Statins reduce neuronal alpha-synuclein aggregation in in vitro models of Parkinson's disease

Abstract

Aggregation of alpha-synuclein (alpha-syn) is believed to play a critical role in the pathogenesis of disorders such as dementia with Lewy bodies and Parkinson's disease. The function of alpha-syn remains unclear, although several lines of evidence suggest that alpha-syn is involved in synaptic vesicle trafficking probably via lipid binding. Moreover, interactions with cholesterol and lipids have been shown to be involved in alpha-syn aggregation. In this context, the main objective of this study was to determine if statins--cholesterol synthesis inhibitors--might interfere with alpha-syn accumulation in cellular models. For this purpose, we studied the effects of lovastatin, simvastatin, and pravastatin on the accumulation of alpha-syn in a stably transfected neuronal cell line and in primary human neurons. Statins reduced the levels of alpha-syn accumulation in the detergent insoluble fraction of the transfected cells. This was accompanied by a redistribution of alpha-syn in caveolar fractions, a reduction in oxidized alpha-syn, and enhanced neurite outgrowth. In contrast, supplementation of the media with cholesterol increased alpha-syn aggregation in detergent insoluble fractions of transfected cells and was accompanied by reduced neurite outgrowth. Taken together, these results suggest that regulation of cholesterol levels with cholesterol inhibitors might be a novel approach for the treatment of Parkinson's disease.

Fig. 1

Effects of statins on cell viability and α-syn accumulation in stably transfected B103 neuronal cells. (a) Analysis of cell viability by MTT assay in α-syn transfected B103 cells treated with lovastatin (LVT), simvastatin (SVT), or pravastatin (PVT). (b) Analysis of levels of cytotoxicity by LDH assay in α-syn transfected B103 cells treated with LVT, SVT, or PVT. LDH was measured in aliquots taken from the media of treated samples and values are expressed as percentage cell death in treated samples compared with total levels of LDH in a lysed control sample. (c) Cellular cholesterol levels in α-syn transfected B103 cells treated with LVT, SVT, or PVT. (d) Immunoblot analysis was performed with detergent soluble and insoluble cell lysate fractions (20 µg total protein per sample). Blots were probed with a rabbit polyclonal anti-α-syn antibody (Chemicon). Immunoblot analysis of fractions from α-syn transfected B103 cells treated with LVT, SVT, or PVT. Levels of α-syn immunoreactivity are expressed as a ratio compared with levels of actin. (e) Quantitative analysis of α-syn levels in detergent soluble fraction. (f) Quantitative analysis of α-syn levels in detergent insoluble fraction showing reduced α-syn accumulation in cells treated with statins. *p < 0.05 compared with vehicle-treated α-syn transfected controls; one-way anova with post hoc Dunnett’s test.

Fig. 2

Immunocytochemical analysis of α-syn immunoreactivity in stably transfected B103 neuronal cells treated with statins. Cells were double-labeled with antibodies against microtubule-associated protein 2 (MAP2) (green channel) and α-syn (red channel) and analyzed by laser scanning confocal microscopy. (a–c) Control vehicle-treated, untransfected B103 cells. (d–f) Vehicle-treated α-syn transfected B103 cells. (g–o) Reduced α-syn immunoreactivity in α-syn transfected B103 cells treated with lovastatin (LVT; g–i), simvastatin (SVT; j–l) or pravastatin (PVT; m–o). (p) Levels of α-syn immunoreactivity in α-syn transfected B103 cells treated with vehicle (veh), LVT, SVT, or PVT. (q) Measurements of neurite lengths in control untransfected and α-syn transfected B103 cells treated with vehicle (veh), LVT, SVT, or PVT. *p < 0.05 compared with vehicle-treated α-syn transfected controls; **p < 0.05 compared with vehicle-treated untransfected controls; one-way anova with post hoc Dunnett’s test. Scale bar: 10 µm (a–o).

