Depopulation of dense α-synuclein aggregates is associated with rescue of dopamine neuron dysfunction and death in a new Parkinson’s disease model

This study aimed to generate and characterize a novel transgenic mouse model of PD that would enhance our understanding of the relationship between progressive αSyn aggregation and mechanisms of disease and allow testing prospective therapies for α-synucleinopathies. The homozygous line expressing 1–120 hαSyn under the TH promoter, in a null endogenous αSyn background was generated and used for the experiments. The αSyn-null C57Bl/6S strain was used as a control line. C57Bl/6J mice, expressing endogenous mouse αSyn were also included in the experiments where transgenic protein expression was compared to physiological mouse αSyn. Moreover, DA characteristics in MI2 mice were compared with both C57Bl/6S and C57Bl/6J mice to confirm that differences between C57Bl/6S and MI2 mouse lines were due to the expression of the transgene rather than the absence of αSyn in the C57Bl/6S animals.

MI2 and control mice were obtained from separate lines, therefore their genotypes were known at the time of experimental design. However, for behavioral experiments and anle138b-treatment mice were assigned randomly to the experimental groups. For each experiment, mouse numbers and statistical tests are described in the figure legends and in Online Resource. Analysis of dopaminergic impairment, namely measurements of DA in striatal homogenates and microdialysates, stereological counting of nigral cells and striatal innervation were performed by researchers blinded to experimental conditions.

The MI2 mouse line was generated using the same procedures as described by Tofaris et al. for α-Syn120 mice [35]. Briefly, a transgene construct containing human truncated 1–120 αSyn, subcloned downstream of the rat TH promoter (Fig. 1a) was injected into αSyn-null C57Bl/6OlaHsd (C57Bl/6S) mouse [31] pronuclei. The presence of the transgene was detected in the founders and progeny by PCR. Founders were then bred with αSyn-null C57Bl/6S mice and mouse αSyn-negative/1–120 hαSyn-positive progeny were crossed to homozygosity.

To analyze the solubility of 1–120-hαSyn species, sequential extraction of protein was performed using buffers of increasing strength. Following dissection, tissue was homogenized in TBS, incubated on ice for 15 min and centrifuged at 120,000×g for 30 min, at 4 °C. The supernatant was collected for western blotting analysis, and the pellet was washed three times with TBS, resuspended in TBS + 1% Triton-X100, incubated on ice for 15 min and centrifuged at 120,000×g for 30 min at 4 °C. These steps were then repeated using RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.1% SDS, 0.05% sodium deoxycholate, 1% NP-40), and 8 M urea (Sigma) with 5% SDS (Sigma), with the exception that urea/SDS step was performed at room temperature rather than 4 °C. All the solutions used in this procedure were supplemented with 1 × protease inhibitor cocktail.

Following 5 min at 95 °C in 1 × Laemmli buffer, samples were separated by SDS-PAGE, blotted onto nitrocellulose membranes (Bio-Rad) and proteins were crosslinked to the membrane with 4% paraformaldehyde (PFA) for 30 min. Non-specific background was blocked with 5% milk in TBST before overnight incubation of the membrane at 4 °C with primary antibodies (mouse anti-αSyn (Syn1), BD Biosciences, 1:500; rabbit anti-TH, Abcam, 1:1000; rabbit anti-β-actin, Abcam, 1:10,000) in 5% milk. Membranes were then incubated with peroxidase-conjugated secondary antibodies (GE Healthcare or DAKO, 1:5000) and the blots imaged using a Chemi Doc MP imager (Bio-Rad), using West Dura Extended Duration Chemiluminescent Substrate (Thermo Fisher Scientific). Blots were analyzed using Image Lab 5.1 (Bio-Rad Laboratories).

Bands corresponding to mouse full length monomeric (~ 17 kDa) and 1–120 truncated human αSyn (~ 14 kDa) were observed in C57Bl/6J and MI2 mice, respectively. No αSyn was present in C57Bl/6S (Fig. 1c). Non-specific bands recognized by the Syn1 antibody were previously reported (Fig. 2c) [29].

