Rapid Induction of Dopaminergic Neuron Loss Accompanied by Lewy Body-Like Inclusions in A53T BAC-SNCA Transgenic Mice

Abstract

Parkinson’s disease (PD), the most common neurodegenerative movement disorder, is characterized by dopaminergic neuron loss in the substantia nigra pars compacta (SNpc) and intraneuronal α-synuclein (α-syn) inclusions. It is highly needed to establish a rodent model that recapitulates the clinicopathological features of PD within a short period to efficiently investigate the pathological mechanisms and test disease-modifying therapies. To this end, we analyzed three mouse lines, i.e., wild-type mice, wild-type human α-syn bacterial artificial chromosome (BAC) transgenic (BAC-SNCA Tg) mice, and A53T human α-syn BAC transgenic (A53T BAC-SNCA Tg) mice, receiving dorsal striatum injections of human and mouse α-syn preformed fibrils (hPFFs and mPFFs, respectively). mPFF injections induced more severe α-syn pathology in most brain regions, including the ipsilateral SNpc, than hPFF injections in all genotypes at 1-month post-injection. Although these Tg mouse lines expressed a comparable amount of α-syn in the brains, the mPFF-injected A53T BAC-SNCA Tg mice exhibited the most severe α-syn pathology as early as 0.5-month post-injection. The mPFF-injected A53T BAC-SNCA Tg mice showed a 38% reduction in tyrosine hydroxylase (TH)-positive neurons in the ipsilateral SNpc, apomorphine-induced rotational behavior, and motor dysfunction at 2 months post-injection. These data indicate that the extent of α-syn pathology induced by α-syn PFF injection depends on the types of α-syn PFFs and exogenously expressed α-syn in Tg mice. The mPFF-injected A53T BAC-SNCA Tg mice recapitulate the key features of PD more rapidly than previously reported mouse models, suggesting their usefulness for testing disease-modifying therapies as well as analyzing the pathological mechanisms.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13311-021-01169-5.

Introduction

Parkinson’s disease (PD) is the most common neurodegenerative movement disorder [1]. The pathological findings include the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and the presence of α-synuclein (α-syn) inclusions in the form of Lewy bodies (LBs) and Lewy neurites. Clinically, dopamine depletion in the nigrostriatal system causes motor symptoms, which can be partially relieved by dopamine replacement therapy. Although this symptomatic therapy is initially effective for motor symptoms, most patients suffer from treatment-resistant motor symptoms, dysphagia, autonomic failure, psychosis, and dementia years after the onset of PD [1, 2]. The pathological changes are also relentlessly progressive, seemingly associated with the progression of clinical symptoms. For instance, the motor dysfunction scores of the Unified Parkinson’s Disease Rating Scale (UPDRS) are linearly related to the neuronal density in the SNpc, which decreases with time [3]. Lewy pathology is also seen in the widespread brain regions in the late stage of PD, and its severity in the cortical areas and hippocampus correlates with cognitive decline in PD [4–6]. Thus, one of the greatest unmet therapeutic needs in PD is the development of disease-modifying therapies that could slow down the disease progression or change the course of the disease.

To date, all attempts to develop disease-modifying therapies for PD have failed. One of the biggest reasons for the past failures is the discrepancy between human PD and the animal models used for testing the candidate drugs. Toxin-based animal models, i.e., animals administered with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine or 6-hydroxydopamine, which rapidly induce dopaminergic neuron loss, have been often used to identify the potential disease-modifying therapies [7, 8]. However, their neurotoxic mechanisms are distinct from α-syn–induced cellular dysfunction and death. Indeed, only a few of the candidates identified in toxin-based animal models are still considered to have a potential for disease modification [7]. Meanwhile, certain point mutations in the α-syn gene (SNCA) and genomic duplication and triplication that contain the SNCA locus lead to autosomal dominant familial PD [9–12]. Based on these genetic findings, many α-syn transgenic (Tg) rodent models have been generated to recapitulate the α-syn–driven pathological changes in PD [7, 13]. Although these models have enhanced our understanding of fundamental pathological processes of PD, their usage has limitations for the development of disease-modifying therapies: none of them exhibit α-syn aggregation followed by dopaminergic neuron loss in the SNpc or behavioral abnormalities within several months of age [7]. To overcome their shortcomings, viral vector-based α-syn overexpressing rodent models have been also generated [14, 15]. They show PD-like phenotypes on the scale of weeks, the timeframes applicable to drug discovery. However, one criticism is that this is only achieved with extremely high α-syn expression levels in the SN (several to 20 times higher than the endogenous level), which far exceed both physiological and disease conditions.

