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

Abstract

Parkinson's disease (PD) is characterized by the presence of α-synuclein aggregates known as Lewy bodies and Lewy neurites, whose formation is linked to disease development. The causal relation between α-synuclein aggregates and PD is not well understood. We generated a new transgenic mouse line (MI2) expressing human, aggregation-prone truncated 1-120 α-synuclein under the control of the tyrosine hydroxylase promoter. MI2 mice exhibit progressive aggregation of α-synuclein in dopaminergic neurons of the substantia nigra pars compacta and their striatal terminals. This is associated with a progressive reduction of striatal dopamine release, reduced striatal innervation and significant nigral dopaminergic nerve cell death starting from 6 and 12 months of age, respectively. In the MI2 mice, alterations in gait impairment can be detected by the DigiGait test from 9 months of age, while gross motor deficit was detected by rotarod test at 20 months of age when 50% of dopaminergic neurons in the substantia nigra pars compacta are lost. These changes were associated with an increase in the number and density of 20-500 nm α-synuclein species as shown by dSTORM. Treatment with the oligomer modulator anle138b, from 9 to 12 months of age, restored striatal dopamine release, prevented dopaminergic cell death and gait impairment. These effects were associated with a reduction of the inner density of large α-synuclein aggregates and an increase in dispersed small α-synuclein species as revealed by dSTORM. The MI2 mouse model recapitulates the progressive dopaminergic deficit observed in PD, showing that early synaptic dysfunction is associated to fine behavioral motor alterations, precedes dopaminergic axonal loss and neuronal death that become associated with a more consistent motor deficit upon reaching a certain threshold. Our data also provide new mechanistic insight for the effect of anle138b's function in vivo supporting that targeting α-synuclein aggregation is a promising therapeutic approach for PD.

Keywords: Alpha-synuclein; Anle138b; Mouse model; Parkinson’s disease; Striatum; Substantia nigra; dSTORM.

Conflict of interest statement

A. G. and C. G. are co-founders of MODAG. A. L. is partly employed by MODAG.

Figures

Fig. 1

Expression of transgenic 1–120 hαSyn in MI2 mice. a 1–120 hαSyn transgene construct used for generating the MI2 mice. b Immunoblots showing αSyn in lysates from cerebellum (Cbl), cortex (Ctx), substantia nigra (SN), striatum (Str) and olfactory bulbs (OB) of 1.5 month-old mice following short (short exp) and long (long exp) exposure times. Quantification was performed for SN, Str and OB, but not for Cbl and Ctx, where the signal was negligible. Data are expressed as fold difference compared to SN (mean ± SEM, n = 3 mice, one-way ANOVA with Bonferroni correction (**p < 0.01, ***p < 0.001) (detailed statistics in Online Resource). c Immunoblots comparing expression levels between control and MI2 mouse lines. Expression of 1–120 hαSyn (αS(1-120)) in MI2 mice is much lower than that of endogenous full-length αSyn (fl) in WT C57Bl/6J in the OB. Interestingly some truncated αSyn with a similar size to the transgenic 1–120 hαSyn can be seen in protein extracts of SN and Str of WT C57Bl/6J mice. d Immunohistochemistry of brain sections of 1.5 month-old MI2 mice detected with the Syn1 antibody shows 1–120 hαSyn protein in neuronal cell bodies and processes in SNpc and ventral tegmental area (VTA) and in neuropil in striatum (see also Supplementary Fig. S1a, Online Resource 1). In C57Bl/6J mice, endogenous αSyn, is also found in SN pars reticulata (SNpr) and Ctx besides SNpc, VTA and Str. The specificity of Syn1 antibody for αSyn was confirmed by the absence of staining in C57Bl/6S mice that lack endogenous αSyn

