Figure 4
From: Cyclized NDGA modifies dynamic α-synuclein monomers preventing aggregation and toxicity
NDGA pretreatment prevents α-synuclein aggregation. (a) α-Synuclein did not aggregate after pretreatment with NDGA. α-Synuclein was incubated 1:1 with small molecules for 24 hours then dialyzed against PBS for 24 hours. After aggregation for 3 days, PBS-insoluble α-synuclein was separated by centrifugation (21k g for 10 min). Soluble and insoluble fractions were boiled in SDS, run by SDS-PAGE, and colloidal stained. α-Synuclein in each fraction was quantified by in-gel densitometry (n = 3, ***p < 0.001). (b) Both oligomeric and monomeric α-synuclein species induced by NDGA treatment resist aggregation. α-Synuclein was incubated 1:1 with small molecules for 24 hours then subjected to native state size exclusion chromatography. Oligomeric (≥51 Å) and Monomeric (<51 Å) α-synuclein fractions were collected and aggregated separately for 3 days. Soluble and insoluble species were separated and quantified as above (n = 3). (c) NDGA-treated α-synuclein resisted fibrillization in the presence of 5% fibril seed. α-Synuclein was incubated 1:1 with small molecules for 24 hours then dialyzed against excess PBS for 24 hours. Untreated α-synuclein fibrils equal to 5% total protein was added immediately before mixtures were aggregated for 3 days. Soluble and insoluble species were separated and quantified as above (n = 3, ***p < 0.001). (d) NDGA pretreated, dialyzed α-synuclein inhibited aggregation of untreated α-synuclein. α-Synuclein was incubated 1:1 with small molecules for 24 hours then dialyzed against excess PBS for 24 hours. Pretreated, dialyzed α-synuclein was then mixed with untreated monomeric α-synuclein at the indicated ratios. Mixtures were aggregated for 3 days. Soluble and insoluble species were separated and quantified as above (n = 3, ***p < 0.001).
