Extended Data Fig. 7: Human-specific regulation of NPTX2 via TDP-43 binding on its 3’UTR.
From: A model of human neural networks reveals NPTX2 pathology in ALS and FTLD
a, Genomic track depicting the Nptx2 gene structure in mouse, the lack of TDP-43 binding in adult mouse brain (iCLiP crosslinks11), and the sequence conservation across placental mammals95 (top). Genomic track depicting the NPTX2 (human) gene structure, TDP-43 iCLiP crosslinks in mouse brain (after liftover)11, TDP-43 iCLiP crosslinks in human brain13, ‘GU’ repeats, sequence conservation in placental mammals95, and sequence conservation in vertebrates (middle). Zoom-in on 3’UTR (bottom). b, Compared to non-transduced neurons, there is no increase in NPTX2 immunofluorescence labeling in primary neurons overexpressing TDP-43-HA or GFP-FUS (note that HA-FUS also does not induce NPTX2 upregulation in iNets, see Extended Data Fig. 11). Primary neuron experiment was performed once. Scale bars, 20 µm. c, SH-SY5Y were transduced with lentivirus coding for TARDBP shRNAs or Ctrl shRNAs and the effect on protein levels was checked 4 days later by Western blot. d, Quantification of (c) showing significant TDP-43 protein lowering. 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 (1 for not treated). One experiment. Data shown are mean with SD. Ctrl1 shRNA vs TARDBP shRNAb p < 0.0001; Ctrl1 shRNA vs TARDBP shRNAc p < 0.0001; Ctrl2 shRNA vs TARDBP shRNAb p = 0.0003; Ctrl2 shRNA vs TARDBP shRNAc p = 0.0002. e, Dual-Glo Luciferase Assay demonstrating the rescue of TARDBP and NPTX2 3’UTR reporter signal induced by loss of TDP-43 in KD conditions via temporary overexpression of TDP-43-HA and the opposite effect of RNA Recognition Motif mutant (RRMm) TDP-43-HA. Pairs analyzed by unpaired t test. Data shown are mean with SD and p values are two-tailed. Ctrl shRNA + TDP-43-HA WT OFF vs Ctrl shRNA + TDP-43-HA WT ON TARDBP 3’UTR p < 0.0001; TARDBP shRNA + TDP-43-HA WT OFF vs TARDBP shRNA + TDP-43-HA WT ON TARDBP 3’UTR p = 0.0467; Ctrl shRNA + TDP-43-HA RRMm OFF vs Ctrl shRNA + TDP-43-HA RRMm ON TARDBP 3’UTR p = 0.0009; Ctrl shRNA + TDP-43-HA OFF vs Ctrl shRNA + TDP-43-HA WT ON NPTX2 3’UTR p = 0.0203; Ctrl shRNA + TDP-43-HA RRMm OFF vs Ctrl shRNA + TDP-43-HA RRMm ON NPTX2 3’UTR p = 0.0002; TARDBP shRNA + TDP-43-HA RRMm OFF vs TARDBP shRNA + TDP-43-HA RRMm ON NPTX2 3’UTR p = 0.0325n = 6 independently treated wells (3 for OFF conditions).
