Brain tissues from patients suffering from PD, MSA, and DLB as well as age-matched healthy controls were obtained at autopsy from four to six patients per condition through the UK Brain Bank (Imperial College London, UK). The clinicopathological description of the 19 patients is summarized in Table 1. After removal, the brains were hemisected using a mid-sagittal incision. One hemibrain was dissected fresh and the tissue blocks were frozen. The other hemibrain was formalin fixed for diagnostic neuropathological assessment. The extent of αSYN and tau pathology was assessed using Braak staging criteria and Thal amyloid phases were described [3, 4]. Brain regions enriched with αSYN aggregates were identified by histological examination, isolated and processed into brain homogenates. The cingulate cortex was isolated from the brains of the patients with PD and DLB. The cerebellum was obtained from the brains of the patients with MSA. Both regions were also extracted from the healthy control brains.
Brain tissue homogenization
Frozen brain tissues were weighed in falcon tubes (15 or 50 ml depending on the total weight). The samples were diluted five times in PMCA buffer (150 mM KCl, 50 mM Tris–HCl pH 7.5) to obtain a homogenate at 20% (weight:volume). The homogenization was performed by sonication using the SFX 150 Cell Disruptor sonicator with a 3.17 mm microtip probe (Branson) for 1 min, with 10 s pulses followed by 10 s pauses in a biosafety level 3 environment (BSL-3). The homogenates were aliquoted and immediately frozen in liquid nitrogen before storage at − 80 °C. All contaminated surfaces were cleaned with SDS (1%) .
Aggregated αSYN quantification in patients brain homogenates
Aggregated αSYN was quantified using a filter retardation assay and immunoblotting. To this aim, 0.5 mg of brain homogenates were immobilized on cellulose acetate membranes (0.2 μm pore size, Millipore Corp., Bedford, MA) by filtration using a 48-slot slot-blot filtration apparatus (GE Healthcare). The membranes were blocked in 5% dried skimmed milk and probed with either the 4B12 (Biolegend, cat # 807801) and P-S129 αSYN antibody (mouse 11A5, provided by Elan Pharmaceuticals, Inc., Dublin, Ireland). Following wash with tris buffered saline with triton (TBST), the membranes were incubated with horseradish peroxidase (HRP) conjugated goat-anti-mouse IgG3 secondary antibody (Thermo, cat #M32707) for 1 h at room temperature. Proteins were visualized using ECL reagents (Pierce, USA).
The Cisbio fluorescence resonance energy transfer (FRET) assay (Cisbio, France, cat # 6FASYPEG) was also used to quantify aggregated αSYN in patients’ brain homogenates, following the manufacturer’s recommendations. Briefly, patient brain homogenates were diluted to 2.5% (W:V) in lysis buffer provided in the HTRF kit. 10 µl of each diluted brain homogenates were loaded into a 96 well plate and mixed with 10 µl of the FRET donor and acceptor antibodies in the kit. The plate was sealed with a film (CmlAB, Danemark, cat#13076-9P-500) and incubated for 20 h at 20 °C without shaking in a Thermomixer comfort (Eppendorf, Montesson, France). After incubation, time-resolved FRET was measured upon excitation at 337 nm using a plate reader (CLARIOstar, BMG Labtech, Germany) . The HTRF signal was recorded at two different wavelengths (665 nm and 620 nm). The amount of aggregated αSYN was derived from the 665/620 nm fluorescence ratio and multiplied by 10.000.
Protein misfolding cyclic amplification assay
All operations were performed in BSL-3. Brain homogenates were diluted in PMCA buffer (150 mM KCl, 50 mM Tris–HCl, pH 7.5) containing monomeric αSYN (100 µM) to a final concentration of 2% (W:V), equivalent to 6 mg of brain tissue, as described previously for other tissues . The sample was split in two tubes of PCR strips (BIOplastics, Landgraaf, The Netherlands). PMCA amplification was performed in quadruplicates for each patient using the Q700 generator and a 431MPX horn (Qsonica, Fisher scientific, Illkirch, France). The power of the horn was set to 30% of maximal amplitude. The program of amplification consisted in 15 s of sonication and 5 min pause at 31 °C. Every hour, 5 µl were withdrawn from each tube and diluted in 300 µl of 10 µM of Thioflavin T. The amplification was monitored by measuring Thioflavin T fluorescence using a Cary Eclipse Fluorescence Spectrophotometer (Agilent, Les Ulis, France) with fixed excitation and emission wavelength at 440 nm and 480 nm respectively. Cycle 2, 3 and 4 were performed following the same protocol using 1% of the preceding cycle reaction as seeds for PD and DLB cases, 5% for MSA cases. The amounts of brain homogenates and PMCA amplified assemblies used in each amplification reaction were defined through an optimization study aimed at maintaining high stringency by minimizing the de novo aggregation of αSYN under our experimental conditions. The time at which an aliquot from one amplification reaction was withdrawn for a subsequent amplification reaction was also defined through an optimization study aimed at avoiding de novo αSYN fibrillar assemblies formation.
