Animals and ethics statement
WT (hereafter non-transgenic [nTg]) mice, homozygous WT BAC-SNCA Tg mice, and heterozygous A53T BAC-SNCA Tg mice with C57BL/6J background were used for this study. To genotype the progeny of WT BAC-SNCA Tg mice and A53T BAC-SNCA Tg mice, polymerase chain reaction was performed as described previously [20]. All experiment procedures were in accordance with the national guidelines. The Animal Research Committee of Kyoto University granted ethical approval and permission (MedKyo 17184).
Western blot analysis
Western blot analyses were conducted as described previously [21]. Briefly, phosphate buffer saline (PBS)-perfused brains were homogenized in 10 volumes (w/v) of 2% sodium dodecyl sulfate (SDS) buffer (150 mM NaCl, 1mM ethylenediaminetetraacetic acid, 10 mM Tris-HCl, 1% [v/v] Triton X-100, 2% [v/v] SDS) on ice, followed by sonication with a Bioruptor ultrasonic wave disruption system for 1 min and centrifugation at 20,400 g at 4°C for 15 min. The supernatant was boiled in sample buffer (1% [w/v] SDS, 12.5% [w/v] glycerol, 0.005% [w/v] bromophenol blue, 2.5% [v/v] 2-mercaptoethanol, 25 mM Tris-HCl, pH 6.8). Samples containing 18 μg of protein were loaded in each lane and separated on NuPAGE 4–12% (w/v) gradient gels (Invitrogen). Proteins were transferred to polyvinylidene difluoride membranes with a Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad). The membranes were treated with 4% (w/v) paraformaldehyde (PFA) in PBS for 30 min at room temperature before blocking to prevent detachment of α-Syn from the blotted membrane. For western blot analysis, the following primary antibodies were used: anti-human α-Syn (Invitrogen, #180215 [LB509], 1:500), anti-α-Syn (BD Transduction, #610787 [Syn-1], 1:2000), and anti-β-actin (Sigma-Aldrich, #A1978, 1:5000). After blocking with 5% (w/v) skim milk in PBS for 30 min, the membranes were incubated with primary antibodies at 4°C for 1 to 3 d, followed by reaction with horseradish peroxidase-conjugated secondary antibodies (Rockland Immunochemicals, TrueBlot ULTRA) for 1 h. Immunoreactive bands were detected with Chemi-Lumi One Super (Nacalai tesque), and the chemiluminescent signal was detected with Amersham Imager 600 (GE Healthcare). Densitometric analyses were performed using ImageJ software (National Institutes of Health).
Generation of α-Syn monomer and PFFs
α-Syn monomer and PFFs were generated as described previously with minor modifications [22, 23]. Briefly, mouse α-Syn and WT or A53T human α-Syn were expressed in Escherichia coli BL21 (DE3) (BioDynamics Laboratory) and were purified by boiling and ion exchange using Q sepharose fast flow (GE Healthcare). After dialyzed against dialysis buffer (150 mM KCl, 50 mM Tris-HCl, pH 7.5), α-Syn solution (7 mg/ml) was agitated at 37°C at 1,000 rpm for 10 d. After ultracentrifugation at 186,000 g at 20°C for 20 min, the pellets of α-Syn PFFs were resuspended in PBS (2.5 µg/µl) and then sonicated with a Bioruptor ultrasonic wave disruption system for 5 min before injections. The characterization of α-Syn PFFs before and after sonication was described previously [23, 24].
Stereotaxic surgery
Mice at 2–3 months of age were anesthetized with avertin (2,2,2-tribromoethanol) and then stereotaxically inoculated with 2 µl of α-Syn PFFs or PBS into the left dorsal striatum (coordinates: 0.2 mm relative to the bregma; 2.0 mm from the midline, −2.6 mm from the scull surface) using a 33-gauge Neuros syringe (Hamilton).