Mice were anesthetized by an intraperitoneal administration of pentobarbital (Merial Animal Health) and transcardially perfused with ice-cold PBS, followed by 4% PFA in PBS. Brains were post-fixed overnight in 4% PFA, then transferred to 30% sucrose in PBS with 0.1% NaN₃ (Sigma) and stored at 4 °C. Brains were frozen and 30 µm sections cut using a freezing microtome (Bright). Endogenous peroxidase activity was inhibited by treatment with 3% H₂O₂ containing 20% methanol in PBST. Sections non-specific background was blocked with 5% normal horse serum (NHS, Vector) in PBST. Antigen retrieval was performed before Syn1 staining, using 10 mM sodium citrate buffer pH 8.5 at 80 °C for 30 min. Free floating sections were incubated overnight at RT with primary antibodies diluted in PBST (Syn1, 1:500; rabbit or chicken anti-TH, Abcam, 1:1000; mouse anti-NeuN, Millipore, 1:1000, rabbit anti-VAMP2, Abcam, 1:250; rabbit anti-ubiquitin (Ubi-1), Millipore, 1:500) and then with biotinylated (Vector, 1:2000) or Alexa Fluor-conjugated (Thermo Fisher Scientific, 1:500) secondary antibodies diluted in PBST containing 5% NHS. Peroxidase-based staining was developed using Vectastain Elite ABC HRP Kit (Vector) and DAB Peroxidase Substrate Kit (Vector). Sections were mounted on microscope slides, dehydrated, cleared in xylene and coverslipped using DPX Mountant (Sigma). In some experiments, sections were counterstained with 0.1% cresyl violet (Sigma). For immunofluorescence nuclei were counterstained with DAPI (0.5 µg/ml; Roche) and sections coverslipped using FluorSave mounting medium (Calbiochem).

To determine the presence of filamentous αSyn aggregates, brain sections from MI2 and control mice were incubated with either Thioflavin T (TfT, Sigma; 0.2 μM in deionized water, for 10 min) or pentameric formyl thiophene acetic acid (pFTAA, kind gift from P. Nilsson; 0.5 μM in PBST, for 45 min). αSyn staining with Syn1 antibody, revealed by 647 nm Alexa Fluor-conjugated secondary antibody, was performed before TfT or pFTAA to determine the specificity of the staining. Sections were washed extensively with PBST before incubation with DAPI. To further investigate the solubility of the αSyn aggregates serial sections were treated with proteinase PK (PK; Sigma) prior to αSyn immunohistochemistry to digest soluble αSYN. Prior to the non-specific background blocking step, the slices were incubated with PK (20 μg/ml, 10 min, 37 °C, at RT), and washed twice with PBST supplemented with 5 mM phenylmethylsulfonyl fluoride (PMSF; Sigma) to inhibit residual PK activity. For these experiments immunostaining to detect αSyn was performed with Syn1 antibody as described above. DAB-stained sections were analyzed using Olympus BX 53 light microscope and fluorescent sections using a Leica DMI 4000B epifluorescent microscope.

For dSTORM, 30 µm free floating striatal slices were stained using a similar protocol, but PBST containing 0.25% Tween 20 was used and the non-specific background blocking solution contained 5% goat serum and 1% bovine serum albumin. Sections were stained with Syn204 mouse anti-human αSyn antibody (Abcam, 1:250) and Alexa Fluor 647 secondary anti-mouse antibody (Thermo Fisher Scientific, 1:500). In some experiments striatal sections were co-stained with rabbit anti-synaptobrevin 2 (VAMP2) antibody (Synaptic Systems, 1:500), followed by Cy3B anti-rabbit secondary antibody (1:250) to confirm synaptic localization of 1–120 hαSyn by dSTORM.

dSTORM images were taken using the Vutara microscope as described in [3], at power of 40% 647 and 2% 405 laser, and 3000 frames were taken for each movie. Localizations were gathered following the movie with a ratio of 10 of background to noise. All movies were then post analyzed using a denoise level of 0.5.

Cluster analysis, mean diameter cluster analysis and the number of localizations per clusters were determined using the density based algorithm (dbscan) followed by 2D principal component analysis and outliers detection as described in [3] (see Supplementary Fig. S4, Online Resource 1 for more details). For anle138b vs placebo treatment analysis, clusters in both conditions were additionally filtered according to inner density threshold determined according to the placebo condition per imaging day to avoid large-homogenous distributions that could form artifact clustering. Mean density of cluster was calculated for the placebo condition on the individual day of measurement and clusters with less than 10% of this density were excluded from analysis in both placebo and anle138b treatment conditions measured on the same day. The localizations that were accounted for very low-density clusters were in both cases added to the free (unclustered) populations of the proteins.

Bilateral striata were isolated from frozen brains, weighed and homogenized in 0.2 M perchloric acid. Homogenates were centrifuged at 6000×g for 20 min at 4 °C. The supernatants were resolved by high-performance liquid chromatography with a Hypersil BDS C18 reversed phase column (3 µm particle size, 130 Å pore size, 100 × 4.6 mm; Phenomenex) at a flow rate of 1 ml/min as previously reported [12]. The mobile phase comprised of citric acid (31.9 g/l), sodium acetate (2 g/l), octanesulfonic acid (460 mg/l), EDTA (30 mg/l) and methanol (15%), pH 3.6. DA, and its metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC) were detected by redox oxidation using an ESA 5014 analytical cell and ESA Coulochem II electrochemical detector (Thermo Fisher Scientific) with reducing (E1) and oxidizing (E2) electrodes set at − 200 mV and + 250 mV, respectively. The chromatograms were analyzed using the Chromeleon Chromatography Data System (V 6.2; Dionex). DA and DOPAC levels were normalized to the initial wet tissue weight and expressed as pmol/mg.

