Lovastatin ameliorates alpha-synuclein accumulation and oxidation in transgenic mouse models of alpha-synucleinopathies

Abstract

Alpha-synuclein (alpha-syn) aggregation is a neuropathological hallmark of many diseases including Dementia with Lewy Bodies (DLB) and Parkinson's Disease (PD), collectively termed the alpha-synucleinopathies. The mechanisms underlying alpha-syn aggregation remain elusive though emerging science has hypothesized that the interaction between cholesterol and alpha-syn may play a role. Cholesterol has been linked to alpha-synucleinopathies by recent work suggesting cholesterol metabolites appear to accelerate alpha-syn fibrillization. Consistent with these findings, cholesterol-lowering agents have been demonstrated to reduce alpha-syn accumulation and the associated neuronal pathology in vitro. In this context, this study sought to investigate the in vivo effects of the cholesterol synthesis inhibitor lovastatin on alpha-syn aggregation in two different transgenic (Tg) mouse models that neuronally overexpress human alpha-syn. Lovastatin-treated mice displayed significantly reduced plasma cholesterol levels and levels of oxidized cholesterol metabolites in the brain in comparison to saline-treated controls. Immunohistochemical analysis demonstrated a significant reduction of neuronal alpha-syn aggregates and alpha-syn immunoreactive neuropil in the temporal cortex of lovastatin-treated Tg mice in comparison to saline-treated alpha-syn Tg controls. Consistently, immunoblot analysis of mouse brain homogenates showed a reduction in levels of total and oxidized alpha-syn in lovastatin-treated alpha-syn Tg mice in comparison to saline-treated alpha-syn Tg controls. The reduced alpha-syn accumulation in lovastatin-treated mice was associated with abrogation of neuronal pathology. The results from this study demonstrate that lovastatin administration can reduce alpha-syn aggregation and associated neuropathology and support the possibility that treatment with cholesterol-lowering agents may be beneficial for patients with PD and/or DLB.

Figure 1. Characterization of lovastatin activity

In order to assess the effect of the lovastatin treatment plasma cholesterol levels were analyzed in Non Tg, PDGFβ α-syn Tg and mThy1.2 α-syn Tg mice (A). To assess central effects of lovastatin treatment oxidized cholesterol metabolites were analyzed in the brains of Non Tg, PDGFβ α-syn Tg and mThy1.2 α-syn Tg mice (B). # Indicates a significant difference between saline-treated Non Tg mice and saline-treated α-syn Tg mice (p<0.05, one way ANOVA and post hoc Fisher). * Indicates a significant difference between saline-treated and lovastatin-treated members of the same genotype (i.e. saline-treated PDGFβ α-syn Tg mice Vs lovastatin-treated PDGFβ α-syn Tg mice) (p<0.05, one way ANOVA and post hoc Fisher).

Figure 2. α-syn immunoreactivity in α-syn Tg mice following lovastatin treatment

α-syn immunoreactivity was assessed in the temporal cortex of saline-treated Non Tg, PDGFβ α-syn Tg and mThy1.2 α-syn Tg mice (A, B and C) and lovastatin-treated Non Tg, PDGFβ α-syn Tg and mThy1.2 α-syn Tg mice (D, E, and F). Quantitative analysis of levels of α-syn aggregates (G) and α-syn immunoreactive neuropil (H) was also conducted. Scale bar = 50μM # Indicates a significant difference between saline-treated Non Tg mice and saline-treated α-syn Tg mice (p<0.05, one way ANOVA and post hoc Fisher). * Indicates a significant difference between saline-treated and lovastatin-treated members of the same genotype (i.e. saline-treated PDGFβ α-syn Tg mice Vs lovastatin-treated PDGFβ α-syn Tg mice) (p<0.05, one way ANOVA and post hoc Fisher).