Growing evidence suggests that pathological α-syn aggregates behave in a prion-like manner in terms of templated fibrilization and intercellular dissemination [16]. Injection of α-syn preformed fibrils (PFFs) into animals induces this process, formation of LB-like inclusions, and their subsequent intercellular spread [17, 18]. Particularly, striatal injection of α-syn PFFs recapitulates the features characteristic of PD, i.e., the formation of LB-like inclusions followed by dopaminergic neuron loss in the SNpc and motor dysfunction [17]. In WT mice injected with mouse α-syn PFFs in the striatum, phosphorylated-α-syn (p-α-syn) pathology was observed as early as 30 days post-injection, followed by gradual reduction in the number of TH-positive neurons in the SNpc, 15 and 35% loss at 90 and 180 days post-injection, respectively. Motor impairment was also detected by the wire hang test at 90 days post-injection. However, considering that the effects of candidate disease-modifying therapies are evaluated at the end point of a model when it shows some PD phenotypes, these features are expected to be shortened to accelerate the pace of research. Previous studies showed that the development of α-syn pathology after intracerebral PFF injections was promoted in α-syn Tg mice compared with WT mice [19–22]. However, this pathological acceleration depended on Tg mouse lines and types of α-syn PFFs [23, 24], and the degree of dopaminergic neuron loss was not evaluated.

We previously generated bacterial artificial chromosome (BAC) Tg mice harboring human α-syn gene (SNCA) and SNCA with an A53T mutation (hereafter, BAC-SNCA Tg mice and A53T BAC-SNCA Tg mice, respectively) [25, 26]. These Tg mice overexpressed exogenous human α-syn in the brains in a physiological pattern seen in WT mice, with a mild (~ 1.2–1.5-fold) increase of α-syn expression in the SN. The BAC-SNCA Tg mice did not exhibit any PD-related pathological or behavioral phenotypes, whereas the A53T BAC-SNCA Tg mice exhibited REM sleep behavior disorder (RBD)-like behavior and hyposmia, both of which are prodromal symptoms of PD, with proteinase K-resistant α-syn aggregation and slight dopaminergic neuron loss (17% at 18 months of age compared with that of age-matched WT mice). In the present study, we applied striatal injection of human and mouse α-syn PFFs (hPFFs and mPFFs, respectively) to these two SNCA Tg mouse lines to generate a novel mouse model showing key pathological and behavioral phenotypes of PD within a short period amenable to a rapid investigation of the pathological mechanisms and drug testing.

Methods

Animals and Ethics Statement

WT mice, homozygous BAC-SNCA Tg mice, and heterozygous A53T BAC-SNCA Tg mice with C57BL/6 J background were used for this study. To genotype the progeny of BAC-SNCA Tg mice and A53T BAC-SNCA Tg mice, polymerase chain reaction was performed as described previously [26]. All experiment procedures were in accordance with the national guidelines. The Animal Research Committee of Kyoto University granted ethical approval and permission (MedKyo 17,184).

Western Blot Analysis

Western blot analyses were conducted as described previously [27]. Briefly, phosphate buffer saline (PBS)–perfused brains were homogenized in 10 volumes (w/v) of 2% sodium dodecyl sulfate (SDS) buffer (150 mM NaCl, 1 mM 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-α-syn (BD Transduction, #610,787 [Syn-1], 1:2000), anti-human α-syn (Invitrogen, #180,215 [LB509], 1:500), anti-mouse α-syn (Cell Signaling, #4179 [D37A6], 1:1000), 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 days, 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 [28, 29]. 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 days. 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 [29, 30].

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: AP + 0.2 mm relative to the bregma, ML + 2.0 mm from the midline, DV − 2.6 mm from the skull 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-p-α-syn (Abcam, #ab51253 [EP1536Y], 1:5000), anti-p-α-syn (Wako, #015–25,191 [#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, #019–19,741, 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 days and then processed for visualization. As secondary antibodies, Histofine (Nichirei Bioscience) and Alexa Fluor 488 or 594-conjugated antibodies (Invitrogen) 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 Sects. (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; and 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 and averaged Iba1-positive area per microglia 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 [31]. 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 [32]. Male mice were used for all the behavioral analyses. For the comparisons among PBS-injected WT 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 [33]. 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 lx), whereas the other chamber was dark (2 lx). 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 2 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 ~ 1 cm 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, 0.35 mA) 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.

