Extended Data Fig. 13: Silencing NPTX2 after onset of TDP-43 pathology alleviates neurotoxicity in iNets.
From: A model of human neural networks reveals NPTX2 pathology in ALS and FTLD
a, HA and NPTX2 immunofluorescence in EF1α-driven NPTX2-HA overexpressing iNet neurons at 1 week results in an accumulation throughout the soma and processes of transgenic iNet neurons, similar to NPTX2 accumulation induced by TDP-43-HA overexpression. One experiment. b, Correlation plot of averaged (per well) mean nuclear TDP-43-HA intensities (1 week) and the % loss of transgenic TDP-43-HA iNet neurons at 2 weeks. TDP-43-HA nuclear intensity was measured in 2852-3749 cells per condition and between 281-2265 cells were analyzed per data point. c, Western blot demonstrating the effect of expression of NPTX2 shRNAs (vs Ctrl shRNA) in transduced SH-SY5Y cells after 4 days. d, Signal quantification of (c). One-way ANOVA followed by Tukey’s multiple comparison test (mean of each dataset compared with the mean of every other dataset). n = 3 individually transduced well. Data shown as mean with SD. Ctrl shRNA vs NPTX2 shRNAa,b,c and d p < 0.0001. e, Western blot showing NPTX2 upregulation in iNets upon TDP-43-HA overexpression which is corrected by co-expression of NPTX2 shRNAs. f, Signal quantification of (e). One-way ANOVA followed by Tukey’s multiple comparisons test (mean of each dataset compared with the mean of every other dataset, statistics not performed for n < 3). n = 3 individually transduced wells (2 for TDP-43 OE only). Data are shown as mean with SD. TDP-43-HA OE + Ctrl shRNA vs TDP-43-HA OE + NPTX2 shRNAc p < 0.0001 or shRNAd p < 0.0001. g, Representative immunofluorescence of TRE-driven TDP-43-HA-transduced iNets at 1 week, showing clear NPTX2 upregulation and accumulation in both TDP-43-HA only and Ctrl shRNA conditions, which was corrected by NPTX2 KD. Note that the panel of TDP-43-HA + Ctrl shRNA is derived from a separate experiment. h, Representative immunofluorescence (out of 3 experiments) of TRE-driven TDP-43-HA + shRNA NPTX2 C expressing neurons at 2 weeks. i, Quantification of TDP-43-HA-transgenic cell counts in the conditions shown in (g,h). Each data point represents a sum of all HA+ cells counted from 182 fields of view of an independent well and normalized with DAPI, and shown as % of 1 W week condition. Between 189 - 16453 cells were analyzed per data point. Only significant differences are shown. Two-way ANOVA (mixed model) followed by Tukey’s multiple comparison test (compare each dataset mean with every other dataset mean). Differently gray scaled data points are independent experiments. Data shown are mean with SD. TDP-43-HA only vs TDP-43-HA + NPTX2 shRNAd p = 0.0356; TDP-43-HA + Ctrl shRNA2 vs TDP-43-HA + NPTX2 shRNAd p = 0.0316. Scale bars, (a, g, h) 20 µm.