Figure 3. Immunoblot analysis of total and oxidized α-syn in α-syn Tg mice following lovastatin treatment – soluble fraction

In order to examine the effect of lovastatin on α-syn levels, soluble fractions isolated from mouse brain homogenates were assessed by immunoblot with antibodies against total and oxidized α-syn (A). Quantitative analysis of bands at 14kDa and 28kDa (corresponding to monomeric and dimeric species of α-syn) were analyzed for total α-syn (B and E) and oxidized α-syn (C and F). In order to investigate the effect of lovastatin on relative levels of total and oxidized α-syn, ratios were calculated at 14kDa (D) and 28kDa (G). # Indicates a significant difference between saline-treated Non Tg mice and saline-treated α-syn Tg mice (p<0.05, one way ANOVA and post hoc Fisher). * Indicates a significant difference between saline-treated and lovastatin-treated members of the same genotype (i.e. saline-treated PDGFβ α-syn Tg mice Vs lovastatin-treated PDGFβ α-syn Tg mice) (p<0.05, one way ANOVA and post hoc Fisher).

Figure 4. Immunoblot analysis of total and oxidized α-syn in α-syn Tg mice following lovastatin treatment – insoluble fraction

In order to examine the effect of lovastatin on α-syn levels, insoluble fractions isolated from mouse brain homogenates were assessed by immunoblot with antibodies against total and oxidized α-syn (A). Quantitative analysis of bands at 14kDa and 28kDa (corresponding to monomeric and dimeric species of α-syn) were analyzed for total α-syn (B and E) and oxidized α-syn (C and F). In order to investigate the effect of lovastatin on relative levels of total and oxidized α-syn, ratios were calculated at 14kDa (D) and 28kDa (G). # Indicates a significant difference between saline-treated Non Tg mice and saline-treated α-syn Tg mice (p<0.05, one way ANOVA and post hoc Fisher). * Indicates a significant difference between saline-treated and lovastatin-treated members of the same genotype (i.e. saline-treated PDGFβ α-syn Tg mice Vs lovastatin-treated PDGFβ α-syn Tg mice) (p<0.05, one way ANOVA and post hoc Fisher).

Figure 5. NeuN immunoreactivity to assess neuronal pathology in α-syn Tg mice following lovastatin treatment

Neuronal density, as evidenced by NeuN immunoreactivity, was examined in the temporal cortex of saline-treated Non Tg, PDGFβ α-syn Tg and mThy1.2 α-syn Tg mice (A, B and C) and lovastatin-treated Non Tg, PDGFβ α-syn Tg and mThy1.2 α-syn Tg mice (D, E, and F), quantitative analysis (G). Scale bar = 50μM # Indicates a significant difference between saline-treated Non Tg mice and saline-treated α-syn Tg mice (p<0.05, one way ANOVA and post hoc Fisher). * Indicates a significant difference between saline-treated and lovastatin-treated members of the same genotype (i.e. saline-treated PDGFβ α-syn Tg mice Vs lovastatin-treated PDGFβ α-syn Tg mice) (p<0.05, one way ANOVA and post hoc Fisher).

Figure 6. MAP2 immunoreactivity to assess dendritic pathology in α-syn Tg mice following lovastatin treatment

Dendritic complexity, as evidenced by MAP2 immunoreactivity, was examined in the temporal cortex of saline-treated Non Tg, PDGFβ α-syn Tg and mThy1.2 α-syn Tg mice (A, B and C) and lovastatin-treated Non Tg, PDGFβ α-syn Tg and mThy1.2 α-syn Tg mice (D, E, and F), quantitative analysis (G). Scale bar = 50μM # Indicates a significant difference between saline-treated Non Tg mice and saline-treated α-syn Tg mice (p<0.05, one way ANOVA and post hoc Fisher). * Indicates a significant difference between saline-treated and lovastatin-treated members of the same genotype (i.e. saline-treated PDGFβ α-syn Tg mice Vs lovastatin-treated PDGFβ α-syn Tg mice) (p<0.05, one way ANOVA and post hoc Fisher).