Animals were anesthetized with Caprieve and isoflurane and placed in a stereotaxic frame under general anaesthesia. A craniotomy was drilled in the skull according to the following coordinates relative to bregma: + 0.8 anteroposterior, + 2.1 lateral [28]. The guide cannula CMA7 (CMA Microdialysis) was implanted in the brain, so that its tip reached the calloso-striatal border, − 2.3 mm dorsoventral relative to the skull surface [28], and was secured to the skull using glass ionomer luting cement FujiCEM 2 (GC Corporation) and two anchor screws CMA7431021 (CMA Microdialysis). Microdialysis was performed 24 h following the surgery when the mice had recovered. A microdialysis probe CMA7 (0.24 mm × 2 mm membrane, 6 kDa cut-off; CMA Microdialysis) was inserted through the guide cannula, and freely-moving mice were infused with artificial cerebro-spinal fluid (ACSF; 140 mM NaCl, 7.2 mM glucose, 3 mM KCl, 1 mM MgCl₂, 1.2 mM CaCl₂, 1.2 mM Na₂HPO₄, 0.27 mM Na₂HPO₄, pH 7.4) at 2 µl/min. An equilibration period of 30 min was allowed to recover basal levels of extracellular DA in striatum, and then dialysates were collected every 20 min in ice-cold tubes containing 5 µl of 0.2 M perchloric acid. After 60 min, ACSF was replaced with high-potassium ACSF (93 mM NaCl and 50 mM KCl), to induce neurotransmitter release, and a further 3 samples were collected, after which the physiological ACSF was infused again. Each sample was snap frozen in dry ice and stored at − 80 °C until HPLC analysis was performed as described above. Mice were sacrificed and the microdialysis probe position was verified in the striatum. For each individual mouse, DA release was normalized to the baseline fraction (0 min) and expressed as fold difference relative to the average DA release directly following K⁺ stimulation (60 min fraction) in the respective control C57Bl/6S group. DA release was compared between MI2 and C57Bl/6S mice and between anle138b- and placebo-treated mice.

Six 30 µm-thick coronal sections, evenly spaced at 180 µm within the range covering the whole SNpc (between − 2.8 mm and − 3.85 mm AP from bregma [28]), were investigated in each mouse. TH immunohistochemistry was performed as described above, and tissue sections were analyzed with the optical fractionator probe using Stereo Investigator 11.07 (MBF Bioscience) with Olympus BX53 microscope equipped with QImaging Retiga camera and X-Y step motorized stage controlled by MAC 6000 System controller. The SNpc area was outlined under a 4x objective and cells were counted under a 100 × objective using the following parameters: counting grid size—220 × 220 µm, counting frame—85 × 85 µm. The thickness of the section was measured at each counting site. Data were processed using built-in software and the optical fractionator formula to estimate the total number of TH-positive neurons in SNpc.

In order to confirm neurodegeneration in SNpc of MI2 mice, stereological counting of all nigral neurons was performed after staining the sections with the NeuN pan-neuronal marker. In order to contour SNpc for NeuN cell counting, TH immunofluorescence was performed in the same sections and staining visualized with Alexa Fluor 488 secondary antibody. Sections were visualised at 488 nm using an epifluorescent microscope and the SNpc was outlined based on TH immunofluorescence. The resulting contour was super-imposed in the bright field image of NeuN immunohistochemistry. The slides were transferred to a stereology microscope, and the contour of SNpc was manually reconstructed in bright field in Stereo Investigator 11.07 before stereological counting of NeuN-positive cells (see also Supplementary Fig. S6, Online Resource 1).

Density of dopaminergic fibers in the striatum was estimated by the modified spherical probe counting method [26] in two 30 µm-thick coronal sections, representing medial and caudal striatum (+ 0.12 mm and − 0.66 mm AP from bregma [28]). The tissue was processed for TH immunohistochemistry as described above and analyzed using Stereo Investigator 11.07. A series of hemispherical (10 µm radius) probes, arranged in a 2 dimensional array, spaced at 250 µm intervals was superimposed on the striatum, and the total number of TH-positive fibers that cross the surface of the probe was counted under a 100 × objective. The thickness of the section was measured at each counting site. The estimated total length of TH-positive fibres in individual sections was normalized to the measured volume of the section and averaged between counting sites. Data are expressed as nm of length of TH-positive fibers in µm³ of striatal volume.

