Mouse husbandry
Young adult wild-type mice (C57BL/6N, male) aged 2.5 months with a weight average of 23.5 g (Young Bio Company, Korea) were used in this study. All animals were housed at a constant room temperature with a 12-hour light–dark cycle in the animal facility of Ilsong Institute of Life Science, Hallym University, Korea. New bedding and food were replaced every week until sacrifice. The Animal Research Committee of Hallym University Sacred Heart Hospital granted ethical approval and permission to this study (HMC2019-1-1130-42). All methods were performed in accordance with the relevant guidelines and regulations in the methods section. The study is in accordance with ARRIVE guidelines.
Production of recombinant αSyn monomer and generation of αSyn preformed fibril
PD434-SR mouse αSyn vector was purchased from Addgene (Addgene, plasmid #89073, https://www.addgene.org/89073). Mouse αSyn vector was then amplified in E. coli DH5α; later on, the expression vector was transformed into E. coli BL-21 bacteria. A single colony was selected for growth in 2xYT medium supplemented with 100 µg/ml ampicillin. Growth was monitored to log-phase until the bacterial density of OD600 reaches to 0.3–0.6, which was followed by protein induction using 0.5 mM IPTG for 5 hours at 37°C on a shaker at 100 rpm. Harvest cells were then performed by centrifugation at 3000 rpm for 15 min at 4°C. Pellet was then collected, and approximately 5 g of cells was resuspended in 5 mL distilled water, which was supplemented with 50 µL of saturated MgCl2. Next, cell solutions were aliquoted into 1.5 mL Eppendorf tubes, and these were kept at −80°C overnight. After that, homogenates were boiled at 98°C for 30 min and were centrifuged at 13000 rpm for 20 min. The supernatant was then collected, and further dialysis was performed. Briefly, αSyn protein sample was placed into 2 L ice-cold 20 mM Tris-HCl with a pH of 8.0 buffer overnight. Next, the solution was filtered using 0.2-µm membrane to discard the aggregated protein; then, the protein was purified using the HiTrap column with buffer A containing 20 mM Tris-HCl, with a pH of 8.0, and buffer B containing 20 mM Tris-HCl and 1M NaCl, with a pH of 8.0. Eluted fraction containing αSyn was collected and was further dialyzed in the water at 4°C overnight. Dialyzed protein sample was then frozen until completely dry, and αSyn protein was resolved in 20 mM Tris-HCl and 150 mM NaCl, with a pH of 8.0. Finally, the protein sample was centrifuged at 13000 rpm at 4°C, and the supernatant containing αSyn was collected and was kept at −80°C until investigation.
For αSyn PFF production, a siliconized 1.5 mL Eppendorf tube was prepared to prevent surface absorption of protein using Sigmacote. Next, 1 mL of monomeric αSyn was added to the tube, coupled with 10 µL 5% sodium azide and two 5 mm glass beads. The tube was shaken at 800 rpm at 37°C for 10 days on Thermoshaker. PFF was collected, and protein concentration was measured.
Production of PD mouse model
The mouse αSyn PFF (2.5 mg/mL) prepared for this study was stored at –80°C before use. PFF was taken out from the freezer and was put on ice. A well-constructed αSyn PFF was shown using transmission electron microscope, and Thioflavin T assay was performed for PFF quality control. The PFF was then sonicated for 10 min at 37°C in an ultrasonic water bath before use (Supplementary Figs. S1a and b).