Fig. 2

Aggregation of 1–120 hαSyn protein in MI2 mice. a Top panel: progressive accumulation of 1–120 hαSyn protein with age in SNpc of MI2 mice in cell bodies (arrows) and processes (arrowheads). Middle panel: abundant small inclusions of 1–120 hαSyn protein in SNpc cell bodies are present at 1.5 months and large, LB-like aggregates in SNpc neurons (arrows) among cells with 1–120 hαSyn punctate staining (arrowhead) are found at 12 months of age. Bottom panel: large 1–120 hαSyn puncta are distributed along the processes in SNpc at 1.5 and 12 months of age (arrows). At 12 months of age, processes uniformly filled with 1–120 hαSyn (triangle) and round inclusions containing condensed 1–120 hαSyn protein (arrowheads) are also visible. The staining of αSyn in WT C57Bl/6J mice is much less intense and more homogenous. Middle right panel: less αSyn is present in cell bodies, and no cellular inclusions are found (arrow) in control mice. Bottom right panel: αSyn puncta in the nigral processes are much less numerous and smaller in C57Bl/6J mice than in MI2 mice (arrowheads), (see also Supplementary Fig. S2, Online Resource 1). b Progressive accumulation of 1–120 hαSyn puncta in MI2 striatal neuropil in 1.5, 6 and 12 month-old MI2 mice. In 12 month-old C57Bl/6J mice striatal αSyn is distributed more homogenously and large αSyn-positive puncta are not present (bottom panel). c Immunoblotting of brain lysates from MI2 mice as a function of age. The levels of monomeric 1–120 hαSyn (~ 14 kDa, arrow) shown in the western blots were quantified and normalized to either β-actin or TH (right panels). Data are expressed as fold difference compared to 1.5 month-old animals (mean ± SEM, n = 3 mice, one-way ANOVA, multiple comparison with Bonferroni corrections). In SN, a reduction of monomeric 1–120 hαSyn was found as a main effect of age [statistically significant differences between 1.5 and 12 months and between 6 and 12 months (αSyn/β-actin or αSyn/TH; *p < 0.05)] for all comparisons. In the striatum there was a significant increase of 1–120 hαSyn between 1.5 and 6 months of age (*p < 0.05) for both αSyn/β-actin and αSyn/TH. There was no significant change in OB. In all the three brain regions increased amounts of high molecular weight (HMW) 1–120 hαSyn bands (~ 55 kDa, arrowhead) were present in MI2 mice at 12 months compared to 1.5 and 6 months of age (see also Supplementary Fig. S3, Online Resource 1). Stars denote the non-specific bands recognized by Syn1 antibody in both MI2 and αSyn-null C57Bl/6S mice [29] (see detailed statistical evaluation in Online Resource)

Fig. 3

dSTORM analysis of the progression of 1–120 hαSyn aggregation in the striatum of MI2 mice. a Representative dSTORM images of 1–120 hαSyn staining in the striatum of MI2 mice at 1.5, 6 and 12 months of age showing increasing number of aggregates with age. b Quantification of dSTORM data (see also Supplementary Fig. S4) (mean ± SEM, n = 3 mice, one-way ANOVA, multiple comparisons with Bonferroni correction). The main effect of age is the increasing number of aggregates (clusters) (*p < 0.05). No differences in the aggregate median size or number of localizations per cluster (e.g. the inner density which measures the number of fluorescent flashes in a cluster) were found. c Analysis of cluster size distribution shows a statistically significant increase in the number of small-size aggregates (20–100 nm, t test #p < 0.05), medium size aggregates (100–300 nm, one way ANOVA, *p < 0.05) and large-size aggregates (300–500 nm, one way ANOVA, *p < 0.05), between 1.5 and 12 months of age (see detailed statistical evaluation in Online Resource)

Fig. 4

Striatal dopaminergic deficit in MI2 mice. a DA was measured in striatal lysates of MI2 and C57Bl/6S mice at 3, 6 and 12 months of age (mean ± SEM, n = 5–8 mice per group). A main effect of age and interaction between genotype and age was identified by two-way ANOVA with Bonferroni correction, showing a significant reduction of DA levels in MI2 mice at 12 months compared to 12 month old C57Bl/6S mice, and to 3 month old and 6 month old MI2 mice (*p < 0.05, ***p < 0.001) (see also Supplementary Fig. S5a, Online Resource 1 and for DDPAC content S5c). b Striatal DA release was measured by in vivo microdialysis following the infusion of 50 mM KCl for 60 min between 40 and 100 min of the experiment. Data are expressed as a fold difference compared to the baseline fraction (0 min), normalized to the value obtained in age-matched C57Bl/6S controls at 60 min (mean ± SEM, n = 4–6 mice). At 3 months of age no difference between MI2 and C57Bl/6S mice was observed, but a significant progressive decrease in DA release was found at 6, 9 and 12 months of age in MI2 compared with C57Bl/6S mice (see also Supplementary Fig. S5b, Online Resource 1). Two-way mixed ANOVA revealed a significant interaction between genotype and sample time at 6, 9 and 12 months of age (*p < 0.05, **p < 0.01, t test for individual sampling time points) (see detailed statistical evaluation in Online Resource)