At cycle 4, PMCA reactions products were spun for 30 min at 50,000g, the amount of monomeric αSYN in the supernatant was assessed spectrophotometrically and the pelleted assemblies were resuspended in phosphate-buffered saline (PBS) buffer. All resuspended assemblies, at a final concentration of 100 µM αSYN, were fragmented prior to in vivo use by sonication for 20 min in 2-ml Eppendorf tubes in a Vial Tweeter powered by an ultrasonic processor UIS250v (250 W, 2.4 kHz; Hielscher Ultrasonic, Teltow, Germany), aliquoted, flash frozen in liquid nitrogen and stored until use at − 80 °C.
De novo assembled αSYN fibrils and ribbons as well as PD, MSA and DLB patients PMCA-amplified αSYN assemblies (1.4 mg/ml) in 150 mM KCl, 50 mM Tris–HCl, pH 7.5, 1 mM EGTA were treated at 37 °C by Proteinase K (3.8 µg/ml) (Roche). Aliquots were removed at different time intervals following addition of the protease and transferred into Eppendorf tubes maintained at 90 °C containing sample buffer (50 mM Tris–HCl, pH 6.8, 4% SDS, 2% beta-mercaptoethanol, 12% glycerol and 0.01% bromophenol blue) to arrest immediately the cleavage reaction. After incubation of each tube for 5 min at 90 °C, the samples were processed to monitor the time course of αSYN cleavage by polyacrylamide gel electrophoresis (PAGE) (15%) after staining with Coomassie blue.
Transmission electron microscopy
The morphology of the de novo assembled and PMCA-amplified αSYN assemblies was assessed by transmission electron microscopy (TEM) in a Jeol 1400 transmission electron microscope following adsorption onto carbon-coated 200 mesh grids and negative staining with 1% uranyl acetate. The images were recorded using a Gatan Orius CCD camera (Gatan).
Conformational characterization using FILA4 antibody
αSYN fibrils, ribbons assembled de novo and PMCA-amplified αSYN assemblies (0.2 µg) in 150 mM KCl, 50 mM Tris–HCl, pH 7.5, were spotted onto single nitrocellulose membranes. The membranes were blocked in 5% dried skimmed milk and probed with the aggregated αSYN specific antibody FILA4 at a concentration of 0.28 µg/ml . Following wash with TBST, the membranes were incubated with HRP-conjugated swine-anti-rabbit secondary antibody (DAKO, Copenhagen, Denmark) for 1 h at room temperature. Proteins were visualized using ECL reagents (Pierce, USA). ECL signal was detected using a ChemiDocTM MP (BioRad) and the protocol “Chemi”. Acquisition was performed using the protocol ‘Signal Acquisition Mode” from 1 to 60 s of exposure. Images were processed and quantified using Image Lab. The FILA4 signal intensity for each spot was integrated using “Lanes & Bands” function.
αSYN seeding assay
Human H4 neuroglioma cells stably expressing αSYN-YFP fusion protein were seeded at 6000 cells per well in a 384-well plate. Cells were incubated with patient-derived homogenates or PMCA-amplified αSYN assemblies (175 nM). Recombinant αSYN fibrils (175 nM) were used as a positive control. The particle concentration of patient-derived or de novo assembled fibrillar αSYN was obtained by dividing αSYN monomeric concentration by the average number of molecules (as measured by analytical ultracentrifugation (AUC) and TEM). The average number of molecules measured for all amplified αSYN fibrils was 8300 ± 300. The average length of all amplified fibrils was 41 ± 2 nm. Cells were fixed in 10% paraformaldehyde (PFA) 24 h after incubation with the different brain homogenates or PMCA-amplified αSYN assemblies and were conserved in PBS with DAPI (4′,6-diamidino-2-fenylindool, 1/5000) until analysis. The number of αSYN aggregates and PαSYN positive cells was quantified based on YFP or PαSYN fluorescence respectively and the number of cells was determined with DAPI staining using the Operetta High Content Imaging System (PerkinElmer) and Harmony 4.9 software. The width to length ratio and the roundness was determined using the same software. The detailed photographs were taken by a Zeiss LSM 880 confocal microscope.