Motor performance was tested using an accelerating rotarod (Ugo Basile). The test was performed in mice at 6, 12, 15 and 20 months of age using separate cohorts of animals at each time point. Both males and females were tested in MI2 and C57Bl/6S groups, but we observed that some 15 and 20 month-old MI2 females were overweight, therefore, since the body weight may affect motor performance [20, 24], the heaviest individuals from this group were excluded from the test, to obtain no significant difference between the MI2 and C57Bl/6S females mice tested (t test, p = 0.552 at 15 months, p = 0.851 at 20 months). Prior to the test, mice were trained for 2 days. Each training session consisted of four trials at constant speed of 16 rpm, for a maximum of 60 s per trial. Mice were then tested using the acceleration mode (5–40 rpm) for three consecutive days with three trials per day and with at least 30 min intervals between the trials. Mice were placed on the rod and the time that the individual mouse took to fall from the rod was measured. Single trials were considered as technical replicates, and an average of 9 technical replicates (3 days per 3 trials) was calculated for each animal to obtain each biological replicate that was then used for statistical purposes.

The cohort of 20 month-old mice used for the rotarod test were then used for the static rod test performed according to the method of Deacon with modifications [9]. Two 60 cm-long rods of 25 mm and 15 mm diameters were attached to the platform and held horizontally 30 cm above the cushioned surface. Mice were placed at the end of the rod, facing away the platform and were allowed to freely turn back and walk to the platform. Prior to the test animals received training trials using 25 mm and 15 mm rods. On the next day, mice were tested on both 25 mm and 15 mm rods (one trial per each diameter). Two parameters were measured: orientation time (time taken to turn from the initial position towards the platform) and transit time (time taken to traverse the rod from the far end to the platform).

In order to detect the presence of early motor behavior during the progression of αSyn aggregation and synaptic dysfunction in MI2 mice, we used DigiGait automated gait imaging system (Mouse Specifics Inc.) [14, 27]. MI2 and control C57Bl/6S mice were tested at 3, 6, 9, 12, 15 and 18 months of age. Male mice were selected because at older age some female MI2 mice appeared overweight and this could have influenced the results. Individual animals were weighed before the analysis, and placed on the transparent motorised treadmill, that was set at 10 cm/s, the speed was adapted to the performance of aged animals (up to 18 months) that were still able to walk steadily at this rate but not at higher speed. The camera placed under the belt captured the gait of each mouse individually at 150 frames/s, and 3–5 s-long video of steady walking was then selected for further analysis for each mouse. The videos were analyzed by the DigiGait software, that measured the area of paw-belt contact in subsequent frames of the video, and plotted it against time, producing a plot representing stepping cycles for each individual paw. The data were then automatically processed by the DigiGait software to calculate indices for individual paws. These results were analyzed after averaging fore right with fore left paw values and hind right with hind left paw for individual mice.

After the detection of a motor phenotype at early ages the test was repeated to determine whether anle138b had an effect on the motor behavior detected. However, because the number of male mice available at the correct age was not sufficient for statistically significant conclusions we combined males and females taking care to select mice of similar weight. MI2 and C57Bl/6S animals treated with either anle138b or placebo diet were tested at 9 months of age (1 day prior to the treatment start), at 10.5 months of age (in the middle of the treatment period), and at 12 months of age (1 day prior to the treatment end).

Mice were treated from 9 to 12 months of age with anle138b, starting at a time when loss of striatal DA release was already present in MI2 mice but prior to significant nigral neuron loss and completed when MI2 mice presented with no more than 30% cell loss. Drug or placebo was administered in standard diet (2 g/kg of food, ssniff Spezialdiäten GMbH) throughout the whole 3 months study period to different cohorts of MI2 and C57Bl/6S mice. The levels of anle138b in plasma and brains of mice following this type of treatment have been previously reported [36]. At 12 months of age, mice were assessed using in vivo microdialysis. Subsequently, brains were collected for analysis, as described above.

MI2 mice express truncated 1–120 hαSyn under the control of the rat TH promoter (Fig. 1a), but no endogenous mouse αSyn due to a spontaneous deletion of the SNCA gene in the C57Bl/6OlaHsd (C57Bl/6S) mice [31, 35]. A C-terminally truncated form of αSyn was used because it has been shown to aggregate faster than the full-length protein [7] and its presence has been shown in brain extracts from Parkinson’s and dementia with Lewy bodies patients [1]. Immunoblotting revealed the presence of the transgenic protein in brain regions and neurons with TH-expression, such as substantia nigra (SN), striatum and olfactory bulb (OB), while no transgenic protein was expressed in cortex or cerebellum, where TH expression is not prominent (Fig. 1b). Expression of transgenic 1–120 hαSyn appeared significantly lower than that of endogenous αSyn in wild-type (wt) C57Bl/6J mice (Fig. 1c) possibly because αSyn is present in various neuronal populations in wt mice, while its expression in MI2 mice is limited to TH⁺ neurons.

By immunohistochemistry, the distribution of 1–120 hαSyn also followed the TH expression pattern, staining being evident in SNpc and ventral tegmental area (VTA) neurons, while no staining was evident in the SN pars reticulata, where αSyn is instead found in wt C57Bl/6J mice (Fig. 1d). In the SNpc 1–120 hαSyn expression was localized in both neuronal cell bodies and processes, while in the striatum, where nigral neurons project, it appeared as diffuse punctate staining (Fig. 1d).