Results

BAC-SNCA Tg Mice and A53T BAC-SNCA Tg Mice Express an Almost Equal Amount of α-Syn in the Brains

In the present study, we used two SNCA Tg mouse lines, i.e., BAC-SNCA Tg mice and A53T BAC-SNCA Tg mice, both of which we generated previously [25, 26]. We used BAC-SNCA Tg and A53T BAC-SNCA Tg mice in homozygous and heterozygous states, respectively, because the latter line rarely produced homozygous Tg pups. We examined α-syn expression in the brains of these BAC-SNCA Tg mice as well as WT mice. These two SNCA Tg mouse lines expressed an almost equal amount of human and total α-syn in their brains, with the total α-syn overexpressed by 2.9 and 2.7-fold in BAC-SNCA Tg and A53T BAC-SNCA Tg mice, respectively, relative to WT mice (Fig. 1). However, the expression levels of endogenous mouse α-syn in both lines were lower than that in WT mice probably because of the negative feedback response to exogenous α-syn overexpression. Theoretically, the ratios of mouse α-syn to human α-syn expression levels are 1:3 and 1:8 in the BAC-SNCA mice and A53T BAC-SNCA mice, respectively. The expression pattern of the exogenous α-syn expressed by the BAC transgene closely resembled that of the endogenous mouse α-syn in WT mice [26]. Therefore, we presume that the ratios of human α-syn to mouse α-syn expression levels should be almost the same across the brains of each SNCA Tg line. The amount of total α-syn that heterozygous A53T BAC-SNCA Tg mice expressed as compared with that of WT mice was higher than what we reported previously, possibly because of the difference in detergent used to extract proteins (i.e., 1% Triton X-100 buffer for the previous studies [26, 32] and 2% SDS buffer for the present study).

Fig. 1.

α-Syn expression in the brains of BAC-SNCA Tg and A53T BAC-SNCA Tg mice. a Western blot analysis of whole brain lysates of BAC-SNCA Tg (SNCA), A53T BAC-SNCA Tg (A53T-SNCA), and WT mice with Syn-1 (human [Hu] and mouse [Mo] α-syn), LB509 (human α-syn), and D37A6 (mouse α-syn) antibodies. b, c, d Quantification of Syn-1, LB509, and D37A6-positive bands normalized by β-actin (n = 3). Results are shown as values relative to WT (b), A53T-SNCA (c), and WT (d). One-way ANOVA with Tukey’s post hoc test was performed; *p < 0.05, **p < 0.01, ***p < 0.001; n.s., not significant. Data are the mean ± SEM

Comparisons of Pathological Changes Among hPFF-, mPFF-, and PBS-Injected Mice at 1-Month Post-injection

The efficiency of templated fibrillization of α-syn is affected by the species of both monomeric α-syn substrate and fibrillar α-syn seed. Seeded fibrillization of α-syn across species is generally inefficient, which may be attributed to mismatches in amino acid sequences [31]. Because it remains unclear which fibrillar α-syn seed more efficiently induces fibrillization in SNCA Tg mouse lines, we injected hPFFs, mPFFs, or PBS into the unilateral dorsal striatum of WT mice, BAC-SNCA Tg mice, and A53T BAC-SNCA Tg mice. First, we conducted pathological analyses on all the groups at 1-month post-injection. P-α-syn immunohistochemistry revealed stronger p-α-syn immunoreactivity in the Tg mice injected with PBS than that in the WT mice injected with PBS in some brain regions (Fig. 2a, Supplementary file 1: Fig. S1). However, α-syn PFF-induced α-syn inclusions were clearly distinguished based on its even stronger p-α-syn staining intensity and morphology, as we reported previously [32]. The pathological analysis revealed that p-α-syn–positive inclusions in widespread brain regions of both hPFF- and mPFF-injected mice (Fig. 2a, f, Supplementary file 1: Fig. S1). mPFF-injected mice showed comparable to more severe α-syn pathology in most brain regions compared with hPFF-injected mice in all the genotypes (Fig. 2f). Specifically, the number of p-α-syn–positive cells in the ipsilateral SNpc was higher in mPFF-injected mice than that in hPFF-injected mice in all the genotypes despite no significant differences among the mPFF-injected groups (Fig. 2b). We confirmed that p-α-syn–positive inclusions seen in the ipsilateral SNpc were within TH-positive neurons (Fig. 2c). Moreover, most of the p-α-syn inclusions were immunopositive for p62 and ubiquitin (Supplementary file 2: Fig. S2), both of which are also positive in LBs. Since mPFF-injected mice showed more severe α-syn pathology than hPFF-injected mice in all the genotypes, we then evaluated the numbers of TH-positive neurons in the SNpc of mPFF-injected mice (Fig. 2d, e). mPFF-injected WT and BAC-SNCA Tg mice did not show significant change in the number of TH-positive neurons in the ipsilateral SNpc, whereas mPFF-injected A53T BAC-SNCA Tg mice showed significant decrease in the number of TH-positive neurons compared with the contralateral SNpc (Fig. 2e).

Fig. 2.