Primary cortical neuron cultures
Primary cortical neurons were prepared from the cortex of E16 FVB/N mouse embryos. Meninges were removed, cortices were dissected, and subsequently dissociated according to the protocol. Neurons were plated in a 24-well plate with coverslips at a density of 200,000 cells per well. Cells were treated with PMCA-amplified αSYN assemblies (175 nM) in Neurobasal medium supplemented with 2 mM L-glutamine and 2% B27 at DIV 1. Cells were fixed in 10% PFA at DIV 7 for immunocytochemical analysis.
Recombinant AAV production and purification
Vector production and purification was performed as previously described . The plasmids include the constructs for the AAV7 serotype, the AAV transfer plasmid encoding the human mutant A53T αSYN under the control of the ubiquitous CMVie enhanced synapsin1 promoter and the pAdvDeltaF6 adenoviral helper plasmid. Real-time PCR analysis was used for genomic copy determination.
All animal experiments were carried out in accordance with the European Communities Council Directive of November 24, 1986 (86/609/EEC) and approved by the Bioethical Committee of the KU Leuven (ECD project P067-2013 and P085-2014, Belgium). Eight-week-old female Sprague Dawley rats (Janvier, France) weighing about 200 to 250 g were housed under a normal 12-h light and/or dark cycle with free access to pelleted food and tap water. All surgical procedures were performed using aseptic techniques. Rats were anaesthetized with ketamine (60 mg/kg, intraperitoneal (i.p.), Ketalar, Pfizer, Belgium) and medetomidine (0.4 mg/kg, i.p., Dormitor, Pfizer, Belgium). Following anesthesia, the rodents were placed in a stereotactic head frame (Stoelting, IL, USA). Injections were performed with a 30-gauge needle (VWR International, Haasrode, Belgium) and a 10-µl Hamilton syringe (Hamilton, Bonaduz, GR, Switzerland). Animals were stereotactically injected into the SN with 2.5 µl of total brain homogenate (20% W:V) or PMCA-product (100 µM) supplemented with 0.5 µl of A53T αSYN recombinant adeno-associated viral vector 2/7 (rAAV2/7 vector) (5.4E + 11 GC/ml vector dose) or PBS. rAAV control animals were injected with 3 µl of diluted A53T αSYN rAAV2/7 vector in the SN (equal vector dose). Stereotactic coordinates used for the SN were anteroposterior, − 5.3; lateral, − 2.0; and dorsoventral, − 7.2 calculated from the dura using bregma as reference.
To determine the overall amount of aggregated αSYN using the HTRF Cisbio assay in the brains of animals after inoculation with the different PMCA-amplified αSYN assemblies, 5 µl of PMCA-product (100 µM) supplemented with 0.5 µl of A53T αSYN rAAV2/7 vector (5.4E + 11 GC/ml vector dose) were injected over three injection sites in the rat striatum (STR). Stereotactic coordinates used for the STR were anteroposterior, 0.0; lateral, − 2.0; and dorsoventral, − 5.5/− 4.5/− 4.0 calculated from the dura using bregma as reference. The injection rate was 0.25 µl/min and the needle was left in place for an additional five minutes before being retracted.
The cylinder test was used to quantify forelimb use. Contacts made by each forepaw with the wall of a 20-cm-wide clear glass cylinder were scored from the videotapes by an observer blinded to the animal’s identity. A total of 25 contacts were recorded for each animal. The number of impaired forelimb contacts was expressed as a percentage of total forelimb contacts. Non-Lesioned control rats should score around 50% in this test.
Rats were sacrificed with an overdose of sodium pentobarbital (200 mg/kg, i.p., Dolethal, Vetoquinol, Belgium) followed by intracardial perfusion with 4% PFA in PBS. After post-fixation overnight in 4% PFA, 50 µm-thick coronal brain sections were made with a vibrating microtome (HM 650 V, Microm, Germany). IHC was performed on free-floating sections using antibodies against tyrosine hydroxylase (TH, rabbit polyclonal, Ab152, 1:5000, Merck Millipore, Massachusetts, US), phosphorylated αSYN (P-S129 αSYN, mouse 11A5, 1:5000, provided by Elan Pharmaceuticals, Inc., Dublin, Ireland and rabbit, Ab51253, 1:5000, Abcam, Cambridge, UK), Iba1 (goat polyclonal, Ab107159, 1:1000, Abcam), MHC Class II (mouse, MCA46G, 1:250, Serotec), CD4 (mouse, MCA55GA, 1:500, Serotec) and CD8 (mouse, MCA48GA, 1:500, Serotec). Sections were pretreated with 3% hydrogen peroxide for 10 min and incubated overnight with primary antibody in 10% swine, normal goat or rabbit serum (DakoCytomation, Belgium). As secondary antibody (DakoCytomation, Belgium) we used biotinylated anti-rabbit IgG (TH, 1:600), anti-mouse IgG (P-S129 αSYN, 1:600; MHC Class II, CD4 and CD8, 1:300) and anti-goat IgG (Iba1, 1:300), followed by incubation with streptavidin-horseradish peroxidase complex (1:1000, DakoCytomation, Belgium). TH immunoreactivity was visualized using Vector SG (SK-4700, Vector Laboratories, CA) and P-S129 αSYN, Iba1, MHC Class II, CD4 and CD8 immunoreactivity were visualized using 3,3-diaminobenzidine (0.4 mg/ml, Sigma-Aldrich) as a chromogen.