Immunohistochemical analysis
Mice were anesthetized with sevoflurane and transcardially perfused with 4% (w/v) PFA in PBS. The brains were removed and immersed in 4% (w/v) PFA in PBS at 4°C overnight. Paraffinized brains were sectioned with a thickness of 8 μm. For immunohistochemical and immunofluorescent analyses, the following primary antibodies were used: anti-phosphorylated-α-Syn (p-α-Syn) (Abcam, #ab51253 [EP1536Y], 1:5000), anti-p-α-Syn (Wako, #015-25191 [#64], 1:1000), anti-tyrosine hydroxylase (TH, Millipore, #MAB318 [LNC1], 1:5000), anti-TH (Millipore, #AB152, 1:500), anti-p62 (MBL, #PM045, 1:1000), anti-ubiquitin (DAKO, #Z0458, 1:1000), anti-ionized calcium-binding adapter molecule 1 (Iba1, Wako, #019-19741, 1:100), and anti-glial fibrillary acidic protein (GFAP, Sigma-Aldrich, #G3893, 1:500). The sections were incubated at 4°C with the primary antibodies for 1 to 2 d and then processed for visualization. As secondary antibodies, Histofine (Nichirei Bioscience) and Alexa Fluor 488 or 594-conjugated antibodies (Invtrogen) were used for diaminobenzidine staining and immunofluorescence, respectively. To evaluate p-α-Syn–positive pathology and the number of dopaminergic neurons in the SNpc, every 10th section was stained with the anti-p-α-Syn (EP1536Y) and anti-TH (LNC1) antibodies. The numbers of p-α-Syn–positive and TH-positive cells with visible nuclei were manually counted. Semiquantitative analyses were performed on seven coronal sections (2.96, 1.34, 0.14, −1.82, −2.92, −3.88, and −5.34 mm relative to bregma), and color coded onto heat maps. The extent of p-α-Syn–positive pathology was graded as sparse to severe pathology based on the following criteria; sparse, neuritic pathology or 2–3 LB-like inclusions; mild, 4–9 LB-like inclusions; moderate, 10–29 LB-like inclusions; severe, 30 or more LB-like inclusions seen with a 20× objective lens. Scores were determined by the observations of at least 2 mice for each group. To assess microglial activation and reactive astrogliosis, the percentage of Iba1- and GFAP-positive areas in the ipsilateral SNpc were measured with ImageJ (National Institutes of Health). For assessment of nigrostriatal dopaminergic neuron terminals, the TH optical density in the dorsal striatum at −0.22 mm, 0.26 mm, and 0.74 mm relative to the bregma was measured with ImageJ.
In vitro α-Syn fibrillization assay
In vitro α-Syn fibrillization assay was performed as described previously [25]. Briefly, a mixture consisting of mouse α-Syn monomer (0.5 mg/ml), WT or A53T human α-Syn monomer (0.5 mg/ml), and 0.5 μM thioflavin T were seeded with 1% (w/w) hPFF or mPFF, and then continuously agitated at 600 rpm. The fluorescence at 535 nm (excitation 450 nm) was measured every 0.5 h with a multi-label plate reader (PerkinElmer, 2030 ARVO X3). The initiation and elongation rates were defined as the inverse values of time to reach 25% and time from 25% to 75% of maximum thioflavin T fluorescence, respectively.
Behavioral analysis
Behavioral analyses were conducted as described previously [26]. Male mice were used for all the behavioral analyses. For the comparisons among PBS-injected nTg mice, PBS-injected A53T BAC-SNCA Tg mice, and mPFF-injected A53T BAC-SNCA Tg mice, a batch composed of 12, 13, and 13 mice, respectively, was subjected to the behavioral tests at 2 months post-injection. Before every test, the mice were habituated to the experimental environment for more than 30 min.
Apomorphine-induced rotational behavior test
Apomorphine-induced rotational behavior test was performed as previously described with minor modifications [27]. Mice received a subcutaneous injection of apomorphine (0.75 μg/g [body weight]) and were placed in an opaque cylinder of 30-cm diameter. After a 5 min habituation period, movements were recorded for 5 min. Both contralateral and ipsilateral full-body rotations are measured.