Immunohistochemical analysis of 1–120 hαSyn in the nigrostriatal system of MI2 mice showed an age-dependent progressive accumulation of the protein. At 1.5 months, anti-αSyn antibodies stained small puncta that were diffusely distributed throughout the cell body of nigral neurons as well as numerous swollen processes. A more intense pattern was observed at 6 months, and at 12 months of age when, besides the punctate αSyn immunoreactivity, large LB-like structures with condensed αSyn staining were seen, and neuronal processes were uniformly stained, with some containing large 1–120 hαSyn inclusions (Fig. 2a). None of these features were present in wt C57Bl/6J mice, where at 12 months of age, normal αSyn puncta were much smaller and less frequent than the inclusions in the MI2 mice (Fig. 2a).

In the striatum the distribution of 1–120 hαSyn was consistent with its synaptic enrichment, confirmed by co-staining with synaptobrevin (VAMP2), (Supplementary Fig. S1a, Online Resource 1) and followed the pattern of nigrostriatal projections. Staining for transgenic human 1–120 hαSyn appeared as small puncta, accompanied by larger inclusions, whose number increased with age (Fig. 2b), while wild type C57Bl/6J mice displayed a normal diffuse and homogeneous αSyn punctate pattern (Fig. 2b). No αSyn staining was present in wt C57Bl/6S mice with deletion of the endogenous αSyn gene. Staining for VAMP2 showed a progressive redistribution of the protein at the synapse (Supplementary Fig. S1b, Online Resource 1), a pattern similar to that previously reported in our α-Syn120 mice showing SNARE protein redistribution [12].

Staining with anti-ubiquitin antibody was present in some large αSyn aggregates in the neurons of the SNpc starting from 12 months of age in MI2 mice (Supplementary Fig. S2b, Online Resource 1), but was not detected at any age in αSyn presynaptic aggregates in the striatum (data not shown). TfT and pFTAA did not show any specific staining (data not shown). PK digestion of tissue sections from SNpc and striatum of MI2 mice at 1.5 and 12 months of age showed that, while in the SNpc some of αSyn aggregates were resistant to PK treatment and still detectable following treatment, no αSyn staining was found in the striatum indicating that aggregates had been fully digested in this region or if present they were too small to be detected with conventional microscopy (Supplementary Fig. S2a, Online Resource 1).

Immunoblotting of SN extracts showed a significant reduction of monomeric 1–120 hαSyn in 12 month-old MI2 compared with 6 and 1.5 month-old MI2 mice with an associated increase in higher molecular weight (HMW) 1–120 hαSyn species (Fig. 2c). In the striatum, monomeric 1–120 hαSyn increased between 1.5 and 6 months of age and an increase in HMW species was present at 12 months of age, similar to the SN (Fig. 2c).

Serial extraction of αSyn with buffers of increasing strength from both SNpc and striatum showed an increase in the presence of αSyn in TritonX-insoluble fractions between 1.5 and 12 months of age, suggesting increasing aggregates insolubility (Supplementary Fig. S3, Online Resource 1).

No apparent difference in monomeric 1–120 hαSyn was found in the OB between 1.5-, 6- and 12 months of age, although an increase in HMW bands was also present in this region (Fig. 2c).

To obtain a more detailed analysis of synaptic 1–120 hαSyn at a near single-molecule resolution, we imaged the transgenic protein in the striatum of mice at 1.5, 6 and 12 months of age using dSTORM following immunofluorescence staining with a human αSyn specific antibody (Fig. 3a). To perform quantitative analysis of the aggregation profile, size, shape, inner density, local density per region of interest and percentage of aggregated 1–120 hαSyn, a semi-automated software was built that implements a novel set of meta-analysis clustering algorithmic tools for analyzing super-resolution data (Supplementary Fig. S4, Online Resource 1). Using this approach, we observed progressive aggregation of 1–120 hαSyn and followed the size distribution of the aggregates over time in the striatum of 1.5, 6 and 12 month-old MI2 mice. No specific staining was observed by dSTORM in C57Bl/6S control mice (see also Supplementary Fig. S3, Online Resource 1) The population of aggregates was divided into 4 groups according to size: small, with a diameter ranging from 20 to 100 nm, medium, with a diameter between 100 and 300 nm, large, with a diameter of 300–500 nm and very large, with a diameter above 500 nm. dSTORM imaging and analysis demonstrated an age-dependent increase in the overall number of 1–120 hαSyn aggregates from 1.5 to 6 and 12 months of age (Fig. 3b). This increase was mainly due to an age-dependent increase in small (20–100 nm), medium (100–300 nm) and large (300–500 nm) aggregates. The number of very large aggregates (> 500 nm) did not significantly change with age (Fig. 3c).