Sections were rinsed three times in PBS and then incubated overnight in PBS-0.1% triton X-100 with 10% donkey serum (Jackson ImmunoResearch Laboratories, Inc., UK) and the following antibodies: mouse anti-P-S129 αSYN (11A5, 1:5000, Elan Pharmaceuticals), chicken anti-TH (THY, 1:1000, Aves) and rabbit anti-GST-π (312, 1:1000, MBL Life Science), mouse anti-P-S129 αSYN (11A5, 1:5000, Elan Pharmaceuticals), rabbit anti-Olig2 (Ab9610, 1:1000, Merck Millipore) or mouse anti-DARPP-32 (H3, Santa Cruz Biotechnology 1:500). After three rinses in PBS-0.1% triton X-100 the sections were incubated in the dark for 2 h in fluorochrome-conjugated secondary antibodies: donkey anti-mouse Alexa 488 (1:1000, Molecular Probes™, Invitrogen, Belgium), donkey anti-chicken Cy3 (1:500, Jackson ImmunoResearch) and donkey anti-rabbit Alexa 647 (1:1000, Molecular Probes™, Invitrogen) or donkey anti-mouse Alexa 488 (1:1000, Molecular Probes™, Invitrogen, Belgium) and donkey anti-rabbit Alexa 647 (1:1000, Molecular Probes™, Invitrogen). After being rinsed in PBS and mounted, the sections were coverslipped with Mowiol 4–88 (Calbiochem®, California, US) and DAPI (1:1000).
For immunocytochemical analysis, fixed coverslips were incubated overnight in PBS-0.1% triton X-100 with 10% donkey serum (Jackson ImmunoResearch Laboratories, Inc., UK) and primary antibodies against mouse P-S129 αSYN (11A5, 1:5000, Elan Pharmaceuticals) and rabbit β3-tubulin (Ab52901, 1:1000, Abcam). The secondary antibodies used were donkey anti-mouse Alexa 488 (1:1000, Molecular Probes™, Invitrogen, Belgium) and donkey anti-rabbit Alexa 647 (1:1000, Molecular Probes™, Invitrogen, Belgium). Fluorescent stainings were visualized by confocal microscopy with an LSM 880 unit (Zeiss, Belgium).
The number of TH- and P-S129 αSYN positive cells in the SN was determined by stereological measurements using the Optical fractionator method in a computerized system as described before  (StereoInvestigator; MicroBright-Field, Magdeburg, Germany). Every fifth section throughout the entire SN was analyzed, with a total of seven sections for each animal. The coefficients of error, calculated according to the procedure of Schmitz and Hof as estimates of precision , varied between 0.05 and 0.10. All the analyses were performed by an investigator blind to different groups.
Striatal immunoreactivity quantification
Seven sections covering the whole STR were stained using an antibody against TH as previously described. Images were acquired using the LEICA DM6B optical microscope (Leica, Wetzlar, Germany) with a Leica DFC 7000 T digital camera (Leica) and the Leica Application Suite software (Leica). Intensity measurement was performed using the software ImageJ.
Evaluation of the spreading of αSYN pathology
Spreading of the αSYN pathology was analyzed and scored by immunohistochemical analysis using an antibody against P-S129 αSYN as previously described. Data are shown as heat maps to illustrate the distribution of the αSYN pathology throughout seven different brain regions in a semi-quantitative manner. The selected brain regions are the following: (1) and (2) adjacent sections of caudate putamen (CPu) (respectively bregma 2.16; bregma 1.20); (3) lateral caudate putamen (CPu) and internal capsule (ic) (bregma − 1.80); (4) SNpc and cerebral peduncle (cp) (bregma − 4.36); (5) dorsal tier of substantia nigra pars compacta (SNpc) and pars reticulata (SNpr) (bregma − 5.28, injection site); (6) medial and ventral tier of SNpc (bregma − 5.76) and (7) lateral SNpc and SNpr (bregma − 6.24).