Animals were anesthetized via intraperitoneal injection (i.p.) of avertin solution (240 mg/kg, 0.2 ml/10 g, i.p.), and ketoprofen (0.2 cc of 1%, ketoprofen, s.c.) was injected in order to relieve pain. The unilateral intracranial injection was performed into the right striatum with 2 µL recombinant αSyn PFF [final concentration of PFF is 5 µg/2µL, using a 10 µL Hamilton syringe (no.2)], with a speed of 1 µL/min using stereotaxic instrument. Control animals were administered with 5 µg/2 µL monomeric αSyn or 2 µL sterile PBS into the right striatum using the same method with PFF (Fig. 1). The right dorsal striatum was then targeted using the following coordinates from the bregma: anterior–posterior +0.5 mm, mediolateral −2 mm, and dorsoventral in a depth of −3 mm according to the mouse brain atlas. After the injection, the needle was kept for 5 min to fix the solution at the position; then, the syringe was removed slowly by turning upward to −1.5 mm (half of depth) and was kept for 1 min, and the syringe was removed out of brain. After surgery, animals were taken care of using intraperitoneal saline and subcutaneous ketoprofen injection for three consecutive days post injection. The mouse was sacrificed for analysis at baseline without PFF injection (0 day), at 7 days after PFF injection, i.e., 7 days post injection (dpi), 14 dpi, 30 dpi, 60 dpi, 90 dpi, and 120 dpi. Control animals consisted of PBS-injected mice, which were sacrificed at 7 dpi, 30 dpi, 60 dpi, and 90 dpi, and monomeric aSyn-injected mice, which were sacrificed at 120 dpi (Fig. 1; Supplementary Table S1).
Sacrifice and tissue preparation
Prior to sacrifice, the animals were anesthetized using avertin solution (240 mg/kg, 0.2 mL/10 g, i.p.), and they were transcardially perfused with 50 mL of ice-cold 1x PBS, followed by 30 mL of ice-cold 4% paraformaldehyde (4% PFA). The brain was then taken out and was fixed in 4% PFA at4°C overnight. The brains were then sectioned using vibratome at 40-µm thickness, and the cutting speed was 0.055 mm/s. Sections were collected from the olfactory to the substantia nigra, and the sectioned tissues were put in order in the 96-well culture plate. They were stored in the 0.05% sodium azide (0.05% NaN3) at 4°C until they were used for immunostaining.
Immunofluorescence
Free-floating sections were soaked in 1x PBS in 12-well culture plate for 20 min at room temperature (RT) on a shaker before blocking by 0.5% Triton X-100 in PBS with a supplement of normal donkey serum (1:1000) for 1 hour on a shaker at RT. Brain sections were then incubated using the primary antibodies (Supplementary Table S2), which were diluted in 0.5% Triton X-100 in PBS solution overnight at 4°C. Then, the brain tissues were washed thrice for 10 min in PBS at RT and then were incubated with secondary antibodies conjugated to Alexa Fluor 488, Alexa Fluor 555, DyLight 488, and DyLight 550 for 3 hours. To protect the fluorescence, the experimental plate was covered with aluminum foil at RT on the shaker. Sections were then washed three times for 10 min in PBS and were later transferred to the coated slide. Then, the slides were mounted with the DAPI solution using Vectashield (Vector Laboratories) in order to visualize the cell nuclei; these were then stored in a dark box at 4°C.
Immunohistochemistry
For immunohistochemical staining with anti-tyrosine hydroxylase (TH), anti-ionized calcium-binding adaptor molecule 1 (Iba1), anti-glial fibrillary acidic protein (GFAP), and anti-αSyn phospho-Ser129 primary antibodies (pSyn), the sections were washed in 1x PBS for 20 min on a shaker; then, peroxidase endogenous blocking was applied using 0.3% H2O2 at RT for 15 min. For blocking nonspecific binding of immunoglobulin, the sections were continuously incubated in 2% BSA in 0.3 % Triton X/PBS for 1 hour at RT. Primary antibodies were incubated overnight at 4°C and were further incubated with HRP-conjugated secondary antibodies for 2 hours at RT. Next, the brain sections were washed three times using PBS for 10 min. 3,3’-diaminobenzidine (DAB) reagent was then prepared with the following compositions: 2 drops of buffer, 4 drops of DAB, and 2 drops of peroxidase oxide in 5 mL distilled water. The brain sections were then developed in 1 mL DAB reagent in 3 minutes and were then immediately transferred to distilled water. Finally, the tissue was transferred to the coated slide with a mounting solution and was stored at 4°C.
