotorgene 3000 was used for PCR (at 95°C for 10 min, 40 cycles at 95°C for 15 s and at 60°C for 1 min).
For near-infrared Li-cor western blot analysis, kidneys and brains were homogenized in homogenization buffer (10v/w) (additional phosphatase and protease inhibitors: 50 mm Tris–HCl, pH 7.4; 150 mm NaCl; 1.5 mm MgCl2; 5% glycerol; 1 mm Na3VO4; 25 mm NaF; 1 mm dithiothreitol (DTT); 0.8% NP-40 (add NaF, DTT and NP-40 just before use), complete ethylenediaminetetraacetic acid (EDTA)-free protease inhibitors (Roche), 0.5 mm PMSF (Sigma), 5 µg/ml Pepstatin A (Sigma), 5 µg/ml Leupeptin (Sigma), 1:1000 Okadaic acid 100 ng/µl in ethanol (Biomol), 1:1000 Calyculin A 100 ng/µl in ethanol (Biosource), 1:100 Pierce phosphatase inhibitor cocktail ×100 with Precellys24 (Bertin Technologies, rotation speed 5000rpm, number of cycles: brain—2 × 30 s, pause: 10 s; kidney—2 × 50 s, Pause: 10 s), then incubated for 30 min on ice and subsequently centrifuged at 13K rpm at 4°C for 10 min.
Homogenization buffer containing only standard phosphatase and protease inhibitors: 10 v/w; 0.25 M sucrose, 20 mm Tris 20 mm pH 7.4, 1 mm EDTA, 1 mm EGTA, protease inhibitor cocktail (Roche).
Homogenization buffer containing additional phosphatase and but only standard protease inhibitors: 10 v/w; 0.25 M sucrose, 20 mm Tris 20 mm pH 7.4, 1 mm EDTA, 1 mm EGTA, 1:1000 Okadaic acid 100 ng/µl in ethanol, 1:1000 Calyculin A 100 ng/µl in ethanol, 1:100 Pierce phosphatase inhibitor cocktail, protease inhibitor cocktail (Roche).
Supernatant and pellet (resuspended in homogenization buffer in the same volume used for homogenization) was used and protein concentration was determined from the supernatant by Bradford (Bio-Rad). Thirty microgram protein of the supernatant was loaded (LDS Sample Buffer, Sample Reducing Agent, Invitrogen, heated for 10 min at 95°C) on NuPAGE 4–12% Bis–tris gel 1.0 mm (Invitrogen) and ran at 180 V for 40–50 min with NuPAGE MES SDS Running Buffer (Invitrogen) or NuPAGE 3–8% Tris-Acetate gel 1.0 mm ran at 150 V for 60 min with Tris-Acetate SDS running buffer (Invitrogen). Gels were blotted by semi wet transfer (Invitrogen × Cell II Blot Module) with NuPAGE Transfer Buffer, 10% (for one gel) or 20% methanol (for two gels) on polyvinylidene difluoride transfer membrane (Immobilon-P, Invitrogen) at constant 30 V for 1 h. For Li-cor Odyssey detection, membrane was blocked for 1 h at room temperature in Odyssey Blocking Buffer (Odyssey, 1:1 diluted with phosphate buffered saline (PBS)) and then incubated with primary antibody in Odyssey Blocking Buffer (diluted 1:1 with PBS, containing 0.1% or 0.05% Tween 20) overnight at 4°C. Primary antibodies used: mouse anti-β-actin (Sigma, 1:50000); anti-LRRK2 MJJF2 c41 (Michael J. Fox Foundation/Epitomics, 1:2500); mouse anti-α-synuclein (BD Transduction, 1:5000); all subsequent antibodies were from Cell Signaling (rabbit polyclonal, exceptions indicated) and used at 1:1000: anti-S6 (rabbit monoclonal), anti-P-S6(Ser235/236), anti-4E-BP1, anti-P-4E-BP1(Thr37/46), anti-Akt, anti-P-Akt(S473), anti-TSC2, anti-LC3B and anti-mTOR. Membranes were washed four times for 5 min at room temperature in PBS containing 0.1% or 0.05% Tween 20 and then incubated for 45 min (light protected) with second antibodies [Alexa Fluor 680, F(ab′)2 fragment of goat anti-mouse (Invitrogen); IRDye 800CW anti-rabbit IgG (Li-cor); both 1:5000] in Odyssey Blocking Buffer (diluted 1:1 in PBS, containing 0.1% or 0.05% Tween20). Membranes were again washed four times for 5 min at room temperature in PBS containing 0.1% or 0.05 Tween 20, then washed two times for 5 min at room temperature in PBS only and finally scanned on the Odyssey Li-cor System.
