Production and purification of α-Syn
The expression vector for mouse α-Syn was a kind gift from Dr. Virginia M. Lee. It was expressed in BL21(DE3) competent cells and purified as previously described . Briefly, α-Syn purification involved dialysis of proteins against 20 mM Tris pH 6.5 overnight, followed by ion-exchange chromatography on a Poros HQ50 column (Thermo Fisher Scientific) with a 0–2 M NaCl gradient. This method was followed by an additional reverse phase-high pressure liquid chromatography purification step on a Jupiter C18 column (Phenomenex, Torrance, CA) in 0.1% trifluoroacetic acid with an 0–90% acetonitrile gradient. Isolated α-Syn was then extensively dialysed against PBS pH 7.4 overnight followed by an additional dialysis step against 20 mM ammonium bicarbonate overnight. Protein concentration was determined by bicinchoninic acid (BCA) protein concentration assay (Pierce). The proteins were subsequently aliquoted, lyophilized, and stored at − 80 °C until use.
Quantitative α-Syn fibril assembly and sedimentation
Soluble monomeric mouse α-Syn (5 mg/ml) was assembled into preformed fibrils (PFF) by incubation at 37 °C in phosphate-buffered saline (PBS) pH 7.4 (PBS, Gibco) with continuous shaking at 1050 r.p.m. (Eppendorf Thermotop). The generated PFF were harvested by centrifugation at 15,600 g 25 °C for 30 min, and then resuspended in PBS at a concentration of 2 mg/mL, as determined by BCA. Subsequently, PFF were sonicated for 20 min using a Branson 250 Sonifier at 30% intensity, before being aliquoted and frozen at − 80 °C until further use. A fraction of each PFF batch was set aside for SDS-PAGE, (trans,trans)-1-bromo-2,5-bis-(4-hydroxy)styrylbenzene (K114) fluorometry and dynamic light scattering (DLS) analysis of purity, amyloid structure and particle size.
SDS sample buffer (4% SDS, 40% glycerol, 1% bromophenol blue, 50 mM Tris, pH 6.8) was added to mouse PFF, which were heated to 96 °C for 15 min and analysed by SDS-PAGE (sodium dodecyl sulphate–polyacrylamide gel electrophoresis). The Coomassie blue stained SDS-PAGE gels were scanned using Image J software (National Institutes of Health, Bethesda, MD, USA) for quantification.
To quantify the amount of amyloid formation, samples were monitored by (trans,trans)-1-bromo-2,5-bis-(4-hydroxy)styrylbenzene (K114) fluorometry as described previously . In brief, samples were analysed by incubating a fraction of each sample with the K114 (50 µM) in 100 mM glycine, pH 8.5, and measuring fluorescence (λex = 380 nm, λem = 550 nm, cutoff = 530 nm) with an EnSpire 2300 Multilabel Reader (Perkin Elmer).
Dynamic light scattering (DLS)
PFF (2 mg/ml) were subjected to ultrasound breakage for 20 min using a Sonifier (Branson 250; 30% Duty Cycle) equipped with a water jacket cooling system to avoid sample overheating. Then, the size distribution profile of PFF in suspension was measured by DLS using a Wyatt DynaPro NanoStar instrument at 25 °C. Data was processed using the Dynamics 126.96.36.199 software package, with the solvent (PBS) background signal subtracted from each sample.
Seeding test of PFF in organotypic hippocampal slice cultures
Organotypic hippocampal culture slices (OHCS) were created from C57BL/6J pups on post-natal day 7 according to Stoppini et al. . In order to validate the ability of the mouse full-length (1-140) α-Syn PFF for seeding aggregation before using them for in vivo experiments, the PFF were injected into the dentate gyrus of the OHCS, as previously described . Slices were fixed 7 days post injection (dpi), and stained for pathological aggregates using conformation-specific α-Syn antibody MJF-14 (rabbit mAb MJF-14-6-4-2, 1:25,000, Abcam #ab209538) and pSer129 (mouse mAb 11A5, 1:10,000, kindly provided by ImagoPharmaceuticals), as described previously . Alexa Fluor 488 anti-rabbit and Alexa Fluor 568 anti-mouse (Invitrogen, #A11008 and #A11004, 1:2000) were used for detection, along with 4′,6-diamidino-2-phenylindole (DAPI, TH.GEYER, 5 µg/mL) for staining nuclei. As negative controls, C57BL/6J OHCS were injected with either sterile PBS or monomeric α-Syn and processed as above. Furthermore, α-Syn knockout OHCS were injected with human S129A PFF and processed as above.