To establish whether the progressive aggregation of 1–120 hαSyn affected DA neurotransmission in the striatum in MI2 mice, the total content of striatal DA was measured at 3, 6 and 12 months of age. No difference was detected between MI2 and C57Bl/6S control mice at 3 and 6 months of age, while a significant reduction in total DA levels was present at 12 months (control, 65.3 ± 5.8, MI2, 38.1 ± 5.7 pmol DA/mg of tissue, Fig. 4a). DA content in 12 month-old MI2 mice was also reduced compared to MI2 mice at 3 and 6 months of age (87 ± 9.2 and 73.1 ± 11.1 pmol DA/mg of tissue, respectively; Fig. 4a). No difference was found in DA levels at 12 months of age between control mouse lines with (C57Bl/6J) and without (C57Bl/6S) endogenous αSyn (Supplementary Fig. S5a, Online Resource 1), confirming that the absence of endogenous αSyn does not affect striatal DA levels, as previously shown [13].

Dopaminergic deficit in MI2 mice was further investigated by measuring striatal DA release using in vivo microdialysis. DA release was induced in freely moving mice by infusion of 50 mM K⁺. No difference in DA release was observed between MI2 and control C57Bl/6S mice at 3 months of age, while at 6 months, a significant reduction in K⁺ stimulated DA release was present in MI2 mice compared with C57Bl/6S controls [samples were measured at 60, 80 and 100 min, normalized to peak C57Bl/6S value (at 60 min)] (0.57 ± 0.15 vs 1 ± 0.08 at 60 min, 0.38 ± 0.09 vs 0.63 ± 0.07 at 80 min, and 0.34 ± 0.03 vs 0.54 ± 0.05 at 100 min in MI2 vs control mice; Fig. 4b). This impairment was progressive, with the loss of induced DA release being more prominent at 9 months of age (0.24 ± 0.09 vs 1 ± 0.25 at 60 min, 0.17 ± 0.06 vs 0.71 ± 0.17 at 80 min, and 0.17 ± 0.06 vs 0.6 ± 0.14 at 100 min in MI2 vs control mice) and further exacerbated at 12 months of age (0.16 ± 0.06 vs 1 ± 0.24 at 60 min, 0.13 ± 0.05 vs 0.87 ± 0.2 at 80 min and 0.11 ± 0.04 vs 0.61 ± 0.17 at 100 min in MI2 vs control mice; Fig. 4b). No difference was observed in striatal DA release between C57Bl/6S and C57Bl/6J control mice at 12 months of age (Supplementary Fig. S5b, Online Resource 1). These findings indicate that striatal DA release impairment in MI2 mice is associated with the progressive aggregation of 1–120 hαSyn. Importantly, a significant reduction in DA release in MI2 mice appears earlier than a reduction of total DA content in striatal tissue extracts, supporting the presence of a specific synaptic dysfunction (Fig. 4).

Nigral DA neuron death in the MI2 mice was investigated by unbiased stereological counting of cells stained for TH. The results showed a progressive TH⁺ cell loss in SNpc of MI2 mice compared to C57Bl/6S mice starting at 9 months of age (MI2, 11,174 ± 825.1 vs control, 13,222 ± 1658.8 TH⁺ neurons) becoming significant at 12 months of age [31% reduction in MI2 vs control mice: 8530.4 ± 751.9 (MI2) vs 12,398.8 ± 946.1 (C57Bl/6S)] and further progressed at 20 months of age [54% reduction in MI2 vs controls: 6345.3 ± 523.1 (MI2) vs 13,836 ± 616.1 (C57Bl/6S) TH⁺ neurons, respectively] (Fig. 5a, b). The number of TH⁺ cells in SNpc of 20 month-old MI2 mice was also significantly lower (by 43%) compared to that in 9 months-old MI2 mice (Fig. 5b).

To confirm neuronal loss in SNpc and rule out the possibility that the decrease in TH cell number was reflecting a possible reduction in TH expression, cell counting was performed in 12 month-old mice following staining with the neuronal marker NeuN. NeuN and TH staining images were overlapped to delimit the counting area to the SNpc (Supplementary Fig. S6, Online Resource 1). The results showed a 20% reduction of NeuN positive neurons in SNpc of 12 month-old MI2 mice compared to C57Bl/6S [11,877 ± 383.5 (MI2) and 15,633.5 ± 1020.7 (C57Bl/6S) cells; Fig. 5c]. The reason for the reduced proportional neuronal loss observed following NeuN staining compared to TH staining is due to the fact that NeuN recognizes both TH and non-TH neurons in the SNpc, indeed, the absolute number of cell loss was similar for TH and NeuN stained neurons (− 3868 and − 3757 neurons, respectively).

