Fig. 5: Effect of Clusterin on α-Synuclein aggregation and seeding.
From: The extracellular chaperone Clusterin enhances Tau aggregate seeding in a cellular model
a Aggregation of α-Syn (300 μM) without (black) or with Clu (0.3 μM and 3 μM in cyan and red, respectively) was monitored by ThT fluorescence. Molar ratios of α-Syn:Clu are indicated. arb.units, arbitrary units. Averages ± SEM (n = 4 independent experiments). b Effect of Clu on seeding potency of α-Syn aggregation reactions as in (a). Seeding was measured by quantifying the fraction of HEK293T cells stably expressing GFP-α-Syn(A53T) that contained aggregates after 24 h of seeding (200 ng α-Syn) with lipofectamine. Molar ratios of α-Syn:Clu are indicated. Significance is represented relative to α-Syn alone at each time point. Averages ± SEM (n = at least 100 cells examined over three independent experiments). ***p < 0.001 by two-way ANOVA with Sidak post hoc test. (α-Syn 24 h vs. α-Syn/Clu 1000:1 24 h p = 1.9 × 10−8; α-Syn 24 h vs. α-Syn/Clu 100:1 24 h p = 8.4 × 10−13; α-Syn 36 h vs. α-Syn/Clu 1000:1 36 h p = 4.5 × 10−7; α-Syn 36 h vs. α-Syn/Clu 100:1 36 h p = 1.3 × 10−11; α-Syn 72 h vs. α-Syn/Clu 1000:1 72 h p = 1.8 × 10−7; α-Syn 72 h vs. α-Syn/Clu 100:1 72 h p = 2.3 × 10−13). c Representative images of HEK 293 T GFP-α-Syn(A53T) cells seeded with aggregation reactions (200 ng α-Syn after 0 h, 24 h and 72 h aggregation (a)) with or without Clu. GFP-α-Syn(A53T) and DAPI nuclear staining are shown in green and blue, respectively. Arrowheads indicate aggregates. Scale bar, 30 μm. d Heparan sulfate proteoglycan (HSPG) mediated internalization of α-Syn and α-Syn/Clu aggregates. Seeding was measured by quantifying the fraction of GFP-α-Syn(A53T) cells that contained aggregates after 72 h of seeding (50 µg α-Syn after 72 h aggregation (a)) without lipofectamine (−Heparin). GFP-α-Syn(A53T) cells were also treated with α-Syn and α-Syn/Clu aggregates (50 µg) in combination with heparin (200 µg/ml). Molar ratios of α-Syn:Clu are indicated. Data represent the mean ± SEM (n = at least 1000 cells examined over three independent experiments). **p < 0.01, ***p < 0.001 by two-way ANOVA with Sidak post hoc test (−Heparin α-Syn vs. −Heparin α-Syn/Clu 100:1 p = 0.003; −Heparin α-Syn vs. +Heparin α-Syn p = 4.8 × 10−5; −Heparin α-Syn/Clu 1000:1 vs. +Heparin α-Syn/Clu 1000:1 p = 0.007). The significance of +Heparin reactions is relative to the respective −Heparin control.