Comparisons of pathological changes among hPFF-, mPFF-, and PBS-injected mice at 1-month post-injection. a P-α-syn (EP1536Y) immunostaining of the ipsilateral SNpc. Scale bar 100 μm. b Number of p-α-syn–positive cells in the ipsilateral SNpc (n = 3–4). A two-tailed Student’s t-test was performed within each genotype; *p < 0.05, **p < 0.01. One-way ANOVA with Tukey’s post hoc test was performed among mPFF-injected groups; n.s., not significant. c Double immunostaining for TH and p-α-syn (#64) in the ipsilateral SNpc of mPFF-injected A53T BAC-SNCA Tg mice. Scale bar 20 μm. d TH immunostaining of the SNpc of mPFF-injected mice. Scale bar 100 µm. e Number of TH-positive cells in the SNpc of mPFF-injected mice (n = 4). A two-tailed Student’s t-test was performed within each genotype; *p < 0.05; n.s., not significant. Data are the mean ± SEM. f Heat map colors represent the extent of α-syn pathology (light yellow, sparse pathology; yellow, mild pathology; orange, moderate pathology; red, severe pathology). Left is the injection (ipsilateral) side

Collectively, injection of mPFFs induced more severe α-syn pathology in all the genotypes at 1-month post-injection than that of hPFFs. Among mPFF-injected mice, A53T BAC-SNCA Tg mice showed significant decrease in the number of TH-positive neurons in the ipsilateral SNpc as early as 1-month post-injection.

Rapid Formation of α-Syn Pathology in mPFF-Injected A53T BAC-SNCA Tg Mice

Although the severity of α-syn pathology is similar between mPFF-injected BAC-SNCA Tg and A53T BAC-SNCA Tg mice at 1-month post-injection, decrease in the number of TH-positive neurons was already seen in the latter mice at the time point (Fig. 2e). These observations prompted us to go back to an earlier time point to see the extent of α-syn pathology. The pathological analysis of mPFF-injected mice at 0.5-month (2 weeks) post-injection clearly showed more severe α-syn pathology in most brain regions in the mPFF-injected A53T BAC-SNCA Tg mice than those in the mPFF-injected WT and BAC-SNCA Tg mice (Fig. 3a–c). These results suggest that mPFF injection induces a more rapid formation of α-syn pathology and subsequent decrease in the number of TH-positive neurons in A53T BAC-SNCA Tg mice than those in WT and BAC-SNCA Tg mice [34]. Indeed, the α-syn pathology was less severe in most brain regions of the mPFF-injected A53T BAC-SNCA Tg mice at 1-month post-injection than at 0.5-month post-injection (Figs. 2f and 3c).

Fig. 3.

Comparisons of pathological changes in the mPFF-injected mice at 0.5-month post-injection. a P-α-syn (EP1536Y) immunostaining of the ipsilateral SNpc, basolateral amygdala (BLA), and entorhinal cortex (Ent) of mPFF-injected mice. Scale bar 100 μm. b Number of p-α-syn–positive cells in the ipsilateral SNpc of mPFF-injected mice (n = 3–4). One-way ANOVA with Tukey’s post hoc test was performed; **p < 0.01. Data are the mean ± SEM. c Heat map colors represent the extent of α-syn pathology (light yellow, sparse pathology; yellow, mild pathology; orange, moderate pathology; red, severe pathology). Left is the injection (ipsilateral) side

In Vitro Fibrillization Assay Replicates the Rapid Formation of α-Syn Pathology in mPFF-Injected A53T BAC-SNCA Tg Mice

To analyze the kinetics of α-syn fibrillization in the conditions that recapitulate the SNCA Tg mice injected with α-syn PFFs, we conducted an in vitro α-syn fibrillization assay. We prepared two α-syn monomer mixtures: one was a mixture of mouse and WT human α-syn monomers that represent BAC-SNCA Tg mice, and the other was a mixture of mouse and A53T human α-syn monomers that represent A53T BAC-SNCA Tg mice. For simplicity, we used 1:1 ratio of mouse and human α-syn monomers. After seeded with hPFFs or mPFFs, they were constantly agitated, and the fluorescence of thioflavin T was monitored as the extent of α-syn fibrillization. Both mixtures of α-syn monomers seeded with mPFFs showed more rapid initial fibrillization than those seeded with hPFFs (Fig. 4a–d). In addition, the mixture of mouse and A53T human α-syn monomers showed more rapid elongation of fibrillization than the mixture of mouse and WT human α-syn monomers (Fig. 4a–d). These in vitro results are consistent with in vivo results showing mPFF-injected A53T BAC-SNCA Tg mice showed the most rapid and severe pathology among the models tested (Fig. 2a, b, f, Fig. 3, Supplementary file 1: Fig. S1). These results may be partially explained by the fact that 53rd amino acid is homologous between mouse α-syn and A53T human α-syn [31].

