Figure 5.

Treating iPSC-derived human neurons with Lewy-associated brain extracts. (A) Neurogenin-induced human neurons were treated with protein-normalized volumes of sonicated Lewy-associated brain extracts. Neurite morphology was monitored for 88–96 h. Images were analysed by applying a neurite mask (Supplementary Fig. 5) and automatically quantifying mean neurite length. In these representative images, neurons treated with αSyn-enriched control brain extract (top) show largely intact neurites at 52 h, whereas neurons treated with A01-213 DLB αSyn-enriched extract display neurite retraction (bottom). Images shown from each brain sample are taken from identical fields at different time points. Bars are 200 μm. (B) Mean neurite length calculated across four images per well at 88 h was normalized to the mean neurite length at t = 0. While the amount of αSyn levels applied to each well was not normalized, these levels did not correlate with the extent of neurite retraction (not shown). Means with standard deviations are shown. (C) Pooling of data collected from four non-synucleinopathy control extracts and five DLB patient extracts across three separate experiments shows a significantly greater neurite retraction in neurons treated with DLB extracts (P = 0.0066, Mann–Whitney comparison of ranks). Means with SEMs are shown. (D) Human iPSCs were treated with two extracts from five DLB brains, each split into three aliquots: one was applied directly; one was pre-incubated with 2F12, a monoclonal antibody to αSyn; and a third was pre-incubated with 4G8, a monoclonal antibody to Aβ. Immunoneutralization of αSyn in these extracts restored the length of neurons treated compared with neurons treated with non-immunoneutralized aliquots of the same DLB extracts (P = 0.0078, Wilcoxon paired comparison of ranks). Each point represents the mean of technical triplicates from a single experiment. Means with SEMs are shown.