Stabilization of α-Synuclein Fibril Clusters Prevents Fragmentation and Reduces Seeding Activity and Toxicity

Abstract

Protein misfolding results in the accumulation of aggregated β-sheet-rich structures in Parkinson's disease (PD) and Alzheimer's disease. The toxic oligomer hypothesis stipulates that prefibrillar assemblies, such as soluble oligomers or protofibrils, are responsible for the poor prognosis of these diseases. Previous studies demonstrated that a small molecule related to the natural compound orcein, O4, directly binds to amyloid-β fibrils and stabilizes them, accelerating the formation of end-stage mature fibrils. Here we demonstrate a similar phenomenon during O4 treatment of α-synuclein (αsyn) aggregates, the protein responsible for PD pathology. While the drug did not change the kinetics of aggregate formation as measured by the amyloidophilic dye thioflavin T, O4 depleted αsyn oligomers and promoted the formation of sodium dodecyl sulfate and proteinase K resistant aggregates consisting of large fibril clusters. These fibril clusters exhibited reduced toxicity to human neuronal model cells and reduced seeding activity in vitro. The effectiveness of O4 decreased when it was added at later points in the αsyn aggregation pathway, which suggests that the incorporation of O4 into fibril assemblies stabilizes them against chemical, enzymatic, and mechanic degradation. These findings suggest that small molecules, which stabilize amyloid fibrils, can prevent fibril fragmentation and seeding and consequently prevent prion-like replication of misfolded αsyn. Inhibiting prion replication by fibril stabilization could thus be a therapeutic strategy for PD.

Figure 1

O4 does not alter the formation of β-sheet aggregates. (A) ThT fluorescence of αsyn (30μM) aggregation in the presence and absence of 1:1 O4 to αsyn under intermittent shaking conditions. The results represented mean ThT fluorescence intensities (n=3). (B) Normalized ThT fluorescence signals of αsyn samples in (A). Time points for further analysis: 6 h, 20 h, 48 h, 80 h, and 168 h were denoted. (C, D) CD spectra of αsyn incubated with O4 for 0 h and 48 h, respectively.

Figure 2

O4 induces SDS and protease resistant αsyn aggregates. (A) Transmission electron microscopy (TEM) of αsyn time points denoted in Fig. 1 in the presence or absence of O4; scale bar 500 nm. (B and C) Effect of O4 on SDS resistant aggregate formation quantified by FRA. The fluorescent signal was generated by immunostaining using anti-αsyn antibodies on a cellulose acetate membrane. (D) Effect of O4 on αsyn resistance to proteinase K digestion on 1 week time point samples. An additional sample was formed by incubating the αsyn control sample with equimolar O4 for 30 minutes to observe short-term effects of O4 treatment. HMW= High molecular weight, T=trimer, D= dimer, and M=monomer.

Figure 3

O4 Incubation rescues αsyn toxicity in vitro. (A) Time course of ThT fluorescence of αsyn under constant shaking condition. (B) ThT fluorescence data from (A) normalized to end point fluorescence. –(C) Aggregation time course monitored by light scattering. (D) Fraction of soluble αsyn analyzed densitometrically from SDS-PAGE after centrifugation at 200,000 × g. (E and F) Effects of O4 on the formation of αsyn oligomers as assessed by anti-oligomer antibody, A11, dot blot analysis. (G) Assessment of αsyn cytotoxicity using the MTT metabolic assay. SH-EP cells were incubated with αsyn time point samples (2.5μM) used in the A11 dot blot analysis for three days. *P<0.05, **P<0.01, ***P<0.001 (Student’s T-Test, n = 5. (H) Normalized MTT and A11 fluorescent signal data to compare time course dynamics.

Figure 4

O4 Reduces seeding activity. (A) αsyn aggregation (30μM) monitored by Thioflavin T (ThT) fluorescence in the presence of 1:1 O4 to αsyn and 5:1 O4 to αsyn. Samples were taken at 6 h, 20 h, 48 h, and 72 h and sonicated to create seeds. (B - E); αsyn aggregation (30μM) in the presence of untreated seeds and O4 treated seeds created from the 6h, 20 h, 48 h, and 72 h samples, respectively (10% m/m). The results represented mean ThT fluorescence intensities (n=3).

Figure 5

Time dependent effect of O4 on forming SDS resistant aggregates and cellular toxicity. (A) Assessment of time point αsyn cytotoxicity using the MTT metabolic assay. SH-EP cells were incubated with αsyn time point samples (1.25μM) in the presence or absence of O4 for three days. The time points selected correspond to the same time points in Figure 1. *P<0.05, **P<0.01, ***P<0.001 (Student’s T-Test, n = 5). (B, C, and D) αsyn aggregation (30μM) in the presence of untreated seeds and O4 treated seeds created from the 20 h, 48 h, and 72 h samples, respectively (10% m/m). (E) ThT fluorescence of αsyn (30μM) aggregation with O4 added at different time points. The aggregation period lasted for 1 week. The results represented mean ThT fluorescence (n=3). (F) Effect of adding O4 at different time points during the aggregation period on SDS resistant aggregates quantified by FRA. The * on the 168 h time point indicates O4 treatment for 30 minutes. (G) Assessment of αsyn cytotoxicity using the MTT metabolic assay using the same samples (0.3 μM) used in the FRA (F). *P<0.05, **P<0.01, ***P<0.001 (Student’s T-Test, n = 3).

Figure 6

Model of autocatalytic amyloid formation through fibril fragmentation and secondary nucleation. O4 promotes the formation of fibril bundles and inhibits fragmentation and seeding.