Inhibition of Prolyl Oligopeptidase Restores Spontaneous Motor Behavior in the α-Synuclein Virus Vector-Based Parkinson's Disease Mouse Model by Decreasing α-Synuclein Oligomeric Species in Mouse Brain

Abstract

Decreased clearance of α-synuclein (aSyn) and aSyn protein misfolding and aggregation are seen as major factors in the pathogenesis of Parkinson's disease (PD) and other synucleinopathies that leads to disruption in neuronal function and eventually to cell death. Prolyl oligopeptidase (PREP) can accelerate the aSyn aggregation process, while inhibition of PREP by a small molecule inhibitor decreases aSyn oligomer formation and enhances its clearance via autophagy in different aSyn overexpressing cell types and in transgenic PD animal models. In this study, we investigated the impact of chronic PREP inhibition by a small molecule inhibitor, 4-phenylbutanoyl-l-prolyl-2(S)-cyanopyrrolidine (KYP-2047), on aSyn oligomerization, clearance, and underlying spontaneous motor behavior in a virus vector-based aSyn overexpression mouse model 4 weeks after aSyn microinjections and after the onset of symptomatic forepaw bias. Following 4 weeks of PREP inhibition, we saw an improved spontaneous forelimb use in mice that correlated with a decreased immunoreactivity against oligomer-specific forms of aSyn. Additionally, KYP-2047 had a trend to enhance dopaminergic systems activity. Our results suggest that PREP inhibition exhibits a beneficial effect on the aSyn clearance and aggregation in a virus mediated aSyn overexpression PD mouse model and that PREP inhibitors could be a novel therapeutic strategy for synucleinopathies.

Significance statement: Alpha-synuclein (aSyn) has been implicated in Parkinson's disease, with aSyn aggregates believed to exert toxic effects on neurons, while prolyl oligopeptidase (PREP) has been shown to interact with aSyn both in cells and cell free conditions, thus enhancing its aggregation. We demonstrate the possibility to abolish motor imbalance caused by aSyn viral vector injection with chronic 4 week PREP inhibition by a potent small-molecule PREP inhibitor, 4-phenylbutanoyl-l-prolyl-2(S)-cyanopyrrolidine (KYP-2047). Treatment was initiated postsymptomatically, 4 weeks after aSyn injection. KYP-2047-treated animals had a significantly decreased amount of oligomeric aSyn particles and improved dopamine system activity compared to control animals. To our knowledge, this is the first time viral overexpression of aSyn has been countered and movement impairments abolished after their onset.

Keywords: Parkinson's disease; prolyl oligopeptidase inhibitor; protein aggregation; serine protease; synuclein; synucleinopathies.

Figure 1.

The effect of KYP-2047 on mouse brain PREP activity. A–C, KYP-2047 intraventricular administration significantly reduced PREP activity in mouse striatum and cortex 1 week after osmotic minipump implantation (A, B), and PREP inhibition was observed after 4 week intraperitoneal KYP-2047 administration in mouse cortex (C) compared to vehicle administration. Intraventricular VEH, n = 3; intraventricular KYP-2047, n = 5; intraperitoneal VEH, n = 23; intraperitoneal KYP-2047, n = 21). Error bars represent means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 (unpaired Student's t test).

Figure 2.

Unilateral AAV-aSyn virus vector–injected mouse on the cylinder test. Unilateral AAV-aSyn virus vector injection above mouse SN caused increased ipsilateral paw use starting 2 weeks after injection [aSyn-VEH, 0.2% DMSO; aSyn-KYP-2047, 16 mm), while AAV-GFP injection did not cause any changes in paw use. Chronic KYP-2047 administration (aSyn-KYP) started 4 weeks after injection rescued spontaneous forepaw, while vehicle treatment (aSyn-VEH) had no effect. The dashed line separates the numbers of animals per group (left, n = 7–9; right, n = 4–7). Error bars represent means ± SEM. *p < 0.05, aSyn-VEH versus GFP-VEH; ^∧∧p < 0.01, aSyn-KYP versus GFP-KYP; ^##p < 0.01, aSyn-VEH versus aSyn-KYP; ^§§p < 0.01, aSyn-VEH versus aSyn-KYP, GFP-VEH, and GFP-KYP (two-way ANOVA with Tukey's post hoc comparison).

Figure 3.

Total aSyn immunoreactivity in striatum (STR) and SN after AAV-GFP and AAV-aSyn injections. A, aSyn immunoreactivity showed a robust increase 8 weeks after unilateral AAV-aSyn injection but not in AAV-GFP control groups (GFP-VEH and GFP-KYP). B–D, Significant increase in total aSyn OD was observed in STR, SNpc, and SNpr in OD analyses of AAV-aSyn-injected brain. KYP-2047 treatment (aSyn-KYP) did not have an effect on total aSyn amounts (A–D). OD of total aSyn staining is shown as a fraction of the control side. n = 7–9 in each group. Error bars represent means ± SEM. ***p < 0.001; ****p < 0.0001 (one-way ANOVA with Tukey's post hoc comparison).