The Bac-to-Bac Baculovirus Expression System (Invitrogen) was used (according to the protocol of the manufacturer) to express N-terminal HIS-GST-tagged (6×Histidine- and Glutathion-S-Transferase-tag) human LRRK2 constructs. The LRRK2 constructs consisted of a part of LRRK2 (amino acids 1675–2527) which produced stable and active LRRK2 protein (HIS-GST-hLRRK2 1675-2527 G2019S and HIS-GST-hLRRK2 1675-2527 D1994S). Cells were harvested, centrifuged at 3000rpm for 7 min and HIS-GST-hLRRK2 1675-2527 G2019S and D1994S were purified by GST SpinTrap columns (GE Healthcare Life Sciences). Briefly, the cell pellet was lysed in lysis buffer (50 mm
Tis–HCl, 150 mm
NaCl, 2 mm
DTT, 0.02% Tween 20, 0.5 mm
EDTA, 20% glycerol, 1X complete (protease inhibitors, Roche). Centrifugation, sonication and purification were performed according to the protocol of the manufacturer (GE Healthcare Life Sciences). Lysate were applied to the GST SpinTrap column, washed once with PBS, then three times with washing buffer (20 mm
Tris–HCl, 2 mm
DTT, 0.1 mg/ml bovine serum albumin (BSA), 0.5 mm
EDTA, 20% glycerol, 1 mm
, 5 mm
β-glycerophosphate) and eluted with elution buffer (50 mm
Tris–HCl, 0.1 mg/ml BSA, 20% glycerol, 5 mm
β-glycerophosphate, 10 mm
GSH (reduced glutathione, GE Healthcare Life Sciences). The kinase reaction was performed by diluting the components to 3-fold final concentration in 1× kinase buffer (50 mm
Hepes, 1 mm
DTT, 0.6 mm
EGTA, 10 mm
, 3 mm
): ATP to 750 mm
ATP stock; Biolabs), GST-4xLRRKTide long [as a 4× repeat fused to GST, (58
)] to 6 mm
, while GST SpinTrap elution fraction #1 was not diluted. The final reaction volume was 15 µl: 5 µl 3x ATP, 5 µl 3x GST-LRRKtide and 5 µl GST-hLRRK2 1675-2527 constructs. Reaction was incubated for 30 min at room temperature, stopped by adding 5 µl 4xLDS sample buffer (Invitrogen, with 200 mm
DTT) and heating to 90°C for 10 min, analyzed on western blot (4–12% Tris-Acetate gels and semi-dry blotting, Invitrogen) using anti-LRRK2 CK (home-made, directed against the c-terminus of the kinase domain of LRRK2; rabbit, 1:500) and anti-phospho-ezrin (rabbit, Cell Signaling Technology, 1:1000) antibodies and visualized with Li-Cor Odyssey.
Following necropsy, organs were fixed in 4% neutral phosphate-buffered formalin for 1 day, then transferred to PBS, trimmed, embedded in paraffin wax, sectioned and stained with hematoxylin and eosin (H&E) or left unstained for autofluorescence analysis. The tissue slices were evaluated and reviewed by pathologists.
Antibodies/dilutions used for immunohistochemistry and immunofluorescence staining:
primary antibodies: polyclonal rabbit anti-LAMP1 (Abcam ab24210); monoclonal rat anti-LAMP2 (SouthernBiotech; 1:250); polyclonal rabbit anti-MUC1 (Mucin-1) (Abcam ab15481); polyclonal rabbit anti-GFAP (DAKO #Z0334; 1:5000); polyclonal rabbit anti-Iba1 (WAKO chemicals, #019-19471; 1:500); polyclonal rabbit anti-TH (Novus Biologicals NB300-109; 1:500); monoclonal rat anti-DAT (Chemicon MAB369; 1:200); monoclonal rabbit anti-DARPP-32 (Cell Signalling # 2306; 1:500); polyclonal rabbit anti-α-synuclein (Chemicon AB 5038; 1:1000); monoclonal mouse anti-α-synuclein (Abcam 4D6; 1:1000); polyclonal guinea pig anti-p62 (C-terminus) (Progen, 1:100); monoclonal mouse anti-neurofilament (Covance SMI310R; 1:1000). Secondary antibodies: biotinylated goat anti-guinea pig (Vectorlabs, 1:200); biotinylated goat anti-mouse (Jackson ImmunoResearch; 1:1000); biotinylated goat anti-rat (Jackson ImmunoResearch; 1:1000); biotinylated goat anti-rabbit (Jackson ImmunoResearch; 1:1000); Alexa488-labeled goat anti-rat (Invitrogen, 1:500); Alexa488-labeled goat anti-rabbit (Invitrogen; 1:500).