Fig. 5

Loss of dopaminergic neurons in SNpc of MI2 mice. a Immunohistochemistry showed a reduction in TH staining in SNpc of MI2 mice compared to C57Bl/6S animals at 12 and 20 months of age. b Stereological counting of TH-positive neurons in SNpc. Average number of nigral TH-positive cells ± SEM, n = 4–6 mice per group; two-way ANOVA with Bonferroni correction showing a significant reduction of TH+ neurons in MI2 mice compared to C57Bl/6S controls at 12 (*p < 0.05) and 20 (***p < 0.001) months of age. There was also a significant reduction in TH+ cells in MI2 mice between 9 and 20 months of age. c Stereological counting of total, NeuN+ neuron number in SNpc at 12 months of age (see also Supplementary Fig. S6, Online Resource 1). A significant decrease of NeuN+ neurons is present in MI2 mice compared to C57Bl/6S mice (average number of nigral NeuN-positive cells ± SEM, n = 4 mice per group; t test, *p < 0.05). d Reduced TH-positive fiber staining in the striatum of MI2 mice compared to C57Bl/6S mice at 12 months of age. Insets show high magnification images. e TH+ striatal neurites were estimated using the spaceball probe (mean length of striatal TH+ fibers per volume of striatal tissue ± SEM, n = 4–6 mice per group; effect of genotype was identified by two-way ANOVA). Significant decrease in the total length of TH+ fibers was present in 12 month-old MI2 mice compared to 12 month-old C57Bl/6S mice (**p < 0.01) and 12 and 9 month-old MI2 animals (*p < 0.05), multiple comparisons with Bonferroni corrections (see detailed statistical evaluation for 5b, 5c, 5e in Online Resource)

Fig. 6

Motor impairment in MI2 mice. a C57Bl/6S and MI2 mice were analyzed using accelerating rotarod test at 6, 12, 15 and 20 months of age (mean latency to fall from the rod ± SEM, n = 12–18 mice per group). A main effect of age was identified by two-way ANOVA. Statistically significant differences between 6 and 20 months (###p < 0.001) and 12 and 20 months (‡‡‡p < 0.001) in MI2 mice, and between MI2 and C57Bl/6S animals at 20 months (*p < 0.05) were revealed by multiple comparisons with Bonferroni correction. b Motor performance of 20 month-old mice was tested using the static rod test (mean orientation time or mean transit time ± SEM, n = 13–18 mice). No differences were identified between experimental groups using a 25 mm rod or in orientation time using a 15 mm rod, but there was a statistically significant difference in transit time on the 15 mm rod between MI2 and C57Bl/6S mice (*p < 0.05, t test). c Gait pattern of mice was analyzed between 3 and 18 months of age using DigiGait test (see also Supplementary Fig. S7, Online Resource 1). Alterations in individual gait phases were found in MI2 animals compared to control C57Bl/6S mice (mean ± SEM, n = 9–17 male mice per group). Two-way ANOVA identified a main effect of age on forelimb stance and swing duration, and on hindlimb propulsion duration, a main effect of genotype on forelimb stride length and stance, swing and propulsion duration, and two-way interaction between age and genotype for forelimb propulsion duration. Statistically significant differences were identified at 9, 12 and 18 months for forelimb stride length and stance, swing and propulsion duration, and at 18 months for hindlimb propulsion duration (*p < 0.05; **p < 0.01, ***p < 0.001) by pairwise comparisons with Bonferroni correction between MI2 and C57Bl/6S mice (see detailed statistical evaluation in Online Resource)

