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Fig. 8 | Molecular Neurodegeneration

Fig. 8

From: VCP suppresses proteopathic seeding in neurons

Fig. 8

VCP disease mutation expression enhances αS seeding. A αS biosensors were transfected with plasmids expressing VCP-WT, or one of three disease mutations (R95G, R155H and A232E) fused to an mcherry tag for 24 hours and then treated with αS PFF(10nM). FRET efficiency is quantified in mCherry+ and mCherry- cells separately and all normalized to VCP-WT (n=11 repeats for each group. ***p < 0.001; ****p < 0.0001; ns, no significance; two-way ANOVA with Dunnett’s correction). B αS biosensors were transfected with plasmids expressing mCherry or mCherry-VCP-WT for 24 hours and then treated with αS PFF (10nM). FRET efficiency isquantified in mCherry+ and mCherry- cells separately and all normalized to mCherry. (n=11 repeats foreach group. ***p < 0.001; ****p < 0.0001; ns, no significance; two-way ANOVA with Dunnett’scorrection). C Immunofluorescence for phospho-αS and Tuj1 in HNs treated with empty control lentiviral vector, VCP-WT-myc or one of two myc tagged VCP disease mutations (R155H or A232E). HNs were transduced with lentivirus as indicated for 5 days before 10nM αS PFF treatment. HNs were harvested after another 5days. Scale bar=20μm. D Quantitation of phospho-αS/Tuj1 staining in 8C. Experiments were repeated from 3 different cultures. *p < 0.05; ****p < 0.0001; ns, no significance compared with the VCP-WT group by one-way ANOVA. E Immunoblot of lysates from HNs overexpressing lentiviruses expressing empty vector, VCP-WT-myc, VCP-R155H-myc or VCP-A232E-myc using an anti-myc antibody and pan 14-3-3 as a loading control. F Immunofluorescence for phospho-αS and Tuj1 in HNs from wild-type mice or mice carrying a VCP-R155H knock-in allele(VCPR155H/WT) treated with αS PFF (1μg/ml) for 5 days. G Quantitation of phospho-αS/Tuj1 staining in 8F. Neurons came from 10 and 15 independent cultures from WT and VCPR155H/WT embryos. Outlier isremoved by ROUT method, Q=1%, followed by Student’s t test. p<0.0001), Scale bar =20μm. H Immunofluorescence of WT or VCPR155H/WT HNs treated with αS PFF (1μg/ml) and harvested 5 days later with anti-phospho-αS (red) and p62 (green) (upper panels) or anti-phospho-αS (red) LAMP1 (green)(lower panels). Scale bar =10μm

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