Figure 4.

aSyn oligomer immunoreactivity 8 weeks postinjection in AAV-aSyn-injected mouse SN. A, aSyn oligomer-specific particle stereological count was decreased in AAV-aSyn-injected SN after chronic KYP-2047 administration (aSyn-KYP) compared to vehicle treatment (aSyn-VEH). B, No aSyn oligomer staining was observed in AAV-GFP-injected animal SN. C, D, Robust immunostaining of aSyn oligomers was seen in the SNs of vehicle-treated AAV-aSyn-injected animals, while KYP-2047 clearly reduced aSyn oligomer-stained particles (n = 9 in both groups). E, aSyn oligomer-specific particle stereological count after PK treatment did not show significant differences between AAV-aSyn-injected SN after either chronic KYP-2047 administration (aSyn-KYP) or vehicle treatment (aSyn-VEH). G, I, K, M, PK treatment (+PK) only partially cleared total aSyn (G, I) and oligomer-specific aSyn staining (K, M) in AAV-aSyn-treated mouse SN. F–I, Total aSyn staining shows reduced staining after +PK (G, I) compared to staining without proteinase K (−PK; F, H). J–M, A similar effect by PK is seen in oligomer-specific aSyn staining. Less PK-resistant staining, particularly in oligomeric immunohistochemistry, is visually seen in KYP-2047-treated brains (I, M). Error bars represent means ± SEM. ****p < 0.0001 (unpaired Student's t test). Scale bars: 100 μm; insets, 15 μm.

Figure 5.

TH+ cell amount and TH+ immunoreactivity in striatum (STR) and SN showed mild loss of TH+ staining. A, Representative images of TH+ immunoreactivity in STR and SN brain samples 8 weeks after AAV-GFP and AAV-aSyn injections. B, TH+ cell stereology did not yield statistical significance between groups (F_(3,28) = 2.784, p = 0.0593). C, D, TH+ optical density in STR and SN shows significantly decreased TH+ immunoreactivity in the vehicle-treated aSyn injection group in both areas (aSyn-VEH) compared to the GFP-injection control groups (GFP-KYP and GFP-VEH), while a similar decrease was not seen in the aSyn-injected KYP-2047-treated group (aSyn-KYP). n = 7–9 mice/group. Error bars represent means ± SEM. *p < 0.05; **p < 0.01 (one-way ANOVA with Tukey's post hoc comparison).

Figure 6.

Overexpression of aSyn decreases extracellular DA and its metabolites in striatum in mice. A–D, After AAV-GFP (n = 19) or AAV-aSyn (n = 18) injection above the SNpc and 4 weeks of treatment with KYP-2047 or vehicle started 4 weeks postinjection, extracellular concentrations of DA (A), its metabolites DOPAC and HVA (B), 5-HIAA (C), and GABA (D) were measured by striatal microdialysis. Overexpression of aSyn decreased extracellular concentrations of DOPAC and HVA (B), and there was a similar trend for DA (A). Although KYP-2047 elevated the levels of extracellular DA, treatment did not have a statistically significant effect on DA or its metabolites. Virus injection or treatments did not have a statistically significant effect on extracellular 5-HIAA (C) or GABA (D). Error bars represent means ± SEM. *p < 0.05; ^#p = 0.061 (two-way ANOVA).

Figure 7.

Overexpression of aSyn decreases neurotransmitters and their metabolites in nigrostriatal tissue. A–L, HPLC analysis was done after the viral vector injections and the treatment, and the tissue concentrations of DA (A, G), its metabolites DOPAC and HVA (B, H), 5-HT (C, I), its metabolite 5-HIAA (D, J), GABA (E, K), and glutamate (F, L) in striatum (STR) and SN were measured. Overexpression of aSyn decreased the concentration of DA in striatal tissue (A), and there was a similar trend for the metabolites of DA (B) and 5-HIAA (D) in STR. The concentration of DA was not changed in SN tissue (G), but metabolites of DA (H) and 5-HT (I) were decreased. KYP treatment did not have a statistically significant effect on DA, 5-HT, or their metabolites, and virus injection or treatment did not have a statistically significant effect on tissue concentrations of GABA (E, K) or glutamate (F, L). Error bars represent mean ± SEM. *p < 0.05; ***p < 0.001; ^#p = 0.060 (two-way ANOVA).