Mice were transcardially perfused first with PBS and then with 4% paraformaldehyde in PBS. Brains and kidneys were prepared, postfixed for another 2 days at 4°C with 4% paraformaldehyde in PBS and embedded in paraffin according to standard procedures. Four micrometer para-sagittal paraffin sections were mounted on SuperFrost Plus slides and automatically immunostained using the Discovery XT technology (Ventana/Roche diagnostics). Briefly, sections were deparaffinized, rehydrated, subjected to antigen retrieval by heating with CC1 cell conditioning buffer (Ventana/Roche Diagnostics), incubated for 60 min at room temperature with primary antibody diluted in antibody diluent (Ventana/Roche Diagnostics), incubated with the respective biotinylated secondary antibody diluted in Ventana antibody dilution, reacted with DABMab kit (Ventana/Roche Diagnistics) and counterstained with blueing reagent (Ventana/Roche Diagnostics). Digital slides were generated with MIRAX (Zeiss) scanner technology.
Manual immunohistochemistry and immunfluorescence staining of DARPP-32, TH and DAT for quantitative image analysis
Paraffin sections were dewaxed, rehydrated, subjected to antigen retrieval by microwaving the slides for 10 min at 98°C in 0.1 m citrate buffer pH 6.0, incubated for 1 h at room temperature in PBS containing 2% goat serum, reacted over night at 4°C with primary antibody diluted in PBS/2% goat serum, stained with the appropriate Alexa488- or Biotin-labeled secondary goat antibody diluted in PBS. For the biotin-labeled secondary antibody ABC reagent (Vectorlabs) for 1 h at room temperature was applied with subsequent washes in PBS, developed with DAB stain, desalted in 0.01 m Tris (pH 7.8) and mounted with Eu-kitt. Immunofluorescence-stained slides were mounted with ProLong antifade containing 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen). Immunofluorescence in the striatum was imaged, at consistent exposure time for the respective marker, with an Olympus BX51 microscope equipped with appropriate fluorescence filters and colorview III digital camera. Fluorescence intensity in the striatum was analyzed with CellF image analysis software (Soft Imaging Systems/Olympus). Each six sagittal sections were analyzed per animal.
Kidneys and lungs were fixed with 3% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, for about 1 h at 4°C and 1% OsO4 in 0.1 M cacodylate buffer, pH 7.4, for 1 h at 4°C. After post-fixation, the tissues were dehydrated in graded acetone solutions, and embedded in Epon. For microscopic evaluation, semi-thin sections were prepared from tissue blocks, stained with toluidine blue and examined with a Zeiss light microscope. Based on these light microscopic investigations, ultrathin sections from selected tissue blocks were counterstained with uranyl acetate and lead citrate and examined with a Philips CM 10 transmission electron microscope.
Urine samples were collected into plain tubes. Total protein and creatinine were determined, using a Cobas 6000® analyzer. Urine total protein concentration was analyzed with the benzethonium chloride assay with a γ-globulin recovery rate which is 30% less than that of albumin.
Telemetric blood pressure and heart rate measurements
The radio-telemetry system (Data Sciences Int.) used in this study is composed of four basic components: an implantable transmitter (AM unit, model TLM-PAC10, volume: 1.1 cc, weight: 1.4 g) which continuously senses and transmits information from within the animal, one receiver located under the home cage, a matrix interface for coordination of signals and a computer-based data acquisition system for collection, analysis and storage of data. The body of the transmitter was implanted s.c. in aseptic conditions into the flank of the animal under isoflurane anesthesia. The sensing catheter was inserted into the left femoral artery. The total operation time for the implantation of the telemetry transmitter was about 40 min. Postoperative analgesia was provided using Buprenorphin (Temgesic®) injection of 0.05 mg/kg s.c. twice, immediately after surgery and 8–12 h later. Following surgery, the unconscious animal is placed on soft material in a clean cage with water ad libitum. The animals were allowed a period of 2–3 weeks to recover from surgery before starting acquisition of any physiological data. Mean, systolic and diastolic blood pressure and heart rate were recorded continuously in all animals over 24 h up to 8 days, and analyzed in 1 min cyclic runs for 10 s with a 500 Hz sampling rate.
Locomotor activity was measured with the TSE system (process control type 302013-CD, software: Motilitätsmesssystem 4.2) in motility cages (Makrolon Typ III with a lid and without embedding). Cages were changed after each animal. After the habituation phase (30–60 min), animals were taken out of the cage, administered the drug and then placed back into the cage and further monitored for motility (30–60 min). The dopamine-receptor antagonist/agonists SCH23390, haloperidol, quinpirole and SKF38393 (all from Sigma) were administered s.c. in a physiological NaCl solution. D-amphetamine hemisulfate (Sigma) and cocaine-HCl (Sigma) were administered i.p. (10 ml/kg) in a physiological NaCl solution.
Animal maintenance and LRRK2 kinase inhibitor drug administration
The animals were housed in a temperature-controlled room that was maintained on a 12 h light/dark cycle. Food and water were available ad libitum. Mice were administered orally with 30 mg/kg LRRK2 kinase inhibitor compound (dissolved in 0.5% methylcellulose with 0.5% Tween 80, administration volume 10 ml/kg) twice daily (morning/afternoon) for 5 days and were killed 2 h after last dosing. Novartis is willing to provide said LRRK2 kinase inhibitor under a material transfer agreement for academic research and non-commercial purposes.