Imaging and quantification
Fluorescence images were obtained using a confocal microscopy system (LSM 700, Carl Zeiss) or an Olympus BX51 conventional fluorescence microscopy with U-RFL-T power supply equipped with a 1.25x/0.04NA, 4x/0.1NA, 10X/0.3NA, 20X/0.5NA, and 40X/0.75NA objective lens. Fluorescence images were taken in different brain regions including the striatum, substantia nigra, piriform cortex, somatosensory cortex, amygdala, and hippocampus as shown in Supplementary Fig. S1c. Images were processed using the Zen software, Carl Zeiss, and ImageJ Fiji software.
In order to evaluate for TH, GFAP-positive astrocyte, and Iba1-positive microglia immunoactivity in the whole mouse striatum, and TH immunoactivity in the SN, coronal brain section images were obtained using an Olympus BX51 microscope equipped with a PLN 4X/0.1NA objective lens, and all parameters in terms of exposure time, contrast, and resolution were set in a similar manner. Semiquantitative optical density (OD) analysis of DAB images was carried out using ImageJ Fiji software, as per the modified protocol previously described 12–14. Briefly, the color of all images was corrected by applying color deconvolution with the DAB vector function in ImageJ. Next, the striatum of four 40 µm slices from each independent mouse was measured, one in each PFF-injected striatum and one in noninjected striatum at each time point by drawing the region of interest from four sections per brain region in order to yield eight mean gray values with a value of 0 and 255 for black and white, respectively. The observed gray level was converted to relative OD using the following formula OD = log (255/mean gray level) 12,13. Therefore, eight OD values in each animal were obtained. The measurement was then replicated in each time point. The resulting average OD in each time point was represented as a measurement for TH degeneration and microglial activation or astrocytic activation.
To evaluate for pSyn OD, similar method as mentioned above was applied on DAB images using ImageJ Fiji software. Briefly, pSyn OD measurement was carried out on the images that were captured at 10X magnification using a PLN 10X/0.3NA objective lens, and all parameters in terms of exposure time, contrast, and resolution were the same. The same method of OD was also applied to estimate pSyn expression in the striatum. The OD value in each animal and the measurement were replicated in each time point. The resulting average OD value was represented by pSyn OD in each mouse.
Western blot
Biochemical analysis of the mouse brain was performed, as per the modified protocol previously described 15. Briefly, the mouse brain was dissected into two parts, i.e., PFF-injected side and noninjected side of the brain excluding the cerebellum. The brain was homogenized in the nonionic detergent-soluble (NP40-soluble fraction) and ionic detergent (NP40-insoluble fraction). First, the mouse brain was homogenized in a nonionic detergent, which contained of 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 1 × phosSTOP, and 1 × protease inhibitor cocktail. The homogenate was centrifuged at 12000 rpm at 4°C for 20 min (centrifuge 5430R). Then, the resulting pellet and supernatant part (NP40-soluble fraction) were collected. The pellet was washed twice in a nonionic lysis buffer and was further homogenized in the ionic detergent, which contained of 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 1% SDS, 0.5% sodium deoxycholate, 1 × phosSTOP, and 1 × protease inhibitor cocktail. Then, the homogenate was incubated on ice for 5 min and was further centrifuged at 12000 rpm at 4°C for 40 min. Then, the resulting supernatant (NP40-insoluble fraction) was collected. Protein concentrations were determined using BCA assay. Proteins were kept in aliquots at –80°C until investigation.
The protein samples were treated for 3 min at 100°C in a sample-treated dye solution (5x Laemmli sample buffer) containing 2% SDS and 5% β-mercaptoethanol. Equal amounts of the protein samples from each group in soluble fraction or insoluble fraction were subjected to 12% SDS-PAGE for 2 hours at 80 V. The gel was transferred to a methanol-activated PVDF membrane (PVDF membrane was exposed in methanol 100% for 1 min and was soaked in transfer buffer before use) (Immobilon-P, pore size 0.45 µm, and cut size 6cm*8cm) in order to detect the interested proteins using the Mini Trans-Blot electrophoretic transfer cell (Bio-Rad) at 250 mA 90 V constant current for 1h and 30 min using a Model 200/2.0 Power Supply (Bio-Rad). Next, the transferred membranes were soaked in Ponceau S solution to be able to visualize the proteins and were washed three times for 5 min in 1x-TBST buffer.