Fig. 3

Lovastatin reduces α-syn accumulation in primary fetal cortical neuronal cultures. (a and b) Over 80% of the neuronal cells infected with lentiviral (lenti)-GFP expressed the marker. (c) Endogenous levels of α-syn expression in primary neuronal cultures. (d) Abundant α-syn accumulation in neuronal cell bodies and neurites of primary neurons infected with lenti-α-syn. (e and f) Immunoblot analysis was performed with detergent soluble and insoluble cell lysate fractions (20 µg total protein per sample). Blots were probed with a rabbit polyclonal anti-α-syn antibody (Chemicon). There was reduced α-syn accumulation in the detergent insoluble fraction of lenti-α-syn infected primary neuronal cells treated with lovastatin (LVT). (g–i) Treatment with LVT ameliorated the neurite outgrowth deficits in primary neuronal cells infected with lenti-α-syn. No changes in neurite length were detected in primary neuronal cells infected with lenti-GFP and treated with LVT. (j and k) Immunoblot analysis showing unchanged levels of endogenous α-syn in detergent soluble and insoluble fractions of un-infected primary neuronal cells treated with LVT. (l–n) Treatment with LVT had no significant effects on neurite outgrowth in un-infected primary neuronal cells. *p < 0.05 compared with lenti-α-syn infected, vehicle-treated controls; **p < 0.05 compared with lenti-GFP infected, vehicle-treated controls; Student’s t-test. Scale bar: 50 µm (a–d, g, h, l, and m).

Fig. 4

Effects of lovastatin on α-syn distribution in sucrose gradient fractions from stably transfected B103 neuronal cells. Fractions 1–3 contain little protein content, while fractions 4–8 represent the caveolar lipid raft-like, insoluble components, and soluble components are located in fractions 9–12. (a) Representative cholesterol levels in the sucrose gradient fractions of α-syn transfected cells treated with lovastatin (LVT), simvastatin (SVT), or pravastatin (PVT). (b) Reduced total cholesterol levels among all fractions in statin-treated cells. (c) Representative total protein levels in the sucrose gradient fractions. (d) Total protein levels among all fractions. (e and f) Immunoblot analysis was performed with sucrose gradient fractions prepared from cell lysates (20 µL volume per sample). Blots were probed with a rabbit polyclonal anti-α-syn antibody (Chemicon). Immunoblot analysis of levels of α-syn and flotillin in sucrose gradient fractions prepared from α-syn transfected, vehicle-treated B103 cells. (g and h) Immunoblot analysis of levels of α-syn and flotillin in sucrose gradient fractions prepared from α-syn transfected, LVT-treated B103 cells. *p < 0.05 compared with vehicle-treated α-syn transfected controls; one-way anova with post hoc Dunnett’s test.

Fig. 5

Immunoblot analysis of the effects of statins on post-translational modifications of α-syn. B103 neuronal cells expressing α-syn were treated for 24 h with lovastatin (LVT), simvastatin (SVT), or pravastatin (PVT). (a) Representative western blot illustrating the effects of LVT on insoluble, oxidized, and phosphorylated (pSer129) α-syn. (b) Levels of insoluble and oxidized α-syn but not phosphorylated α-syn were reduced with statins. *p < 0.05 compared with vehicle-treated α-syn transfected controls; one-way anova with post hoc Dunnett’s test.

Fig. 6

Analysis of the effects of cholesterol on α-syn accumulation in stably transfected B103 neuronal cells. (a and b) Total cholesterol levels in the media and cell lysates from α-syn transfected B103 cells exposed to 25 µM cholesterol. (c and d) Immunoblot analysis was performed with detergent insoluble fractions (20 µg total protein per sample). Blots were probed with a rabbit polyclonal anti-α-syn antibody (Chemicon). Immunoblot analysis demonstrating increasing levels of α-syn oligomers in α-syn transfected B103 cells exposed to 25 µM cholesterol. *p < 0.05 compared with vehicle-treated α-syn transfected controls; Student’s t-test.

Fig. 7

Immunocytochemical analysis of the effects of cholesterol on α-syn accumulation and neurite extension in stably transfected B103 neuronal cells. Cells were double-labeled with antibodies against microtubule-associated protein 2 (MAP2) (green channel) and α-syn (red channel) and analyzed by laser scanning confocal microscopy. (a–c) Control vehicle-treated, vector transfected B103 cells. (d–f) Vehicle-treated α-syn transfected B103 cells. (g–i) Increased α-syn immunoreactivity in α-syn transfected B103 cells treated with cholesterol (Cholest). (j) Levels of α-syn immunoreactivity in α-syn transfected B103 cells treated with vehicle or cholesterol. (k) Measurements of neurite lengths in vector or α-syn transfected B103 cells treated with vehicle or cholesterol. Dashed line represents average neurite lengths in control untransfected cells. Experiments were performed in triplicate; *p < 0.05 compared with vehicle-treated α-syn transfected controls; #p < 0.05 compared with vehicle-treated vector transfected controls; Student’s t-test. Scale bar: 15 µm (a–i).