Experimental procedures involving mice were approved by The Danish Animal Experiments Inspectorate (license 2017-15-0201-01203) and followed the Danish and European Animal experimentation guidelines and legislations (directive 2010/63/EU).
Mice were housed in a temperature-controlled room under a 12 h light/dark period with water and food ad libitum. Three-month-old heterozygous female mice transgenic for human mutant A53T α-Syn (M83+/−) were bilaterally injected with vehicle (PBS pH 7.4, n = 6), monomeric α-Syn (2 × 5 μL at 2 mg/mL, n = 8) or full-length (1-140) mouse α-Syn PFF (2 × 5 μL at 2 mg/ml, n = 8). An additional control experiment was performed where mice were injected with full-length (1-140) mouse α-Syn PFF (n = 4) in the right hindlimb (ipsilateral) and vehicle in the left hindlimb (PBS, pH 7.4, contralateral). Mice were anaesthetised with isoflurane (3.5%) inhalation. Intramuscular injections were made using different 10-μL Hamilton syringes with a 25-gauge needle to avoid any cross-contamination and performed by inserting a needle ∼1 mm deep into the gastrocnemius muscle, as described elsewhere . Hindlimb clasping behaviour was monitored and scored regularly as previously reported [23, 27]. Briefly, the mouse is lifted by its tail, and the time the hindlimbs are retracted towards the abdomen is scored on a 4-point scale, from 0 to 3. A score of 0 is given if the hindlimbs are continuously spread away from the abdomen; a score of 1 when one hindlimb is retracted towards the abdomen more than 50% of the suspended time; and score of 2 if both hindlimbs are partially retracted for 50% of the time. A maximum score of 3 implies that both hindlimbs were completely retracted and touching the abdomen for more than 5 s of the suspended time [23, 27].
At 45 dpi, while motor function remains intact and before the development of debilitating motor impairments , M83 mice were euthanized with an overdose of isoflurane and perfused with PBS pH 7.4 with phosphatase inhibitors (25 mM β-glycerolphosphate, 5 mM NaF, 1 mM Na3VO4, 10 mM Na-pyrophosphate) before dissecting brain, spinal cord, dorsal roots and lumbar (L3-L5) dorsal root ganglia (DRG). Tissues were snap frozen and stored at − 80 °C until use, or fixed and processed as described below.
Evaluation of mechanical allodynia
Mice were acclimatized in a Plexiglas cage atop a mesh metal grid for approximately 30 min prior to testing. The “ascending stimulus” method was used to determine the mechanical withdrawal thresholds, by manually applying calibrated Semmes–Weinstein monofilaments (Stoelting Co) into the plantar surface of the hind paws . A response is considered positive if the mouse exhibits any nocifensive behaviors, including sudden paw withdrawal, licking, flinching or trembling of the paw, either while applying the stimulus or immediately afterwards . A total of five stimuli per filament were recorded and when at least three out of five trials rendered a positive response, the corresponding force was defined as the tactical threshold (expressed in grams) . The average of both paws per mouse was used for the statistical analysis.
Nerve conduction velocity recordings
Mice were anesthetized with 2% isoflurane for measuring sural sensory and sciatic motor nerve conduction velocities (NCV), using a Viking Quest apparatus (Natus Neurology Incorporated, USA). For recording sural sensory nerve conduction velocity (SNCV), electrodes were placed in the dorsum of the hind paw with supramaximal stimulation at the ankle.
For motor nerve conduction velocity (MNCV), the stimulation needle electrodes (Natus Biomedical, Madison, WI) were set at the ankle with recording electrodes placed about 7–8 mm away from it, in the dorsum of the hind paw. A second measurement was performed with the stimulation set at the sciatic nerve notch. Sciatic-tibial MNCV was then calculated following the formula: [MNCV (m/s) = D/L], where (L) represents the take-off latency (ms) of the sciatic nerve and (D) the distance between the stimulating and recording electrodes (mm). After the electrophysiological recordings, mice were terminally anaesthetized with an overdose of isoflurane for tissue dissection.