For αSyn detection, the transferred membranes were treated using 0.4% PFA in PBS for 30 min at RT as previously described 16 to increase detection sensitivity. After the incubation, the membrane was washed thrice in 1x-TBST for 5 min and in 5% (w/v) nonfat dry milk blocking for 1 hour at RT. The membranes were then incubated with primary antibodies (ab212184, ab51253, Abcam) in a blocking buffer at 4°C overnight and were washed three times for 5 min in 1x-TBST buffer, which was followed by incubation with secondary antibodies (HRP conjugate) in a blocking buffer for 1h at RT.
To be able to detect the presence of other proteins, the transferred membranes were blocked in 5% (w/v) nonfat dry milk for 1 hour at RT. The membranes were then washed three times in 1x-TBST for 5 min, and they were then incubated in primary antibodies at 4°C overnight. Next, the membranes were washed three times for 5 min in 1x-TBST buffer and were then incubated in secondary antibodies (HRP conjugate) for 1 hour at RT. Finally, protein detections were conducted using ECL reagent and then were detected using ChemiDoc MP Imaging System. Quantitative comparisons between samples on the blots were processed in parallel. All raw blot and gel images are available in Supplementary Fig. S5.
Behavior testing
To assess the deterioration of motor symptoms, behavioral tests including rotarod, wire hang, and clasping tests were conducted at two time points –baseline (before injection at 2.5 months old) and at sacrifice day.
Rotarod test
Rotarod test was performed following the previous report with some modifications 17. Briefly, mice run on a 2.5-cm-diameter rotating apparatus at a speed of 40 rpm, and four initial training sessions composed of 5-min run and 5-min break were conducted. The rotarod machine was then cleaned with 70% ethanol in between each mouse. An hour later, the test trials were conducted, and the time each animal remained on the rotarod was recorded. Each mouse was able to stay on the rotarod for at least 30 s, and animals not falling off the rotarod after 300 s were given a maximum score of 300 s (≤30 s x ≤300 s). Each mouse was tested three times, and the average time was used for statistical analysis.
Wire hang test
The wire hang test was also performed 17. To summarize this, the mice were hanged on a 55-cm-long 2-mm-thick wire at a height of 90 cm. Four initial training sessions composed of 5-min hang and 5-min break were conducted. An hour later, the test trials were conducted, and the latency of mice to fall off the wire was recorded. Each mouse should stay on the wire for at least 10 s, and animals not falling off after 300 s were given a maximum score of 300 s (≤10 s x ≤300 s). The average values from the three trials were then used for analysis.
Clasping test
Hindlimb clasping during tail suspension was recorded in order to assess the behavioral phenotype and disease progression during the αSyn propagation. It was recorded for 10 s for each mouse using a hand camera, and the score was measured as described in the following 18. The score of hindlimb clasping was assigned from 0 to 4: 0, no limb clasping, normal escape extension; 1, one hindlimb exhibits incomplete splay with toes exhibiting normal splay; 2, both hindlimbs exhibit incomplete splay with toes exhibiting normal splay; 3, both hindlimbs exhibit clasping with curled toes and immobility; and 4, forelimbs and hindlimbs exhibit clasping and are crossed with curled toes and immobility 18.
Statistical analysis
In this study, statistical analysis of data was performed using Microsoft Excel and GraphPad Prism version 5 software (https://www.graphpad.com/). The mean value of OD in different groups and the score of clasping test were compared between groups using nonparametric ANOVA (Kruskal–Wallis test), which were then followed by post hoc analysis (Dunn’s test). Densitometric analysis of western blot was performed using Fiji (ImageJ) software, and the final values were compared using nonparametric ANOVA (Kruskal–Wallis test), which were followed by a post hoc analysis. The comparison of rotarod and wire hang test results at baseline and each time post injection were performed using Wilcoxon signed-rank test. The results were considered statistically significant for *p < 0.05, **p < 0.01, and ***p < 0.001.