Fig. 2

Aggregation-dependent neurotoxicity of different neurodegeneration-related peptides and proteins in cell culture. To test the previously described [10, 11] toxic effect of neurodegeneration-related aggregates, we used a differentiated SH-SY5Y human neuroblastoma cell model. The EZ4U and Calcein-AM cell viability assays were used to detect the NADH- and esterase-activity-dependent cell viability of the cultures (presented in orange and green columns of the chart, respectively). The mean viability of the untreated control wells was regarded as 100% (the S.E.M. of the mean was ±2.8%). The aggregation level of the proteins was measured with Congo red spectrophotometric assay (red line in the background of the chart). The length of incubation and aggregation time (3 h = 3 h and 3d = 3 days) influenced the toxicity of the treatment in most of the peptides and proteins tested. Significant differences (p ≤ 0.01) were detected between the 3 h- and 3d–groups (no markers on columns) in both viability assays. Significant differences could be detected between the 3 h- and 3d–aggregated forms in the Congo red assay for all Aβs, except for Aβ11–42, Aβ1–28, Aβ1–42 S1, and Aβ1–42 S2. Alpha-synuclein (α-Syn, type E46K) and prepared ‘scrapie’ (PrPSc) form of prion protein also showed significant in vitro toxicity, contrasting with the ineffective normal cellular prion (PrPC). The aggregation time of the prions was 1 day (1d). Each molecule was used in 100 μg/mL concentration; n = 8 (for EZ4U and Calcein-AM) and 20 (for Congo red) wells per group. The error bars present the S.E.M. For statistical analysis, one-way ANOVA was used followed by the Bonferroni post hoc test, and the level of significance were p*** ≤ 0.001 (*- the significant rate of deviation from the non-toxic and non-aggregated forms /Aβ 1–28, 1–42 S1, 1–42 S2, PrPC/ in both viability and Congo red assays)