L3-L5 DRG were homogenised and analysed by Western blot as previously described [31, 32]. Briefly, samples were homogenised in lysis buffer (approx. 10 weight/volume ratio; 20 mM Tris pH 7.4, 0.32 M sucrose, 5 mM EDTA and 1 cOmplete™ proteinase inhibitor tablet/10 mL (Roche), 25 mM sodium fluoride, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate). Homogenates were then centrifuged at 25,000× g for 30 min at 4 °C. The resulting supernatant was saved as the whole-tissue homogenate. Protein concentration was determined by BCA (Sigma, MO, USA). Whole-tissue homogenate (20 μg protein) was dissolved in loading buffer (100 mM Tris–HCl, 8% SDS, 24% glycerol, 0.02% bromophenol blue, pH 6.8) and the samples were then denatured at 95 °C for 10 min. After centrifugation for 5 min at 25,000× g, the supernatant was loaded into 16% Tricine gels (Novex) or 8–16% polyacrylamide gel (GenScript). Proteins were blotted into PVDF membranes using iBlot® 2 Dry Blotting System (Thermo Fischer). The membranes were then fixed with 4% paraformaldehyde (PFA) in PBS for 30 min; then boiled in PBS for 5 min. After being blocked for 1 h (TBS, 0.01% Tween, skimmed milk powder, pH 7.6), membranes were incubated with primary antibodies, mouse mAb pSer129-α-Syn (11A5, 1:2,000), mouse Syn-1 (BD Biosciences #610787, 1:1,000), mouse anti-actin (Sigma A5441, 1:5000), or mouse anti-β-III tubulin (Sigma, T5076, 1:5000), ON at 4 °C, and subsequently incubated with secondary HRP conjugated mouse immunoglobulins (Dako, Denmark) for 1.5 h at RT. Protein bands were visualised with ECL® (GE Healthcare, UK) and image acquisition performed with Fuji LAS-3000 intelligent dark box (Fujifilm, Japan).
Immunofluorescence for frozen DRG sections
Dorsal roots and L4 DRG (n = 4 per group) were fixed in 4% paraformaldehyde (PFA) overnight before being transferred to the 30% sucrose cryoprotection solution, where they stayed overnight at 4 °C. Tissue was then embedded in Tissue-Tek (Sakura) and snap frozen in isopentane on dry ice. Longitudinal sections were cut at 10 μm in a cryostat. After drying, tissue sections were incubated in 20% methanol for 5 min, blocked with a buffer containing 10% fetal bovine serum in 0.3% Triton X-100 in PBS for 1 h at room temperature, followed by overnight incubation with the primary antibodies against mouse mAb pSer129-α-Syn (11A5, 1:5,000) and neurofilament M (NF-M) (Millipore AB1987, 1:200) at 4 °C. Alexa Fluor 488 anti-mouse and Alexa Fluor 568 anti-rabbit-labelled secondary antibodies (Molecular Probes, A10037 and A21206 respectively, 1:500) were incubated for 2 h and Hoechst (Sigma, 1:10,000) was used to label the nuclei, visualized in blue. Slides were mounted with DAKO fluorescent mounting medium and Z-stacked images captured by confocal microscopy (LSM780, Carl Zeiss, Germany).
Immunohistochemistry of paraffin spinal cord and brain sections
Immunohistochemistry on 10 µm thick sections of formalin fixed paraffin embedded spinal cord and brain was performed after deparaffinization and antigen retrieval in citrate buffer pH 6. The following primary antibodies were employed: pSer129-α-Syn (D1R1R, Cell Signaling #23706, 1:500 or mouse mAb 11A5 1:1000). Neuronal nuclei marker (NeuN, Millipore, MAB377, 1:500), and glial fibrillary acidic protein (GFAP, Cell Signaling #12389, 1:200). Alexa-Fluor fluorophore conjugated secondary antibodies (Thermo Fisher) were used (1:1000) for primary antibody detection. High resolution panoramic views were obtained using Olympus VS120 digital slide scanner (equipped with single-band emitters for Hoechst, FITC, Cy3, Cy5 and Cy7), and 20 X views were extracted using OlyVia software (Olympus). Alternatively, sections were imaged using Zeiss observer inverted microscope equipped with colibri 7 LED illumination operated using ZenPro software (Zeiss).
Transmission electron microscopy (TEM)
Dorsal roots (vehicle n = 6, mouse monomeric α-Syn n = 8 and mouse α-Syn PFF n = 8) were dissected, immediately post fixed for 1 h in 2% osmium tetroxide (OsO4) and stained with 2% uranyl acetate in 50% ethanol. Dehydration was performed in a series of ascending concentrations of ethanol and pure acetone (100%). Samples were then embedded in Epoxy (48 h at 60 °C) using Embedding Medium Kit (Sigma-Aldrich, Sweden AB), after a three-step infiltration in a mixture of acetone embedding medium (embedding medium: acetone, 1:3, 1:1, 3:1).
Leica UC7 ultra microtome (Leica Microsystems GmbH, Vienna, Austria) was used for taking 60-nm thick sections that were then collected onto formvar-coated copper slot grids and counterstained with uranyl acetate and lead citrate. A 100 kV transmission electron microscope (JEM 1230, JEOL Ltd., Tokyo, Japan) was used for sample observation.