Fig. 4.

A53T human α-syn fibrillizes more rapidly than WT human α-syn in the presence of mouse α-syn in in vitro α-syn fibrillization assay. a, b Mixture of mouse α-syn and WT or A53T human α-syn monomers seeded with hPFFs, mPFFs, or PBS and then constantly agitated. Fluorescence of thioflavin T was plotted every 2 h (n = 4). c Initiation rates represent the inverse values of time to reach 25% of the maximum fluorescence. d Elongation rates represent the inverse values of time from 25 to 75% of the maximum fluorescence. One-way ANOVA with Tukey’s post hoc test was performed among mixtures of mouse α-syn and WT or A53T human α-syn monomer groups; **p < 0.01, ***p < 0.001; n.s., not significant. A two-tailed Student’s t-test was performed within mPFF-seeded groups; ####p < 0.0001; n.s., not significant. Data are the mean ± SEM

Pathological Characterization of mPFF-Injected A53T BAC-SNCA Tg Mice

The results we obtained so far indicate that mPFF-injected A53T BAC-SNCA Tg mice have the potential as a novel PD mouse model that fulfills the purpose of this study. Therefore, we conducted pathological analyses up to 4 months post-injection to further characterize this model. The number of p-α-syn–positive cells in the ipsilateral SNpc was the highest at 0.5-month post-injection and decreased over time, reaching almost zero at 4 months post-injection (Fig. 5a, d). The number of TH-positive neurons in the ipsilateral SNpc also decreased over time, reaching 38% and 46% reductions at 2 and 4 months post-injection, respectively (Fig. 5b, e). Interestingly, the sum of the number of p-α-syn–positive cells and the decrease in the number of TH-positive neurons was almost constant throughout the time course (Fig. 5d, e), suggesting the loss of α-syn inclusion–bearing dopaminergic neurons. However, we cannot completely exclude the possibility that α-syn inclusion–bearing neurons lost the TH expression [35]. TH immunostaining in the dorsal striatum can be used to assess the degeneration of nigrostriatal dopaminergic neuron terminals [17, 36]. TH optical density in the dorsal striatum was not changed in the PBS-injected A53T BAC-SNCA Tg mice, whereas that was reduced by 42% in mPFF-injected A53T BAC-SNCA Tg mice at 0.5-month post-injection, without further reduction throughout the time course (Fig. 5c, f). We evaluated α-syn pathology throughout the brains and found dynamic changes in the severity of the α-syn pathology, depending on the brain region and time point (Fig. 5g, Supplementary files 3 and 4: Figs. S3 and S4), which could be attributed to the combination of loss of α-syn inclusion–bearing neurons and newly formed α-syn pathology. This fast turnover in the A53T BAC-SNCA mice compared with WT mice reported previously [17] suggests faster interneuronal transmission of α-syn pathology in the A53T BAC-SNCA than WT mice. We also examined glial involvement in this model. Iba1 immunostaining revealed an increase in Iba1-positive area, the number of microglia, and the averaged Iba-1-positive area per microglia with hypertrophic change in the ipsilateral SNpc at 0.5-month post-injection (Fig. 6a–e). GFAP immunostaining also revealed trends toward an increase in the GFAP-positive area in the ipsilateral SNpc especially at 1-month post-injection, suggesting the possible involvement of reactive astrogliosis (Supplementary file 6: Fig. S6).

Fig. 5.

Pathological characterization of the mPFF-injected A53T BAC-SNCA Tg mice. A53T BAC-SNCA Tg mice injected with mPFFs (A53T-mPFF) or PBS (A53T-PBS). Month post-injection is abbreviated as mpi. a, b P-α-syn (EP1536Y) and TH immunostaining of the ipsilateral SNpc. Scale bar 100 μm. c TH immunostaining of the striatum. Left is the injection (ipsilateral) side. d Number of p-α-syn–positive cells in the ipsilateral SNpc (n = 3–4). One-way ANOVA with Tukey’s post hoc test was performed; *p < 0.05, **p < 0.01. e Number of TH-positive cells in the ipsilateral and contralateral SNpc (n = 3–5). Two-way ANOVA with post hoc Sidak’s multiple comparison test was performed; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. f TH optical density ratio of ipsilateral to contralateral side of the dorsal striatum (n = 3–5). One-way ANOVA with Tukey’s post hoc test was performed; *p < 0.05, **p < 0.01. g Heat map colors represent the extent of α-syn pathology (light yellow, sparse pathology; yellow, mild pathology; orange, moderate pathology; red, severe pathology). Asterisks indicate the injection sites. Data are the mean ± SEM

Fig. 6.