Muscular strength test
For the grip strength test, mice were lifted and held by their tail so that they grasped the wire grid of the grip strength meter (O’Hara & Co.) with their forepaws. The mice were then gently pulled backward by their tail with the posture horizontal until they released the grid. The greatest peak force measured among three tests was used for statistical analysis. For the wire hang test, mice were placed on a wire mesh, which was inverted and waved gently so that the mice gripped the wire. The latency to fall was recorded, with a 120-s cut-off time.
Elevated plus maze test
The elevated plus maze apparatus (O’Hara & Co.) consisted of two open arms (25 cm × 5 cm) and two enclosed arms of the same size with transparent walls. The arms were elevated to a height of 55 cm above the floor. Mice were placed in the central square of the maze (5 cm × 5 cm) and allowed to move freely for 10 min. The distance traveled, entries into the arms, and time spent in the arms, were recorded automatically.
Light/dark transition test
The apparatus for the light/dark transition test consisted of a cage (42 × 21 × 25 cm) divided into two sections of equal size by a partition with a door (O’Hara & Co.). One chamber was brightly illuminated (390 lux), whereas the other chamber was dark (2 lux). Mice were placed into the dark area and allowed to move freely between the two chambers through the open door for 10 min. The transitions between chambers, time spent in each chamber, and distance traveled were recorded automatically.
Open field test
Mice were placed at the center of the field inside an open field apparatus (Accuscan Instruments, 42 × 42 × 30 cm) and allowed to move freely for 30 min. The distance traveled, time spent in the center area (20 × 20 cm), vertical activity, and stereotypic movements were recorded automatically.
Rotarod test
The accelerating rotarod (Ugo Basile) was used for the rotarod test. The speed of the rotarod accelerated from 4 to 40 rpm over a 5-min period. Mice were placed on rotating drums (3 cm diameter), and the latency to fall was recorded. Mice were subjected to the test three times a day for two consecutive days.
Porsolt forced swim test
The apparatus for the Porsolt forced swim test consisted of four Plexiglas cylinders (20 cm height × 10 cm diameter). The cylinders were filled with water (23°C), up to a height of 7.5 cm. Mice were placed in the cylinders and allowed to move freely for 10 min. The immobile time and distance traveled were recorded automatically.
Y-maze test
Mice were placed at the end of one arm of the Y-maze apparatus (O’Hara & Co.) and allowed to move freely for 5 min. The distance traveled and series of arm entries were recorded. An alternation was defined as entries into all three arms on consecutive occasions. The number of maximum alternations was therefore the total number of arm entries minus two, and the percentage of alternations was calculated.
Tail suspension test
Mice were suspended 30 cm above the floor in a visually isolated area by adhesive tape placed ~1cm from the tip of the tail. The immobile time was recorded for 10 min.
Contextual and cued fear conditioning test
On day 1, conditioning was performed. Mice were placed in a test chamber inside a sound-attenuated chamber and allowed to explore freely for 2 min. A 55-dB white noise, which is conditioned stimulus (CS), was presented for 30 s, followed by a foot shock (2 s, 0.35mA) serving as unconditioned stimulus (US). Two more CS-US parings were supplied with 2-min interstimulus intervals. On day 2, contextual memory retention test was performed 24 h after conditioning in the same chamber. The immobile time and distance traveled were recorded automatically for 5 min. On day 3, auditory-cued fear retention test under altered context was performed in a different chamber. Mice were allowed to move freely for 3 min, and then CS was presented for 3 min. The immobile time and distance traveled were recorded automatically.
Statistical analysis
Statistical calculations were performed with GraphPad Prism software, Version 5.0. All the statistical tests conducted are described in the figure legends. An F test, a Brown–Forsythe test, and a Bartlett's test were performed to evaluate the differences in variances. Differences with p values of less than 0.05 were considered significant.