We then assessed changes in DA innervation in the striatum of MI2 mice at different ages. We observed a significant decrease in striatal TH⁺ innervation at 12 but not at 9 months in MI2 mice compared to C57Bl/6S mice (Fig. 5d). In order to determine the loss of striatal innervation, we evaluated the density of TH-positive fibers by estimating their length in a defined volume of striatal tissue. We found that MI2 mice had a 40% loss of striatal TH innervation compared to C57Bl/6S mice (14.49 ± 0.77 and 24.29 ± 1.76 nm per µm³, respectively) at 12 months of age while no significant difference was present in 9 month-old MI2 mice compared to controls (21.88 ± 2.48 and 25.6 ± 3.31 nm per µm³, respectively; Fig. 5e). These results indicate that similar to the loss of nigral DA neurons, loss of nigrostriatal DA innervation occurs after the functional synaptic deficit, reflected by a reduction in DA stimulated release that starts at 6 months of age.

An accelerating rotarod test was initially used to analyze motor function in MI2 mice at 6, 12, 15 and 20 months of age. An age-related gradual decline in rotarod performance was present in both C57Bl/6S and MI2 mice, but the reduction was more pronounced in MI2 mice, resulting in a significant loss of latency to fall from the rotarod at 20 months of age compared to their performance at 6 and 12 months of age (127.8 ± 14.9 s, 20 m, 215.8 ± 9.9 s, 12 m, 220.7 ± 11.5 s, 6 m; Fig. 6a). The performance of MI2 mice at 20 months of age was also significantly impaired compared to age-matched C57Bl/6S controls (127.8 ± 14.9 s and 181.7 ± 21.6 s, respectively; Fig. 6a).

The presence of a motor deficit was also tested in 20 month-old MI2 compared to C57Bl/6S mice using the static rod test where the time required to turn 180^o from the initial position (orientation time) and the time required to travel from the far end of the rod to the platform (transit time) were measured. We found no significant differences between transgenic and control mice in either orientation or transit time when a 25 mm diameter rod was used, but when the test was performed with a 15 mm rod, although there was no significant difference in the orientation time, the transit time was longer for MI2 mice compared to controls (12.8 ± 1.6 s and 8.1 ± 0.8 s, respectively), confirming a selective motor impairment in MI2 mice (Fig. 6b).

To determine whether earlier subtle changes were present in motor behavior, we tested MI2 male mice using the more sensitive DigiGait system. The animals were weighed prior to the experiments, and we identified a trend towards increased body weight in MI2 males that however reached the significance only at 18 months of age (Supplementary Fig. S7a, Online Resource 1). The results of the DigiGait assessment show alterations in the gait pattern of MI2 mice in comparison with C57Bl/6S animals starting from 9 months of age, and were manifested by decreased forelimb stride length (4.256 ± 0.178 cm vs. 4.756 ± 0.14 cm) and forelimb stance (0.303 ± 0.014 s vs. 0.334 ± 0.009 s), swing (0.123 ± 0.005 s vs. 0.141 ± 0.007 s) and propulsion (0.191 ± 0.009 s vs. 0.219 ± 0.008 s) duration, that was still present at the end of the test at 18 months of age (Fig. 6c). Impairment of hindlimb gait pattern was less evident, however, a trend towards a decrease in MI2 mice was present at older ages for hindlimb stride length and hindlimb stance and swing duration (Supplementary Fig. S7b, Online Resource 1). Statistically significant differences were found between hindlimb of MI2 mice and C57Bl/6S animals in propulsion duration at 18 months (0.251 ± 0.01 s vs. 0.276 ± 0.005 s) (Fig. 6c), and in brake duration at 6 and 18 months (Supplementary Fig. S7b, Online Resource 1).

Anle138b was administered in the food (2 g/kg of food) for 3 months to MI2 and C57Bl/6S mice. Treatment with anle138b or placebo started at 9 months of age, when striatal DA release impairment is already present in MI2 mice, and ended at 12 months of age when 30% nigral neuron loss is detected in non-treated animals (Fig. 7a). Using immunohistochemistry, we found that anle138b treatment reduced 1–120 hαSyn aggregation in both SNpc cell bodies and synaptic terminals in the striatum (Fig. 7b). Application of dSTORM analysis not only confirmed the reduction in striatal 1–120 hαSyn aggregation but also provided a mechanistic explanation for anle138b activity. dSTORM analysis revealed two opposite but complementary effects of anle138b: we detected an increased percentage of small, non-clustered 1–120 hαSyn species (anle138b, 53.4 ± 2.9%; placebo, 29.9 ± 3%; Fig. 7c, d) that occurred concomitantly with a reduction in the inner density of small and large size aggregates (anle138b, 140.9 ± 12.7; placebo, 233.5 ± 25.3 fluorophore localizations; Fig. 7c, d). The total number of aggregates was only slightly but not significantly reduced following anle138b treatment (Fig. 7d). Analyses of striatal extracts by immunoblotting confirmed this result showing a reduction in HMW 1–120 hαSyn band and an increase in monomeric protein (Fig. 7e). These data show that anle138b substantially affects the aggregation profile of 1–120 hαSyn in MI2 mice.