Microglial activation in the ipsilateral SNpc of the mPFF-injected A53T BAC-SNCA Tg mice. a, b Double immunostaining for p-α-syn (#64) and Iba1 in the ipsilateral SNpc of mPFF-injected A53T BAC-SNCA Tg mice. Scale bar 100 μm (a). Scale bar 50 μm (b). c Percentage of Iba1-positive area in the ipsilateral SNpc of mPFF-injected A53T BAC-SNCA Tg mice (n = 3–4). Kruskal–Wallis test followed by Dunn’s multiple comparisons test: PBS vs. 0.5mpi, *p < 0.05. d Number of microglia/10⁵ μm² in the ipsilateral SNpc of mPFF-injected A53T BAC-SNCA Tg mice (n = 3–4). Kruskal–Wallis test followed by Dunn’s multiple comparisons test: PBS vs. 0.5mpi, *p < 0.05. e Averaged Iba1-positive area per microglia (μm²) in the ipsilateral SNpc of mPFF-injected A53T BAC-SNCA Tg mice (n = 3–4). Kruskal–Wallis test followed by Dunn’s multiple comparisons test: PBS vs. 0.5mpi, *p < 0.05. Data are the mean ± SEM

We also conducted pathological analyses on WT mice injected with mPFFs up to 2 months post-injection and found a constant increase in the number of p-α-syn–positive cells without obvious decrease in the number of TH-positive neurons in the SNpc (Supplementary file 5: Fig. S5a, b, d, e). These results further support the accelerated formation of α-syn pathology and neuron loss in the mPFF-injected A53T BAC-SNCA Tg mice. Indeed, the mPFF-injected WT mice at 2 months post-injection showed a similar number of p-α-syn–positive cells and distribution of α-syn pathology to those seen in the mPFF-injected A53T BAC-SNCA Tg mice at 0.5-month post-injection (Fig. 5d, g, Supplementary file 5: Fig. S5d, g). TH optical density was reduced by 18% in the mPFF-injected WT mice at 0.5-month post-injection, reaching a 30% reduction at 2 months post-injection (Supplementary file 5: Fig. S5c, f). This suggests that the degeneration of dopaminergic neuron terminals is more severe in the mPFF-injected A53T BAC-SNCA Tg mice than in the mPFF-injected WT mice and precedes decrease in the number of TH-positive neurons in the SNpc in both groups.

Behavioral Features of mPFF-Injected A53T BAC-SNCA Tg Mice

We conducted a battery of behavioral analyses including an apomorphine-induced rotational behavior test on the mPFF-injected A53T BAC-SNCA Tg mice together with the PBS-injected WT mice and PBS-injected A53T BAC-SNCA Tg mice as controls at 2 months post-injection. Apomorphine-induced rotational behavior test revealed an increased number of contralateral rotations (away from the injection side) in the mPFF-injected A53T BAC-SNCA Tg mice (Fig. 7a, b, Supplementary files 8–10: Video 1–3), suggesting dopamine hypersensitivity on the ipsilateral side [37]. Latency to fall in rotarod test and total distance in open field test were decreased in the mPFF-injected A53T BAC-SNCA Tg mice, even though both only attained statistical significance when comparing two groups, suggesting possible motor incoordination and reduced locomotion, respectively, in this model (Fig. 7c–e). Wire hang test revealed a decrease in latency to fall in the mPFF-injected A53T BAC-SNCA Tg mice, suggesting muscle weakness in this model (Fig. 7f). We also evaluated memory impairment, anxiety-like behavior, and depression-like behavior by other behavioral tests; however, we found no significant abnormalities in the mPFF-injected A53T BAC-SNCA Tg mice (Supplementary file 7: Fig. S7).

Fig. 7.

Behavioral abnormalities in the mPFF-injected A53T BAC-SNCA Tg mice. WT mice injected with PBS (WT-PBS), A53T BAC-SNCA Tg mice injected with PBS (A53T-PBS), and A53T BAC-SNCA Tg mice injected with mPFFs (A53T-mPFF) at 2 months post-injection (n = 12–13). a, b Number of rotations to the contralateral and ipsilateral directions in apomorphine-induced rotational behavior test. One-way ANOVA with Tukey’s post hoc test was performed; *p < 0.05, ***p < 0.001. c Latency to fall in rotarod test. Two-way repeated measures (RM) ANOVA was performed; Comparing three groups, effect of group, F_2,35 = 2.609, p = 0.088. Comparing A53T-mPFF with WT-PBS, effect of group, F_1,23 = 5.814, p = 0.024. d, e Open field test. d Moving distance plotted every 2 min. Two-way RM ANOVA was performed; comparing three groups, effect of group, F_2,35 = 2.101, p = 0.138. Comparing A53T-mPFF with WT-PBS, effect of group, F_1,23 = 5.31, unadjusted p = 0.031. e Total distance. One-way ANOVA with Tukey’s post hoc test was performed; n.s., not significant. Comparing A53T-mPFF with WT-PBS, a two-tailed unpaired Student’s t-test showed unadjusted p = 0.031. f Latency to fall in wire hang test. One-way ANOVA with Tukey’s post hoc test was performed; *p < 0.05. Data are the mean ± SEM