Fig. 7

Effect of anle138b on αSyn aggregation in MI2 mice. a Mice were treated with anle138b starting at 9 months and analyzed at 12 months of age. b Treatment with anle138b reduces the accumulation of 1–120 hαSyn in SNpc and striatum of MI2 mice as shown by Syn1 antibody immunostaining. c Representative dSTORM images of 1–120 hαSyn staining in the striatum of 12 month old MI2 mice treated with placebo (top panel) or anle138b (bottom panel). Large aggregates appear less dense and an increased number of smaller species are seen following anle138b treatment. d Quantification of dSTORM data revealed a significant decrease in the inner density of aggregates (#localizations/cluster) and a significant increase in the percentage of non-clustered 1–120 hαSyn in anle138b-treated MI2 mice compared to placebo-treated littermates (see also Supplementary Fig. S8, Online Resource 1) (mean ± SEM, n = 3 mice; t test, *p < 0.05, **p < 0.01) while the number of clusters and their size did not change significantly. e Immunoblotting of striatal brain extracts from 12 month-old mice shows a decrease of αSyn high molecular weight (HMW) bands (arrowhead) and an increase of monomeric αSyn (arrow) in the striatum of anle138b-treated MI2 mice compared to MI2 placebo treated. 1–120 hαSyn levels are also shown for 1.5, 9 and 12 month-old untreated MI2 mice. C57Bl/6S (C57S) not expressing endogenous αSyn are shown as negative control. The star denotes non-specific bands recognized by Syn1 antibody. Blot shows levels of αSyn before and after anle138b in 3 mice representative of 5–6 mice per group tested. Right panel—quantification of HMW 1–120 hαSyn and monomeric (mono) 1–120 hαSyn after normalization to the levels of β-actin in all 5–6 mice tested (fold difference compared to placebo-treated mice ± SEM, n = 5–6 mice per group, t test: **p < 0.01)

Fig. 8

Effect of anle138b on striatal DA release and SNpc DA neuron death in MI2 mice. a The effect of anle138b treatment on striatal DA release studied by in vivo microdialysis. Data are normalized to the value obtained in the C57Bl/6S/placebo group at 60 min (mean ± SEM, n = 5–6 mice per group). Three-way mixed ANOVA identified an effect of genotype, sampling time, two-way genotype × sampling time interaction, and sampling time × genotype × treatment three-way interaction. Statistically significant differences in K+-induced DA release at 60, 80 and 100 min were found between placebo-treated MI2 and C57Bl/6S mice (**p < 0.01, ***p < 0.001) and between placebo- and anle138b-treated MI2 mice (#p < 0.01) (multiple comparisons with Bonferroni correction). b Preservation of TH immunoreactivity in SNpc of anle138b-treated MI2 mice compared with placebo-treated littermates. c Effect of anle138b treatment on nigral DA neuronal death determined using stereology (average number of nigral TH-positive cells ± SEM, n = 3 mice per group). A main effect of genotype was identified by two-way ANOVA. Anle138b treatment resulted in increased number of TH+ cells in MI2 mice compared to placebo-treated littermates (*p < 0.05). Placebo-treated MI2 mice had less nigral TH+ cells than placebo-treated C57Bl/6S mice (*p < 0.05, multiple comparisons with Bonferroni correction) (see detailed statistical evaluation in Online Resource)

Fig. 9

Effect of anle138b on gait pattern in MI2 mice (see also Supplementary Fig. S9, Online Resource 1). Placebo- or anle138b-treated C57Bl/6S and MI2 mice were analyzed using the DigiGait test. Treatment with anle138b rescued alterations in the duration of several of gait phases in MI2 mice (mean ± SEM, n = 6–12 mice per group; males and females). Three-way ANOVA revealed a main effect of age and a main effect of genotype on forelimb and hindlimb stride length, and stance and propulsion duration, a main effect of treatment on hindlimb stance and propulsion duration and a three-way age × genotype × treatment interaction for forelimb stance duration. At 10.5 months of age, statistically significant differences were found between placebo-treated C57Bl/6S and MI2 mice in forelimb and hindlimb stride length, and in forelimb and hindlimb stance and propulsion duration (#p < 0.05, ##p < 0.01), and between placebo- and anle138b-treated MI2 mice in hindlimb stance duration and in forelimb and hindlimb propulsion duration (*p < 0.05). At 12 months of age, statistically significant differences were identified between placebo-treated C57Bl/6S and MI2 mice in forelimb and hindlimb stride length and stance duration, and in hindlimb propulsion duration (#p < 0.05, ##p < 0.01). At this age, a difference was found between anle138b-treated C57Bl/6S and MI2 mice in hindlimb stride length and stance duration (‡p < 0.05) and between placebo- and anle138b-treated MI2 mice in forelimb stride length and stance duration (*p < 0.05) (multiple comparisons with Bonferroni correction) (see detailed statistical evaluation in Online Resource)