To determine the effect of anle138b on dopaminergic impairment, we measured striatal DA release in MI2 mice. A 3-month treatment was started at the age of 9 months when the DA release deficit was already present. Released DA was significantly increased in anle138b-treated MI2 mice compared to placebo-treated MI2 animals [Fig. 8a; K⁺-stimulated DA release, relative values of DA release in anle138b- vs placebo-treated MI2 mice sampled at 60, 80 and 100 min, after normalized to peak C57Bl/6S mouse fraction value (at 60 min) reached 0.69 ± 0.14 vs 0.13 ± 0.02 (60 min), 0.36 ± 0.07 vs 0.07 ± 0.01 (80 min) and 0.28 ± 0.05 vs 0.09 ± 0.02 (100 min)]. The DA release values in C57Bl/6S anle138b- vs placebo treated mice were not altered [1.03 ± 0.25 vs 1 ± 0.12 (60 min), 0.41 ± 0.09 vs 0.55 ± 0.05 (80 min) and 0.38 ± 0.09 vs 0.39 ± 0.03 (100 min) (Fig. 8a)]. We then analyzed the effect of anle138b treatment on the number of nigral DA neurons and found a significant increase in number of TH⁺ neurons in SNpc of anle138b-treated MI2 animals compared to placebo-treated mice (Fig. 8b), with anle138b preventing cell loss. The number of nigral TH⁺ cells did not differ between C57Bl/6S mice treated with either placebo or anle138b (10,886.7 ± 706.5 and 10,792 ± 858.9 TH⁺ cells, respectively) and anle138b-treated MI2 mice (9944 ± 567.5 TH⁺ cells), while a substantial DA neuron loss was present in placebo-treated MI2 mice (7465 ± 41.4 TH⁺ cells) (Fig. 8c).

To determine whether anle138b treatment can affect motor dysfunction, we tested the mice before, during and at the end of treatment using DigiGait system. Mice were weighed before each test, and we found no effect of treatment on the body weight, what shows that body weight differences didn’t contribute to behavioral differences between anle138b- and placebo-treated animals over the course of the experiment. An effect of genotype on the body weight was identified only at 12 months of age when MI2 mice were heavier than controls (Supplementary Fig. S9a, Online Resource 1). We found that anle138b rescued several of the parameters associated with the motor impairment (Fig. 9; Supplementary Fig. S9b, Online Resource 1). We observed that MI2 mice treated with anle138b, in comparison with MI2 mice treated with placebo, exhibited increased forelimb stride length (4.417 ± 0.094 cm vs. 4.025 ± 0.131 cm, respectively) and stance duration (0.302 ± 0.006 s vs. 0.279 ± 0.008 s, respectively) at 12 months of age, hindlimb stance duration at 10.5 months of age (0.31 ± 0.009 s vs. 0.275 ± 0.015 s, respectively), as well as forelimb (0.201 ± 0.01 s vs. 0.168 ± 0.0125 s, respectively) and hindlimb (0.262 ± 0.009 s vs. 0.226 ± 0.016 s, respectively) propulsion duration at 10.5 months of age (Fig. 9). When we compared placebo-treated MI2 mice with placebo-treated C57Bl/6S mice, the length of forelimb stride at 10.5 (4.092 ± 0.22 cm vs. 4.659 ± 0.156 cm, respectively) and 12 (4.025 ± 0.131 cm vs. 4.576 ± 0.136 cm, respectively) months of age and hindlimb stride at 10.5 (3.808 ± 0.211 cm vs. 4.345 ± 0.108 cm, respectively), and 12 (3.929 ± 0.133 cm vs. 4.429 ± 0.151 cm, respectively) months of age as well as duration of forelimb stance at 10.5 (0.281 ± 0.015 s vs. 0.321 ± 0.01 s, respectively) and 12 (0.28 ± 0.009 s vs. 0.309 ± 0.009 s, respectively) months of age, hindlimb stance at 10.5 (0.275 ± 0.015 s vs. 0.326 ± 0.008 s, respectively) and 12 (0.287 ± 0.009 s vs. 0.331 ± 0.011 s, respectively) months of age, forelimb propulsion at 10.5 months (0.168 ± 0.012 s vs. 0.204 ± 0.009 s, respectively) of age and hindlimb propulsion at 10.5 (0.226 ± 0.016 s vs. 0.277 ± 0.009 s, respectively) and 12 (0.234 ± 0.009 s vs. 0.29 ± 0.011 s, respectively) months of age was still decreased. However, we also observed statistically significant differences between anle138b-treated MI2 mice and anle138b-treated C57Bl/6S mice in hindlimb stride length (4.144 ± 0.062 cm vs. 4.578 ± 0.169 cm, respectively) and stance duration (0.313 ± 0.006 s vs. 0.341 ± 0.012 s, respectively) at 12 months of age (Fig. 9). Taken together, these results show that targeting αSyn aggregation by anle138b rescues the DA release deficit, several aspects of the gait impairment and protects against cell loss, even when treatment was started when synaptic functional impairment was already present.