Discussion

In the present study, we applied striatal injection of two types of α-syn PFFs, i.e., hPFFs and mPFFs, to two SNCA Tg mouse lines to generate a novel PD mouse model useful for the investigation of pathological mechanisms and drug testing. Among the groups we analyzed, the A53T BAC-SNCA Tg mice injected with mPFFs exhibited the most severe LB-like pathology as early as 0.5-month post-injection. These observations are consistent with our recent report about A53T BAC-SNCA Tg mice injected with mPFFs into the olfactory bulb [32]. The mPFF-injected A53T BAC-SNCA Tg mice exhibited motor dysfunction and a 38% reduction of TH-positive neurons in the SNpc at 2 months post-injection. Given that many clinical trials evaluate the disease-modifying effects of candidate drugs by measuring changes in the UPDRS motor dysfunction scores at the endpoints, the motor dysfunction seen in this model within a short period after α-syn PFF injection could be used as reliable indicators for exploring potential disease-modifying therapies. Since the major pathological processes of this model are assumed to be neuronal uptake of α-syn PFFs, templated fibrillization of α-syn in neurons, interneuronal dissemination of pathological α-syn aggregates, and α-syn aggregation-induced neuron loss, the best application of this model would be testing candidate drugs intervening those processes. Of note, TH-immunoreactivity in the striatum and microglial activation in the SNpc was observed at 0.5-month post-injection, preceding obvious decrease in the number of TH-positive neurons.

Cross-seeded aggregation of human and mouse α-syn is bidirectionally restricted, whereas seeded aggregation of α-syn within the same host is efficiently induced [31]. However, it remains unclear how efficiently α-syn aggregation could be induced by α-syn PFF injection in SNCA Tg mice expressing both human and endogenous mouse α-syn. In the present study, we tested two types of SNCA Tg mice and two types of α-syn PFFs and identified the mPFF-injected A53T BAC-SNCA Tg mice as the model showing the most rapid formation of α-syn pathology and subsequent neuron loss. However, there are possible ways to further accelerate the phenotypes of α-syn PFF-injected mice. For instance, human S87N α-syn PFFs were reported to show greater seeding activity in mouse primary neurons and WT mice than mPFFs [31]. Previous studies reported that the deletion of endogenous mouse α-syn exacerbated the α-syn pathology in SNCA Tg mice [38, 39]. Therefore, it might be worth trying to use different types of PFFs and delete endogenous mouse α-syn in SNCA Tg mice in future studies.

Previous studies with a similar concept to that of the present study reported a rat PD model injected with α-syn PFFs in combination with adeno-associated virus (AAV)-mediated overexpression of α-syn [40, 41]. Thakur et al. first injected human α-syn–expressing AAV at a low dose in the SN and ventral tegmental area of WT rat to avoid the induction of acute dopaminergic neuron loss [40]. They subsequently received injection of human α-syn PFFs into the same brain regions 4 weeks later, showing LB-like inclusions and a 55% reduction of dopaminergic neurons in the SN and impaired motor behavior 3 more weeks later [40]. Hoban et al. injected α-syn–expressing AAV and human α-syn PFFs simultaneously into the two sites of SN, inducing a ~ 60% reduction of dopaminergic neurons in the SN and impaired motor behavior at 4 weeks post-injection [41]. These rat models may have some advantages and disadvantages compared to our mouse model; application of WT human α-syn overexpression and human α-syn PFFs may faithfully replicate pathobiology of human PD, while direct, multiple injections into the SN may cause tissue damage and phenotypic variations among samples. Meanwhile, mPFF-injected WT mice, the current protracted model, are still an option for testing disease-modifying therapies in a sense that they allow longer windows of opportunity to test the therapies. It is necessary to choose the best model for future studies depending on the purposes.

The reduction in TH density in the dorsal striatum is one of the earliest pathological changes in the mPFF-injected A53T BAC-SNCA Tg mice. There was a greater degree of reduction in TH density in the dorsal striatum than that in TH-positive neurons in the SNpc at 0.5-month post-injection. Similar observations have been reported by multiple studies carried out on subjects with PD. Loss of SN neurons and striatal dopamine terminals were estimated to be ~ 30% and ~ 70%, respectively, at the onset of motor symptoms [42]. Consistently, distal-dominant degeneration in Lewy pathology-bearing neurons has been reported. A previous study focusing on the cardiac sympathetic nervous system of cases with incidental LBs and subjects with PD revealed that the accumulation of α-syn aggregates commences in the distal axons, followed by axonal degeneration and the formation of α-syn aggregates in the somata and neurites [43]. In the present study, we conducted pathological analysis from 0.5-month post-injection, when robust p-α-syn–positive somatic inclusions were already observed in the dopaminergic neurons of the SNpc in the mPFF-injected A53T BAC-SNCA Tg mice. Further details of degenerative processes should be examined at earlier time points in future studies.

Microglial activation has been well described in subjects with PD [44–46]. Lines of evidence have suggested that α-syn–induced microglial activation plays both beneficial and detrimental roles in neurodegenerative processes. Activated microglia engulf neuron-released α-syn into autophagosomes for degradation via selective autophagy, which protects against neurodegeneration [47]. Meanwhile, neuron-released oligomeric α-syn induces neurotoxic microglial activation through Toll-like receptor 2 (TLR2) [48]. It has also been demonstrated that recombinant monomeric, C-terminal, and fibrillar α-syn are phagocytosed through TLR4 by microglia, inducing proinflammatory cytokine release and reactive oxygen species production [49]. In the present study, microglial activation shown by hypertrophic change [50, 51] and an increase in the number were observed before obvious dopaminergic neuron loss in the mPFF-injected A53T BAC-SNCA Tg mice, suggesting that microglia may play some roles in the degenerative processes in this model. In this regard, some interventions on microglial functions could be also tested in this model.

One of the caveats about the mPFF-injected A53T BAC-SNCA mice is that this model is generated by a combination of PFF injection, α-syn overexpression, and the A53T mutation, none of which is seen in sporadic PD patients. This suggests that pathological mechanisms seen in this model may differ from that in sporadic PD patients. Therefore, it may be better to test disease-modifying therapies in multiple PD models depending on their therapeutic targets before clinical trials.

Conclusions

We generated a novel mouse model which recapitulates several pathological and behavioral features of PD within a short period by applying α-syn PFF injection to A53T BAC-SNCA Tg mice. This novel model will be useful in the development of disease-modifying therapies as well as the investigation of pathological mechanisms in PD research.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

PD

Parkinson’s disease

SNpc

Substantia nigra pars compacta

α-syn

α-Synuclein

LBs

Lewy bodies

PFFs

Preformed fibrils

hPFFs

Human α-syn preformed fibrils

mPFFs

Mouse α-syn preformed fibrils

BAC-SNCA Tg

Wild-type human α-syn BAC transgenic

A53T BAC-SNCA Tg

A53T human α-syn BAC transgenic

TH

Tyrosine hydroxylase

UPDRS

Unified Parkinson’s Disease Rating Scale

WT

Wild-type

BAC

Bacterial artificial chromosome

RBD

REM sleep behavior disorder

PBS

Phosphate buffer saline

SDS

Sodium dodecyl sulfate

PFA

Paraformaldehyde

p-α-syn

Phosphorylated-α-syn

Iba1

Ionized calcium-binding adapter molecule 1

GFAP

Glial fibrillary acidic protein

CS

Conditioned stimulus

US

Unconditioned stimulus

AAV

Adeno-associated virus

TLR

Toll-like receptor

BLA

Basolateral amygdala

Ent

Entorhinal cortex

mpi

Month post-injection

Ctx

Motor cortex

WT-PBS

WT mice injected with PBS

A53T-PBS

A53T BAC-SNCA Tg mice injected with PBS

A53T-mPFF

A53T BAC-SNCA Tg mice injected with mPFFs

CA1

Cornu ammonis 1

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Funding

This research is supported by the program for Brain Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS) from Ministry of Education, Culture, Sports Science, MEXT, and the Japan Agency for Medical Research and Development, AMED under the Grant Numbers JP18dm0207020 and JP20dm0207070 (RT), JSPS KAKENHI Grant Number JP17H05698 and JP18H04041 (RT), and Japan Science and Technology Agency, JST Moonshot R&D–MILLENNIA Program Grant Number JPMJMS2024 (MI, HY and RT). japan agency for medical research and development,JP18dm0207020,Ryosuke Takahashi,JP20dm0207070,Ryosuke Takahashi,japan society for the promotion of science,JP17H05698,Ryosuke Takahashi,JP18H04041,Ryosuke Takahashi,Strategic Promotion of Innovative R and D,JPMJMS2024,Ryosuke Takahashi

Declarations

Ethics Approval and Consent to Participate

Details of ethical approval and permission about animal research are described in the “Methods” section. All applicable international, national, and/or institutional guidelines for the welfare and usage of animals were followed.

Competing Interests

The authors declare no competing interests.