Fig. 1: TDP-43 pathologies activate the ISR pathway independently of chemical stress.

a Schematic depicting the ISR pathway, including kinases mediating the phosphorylation of eIF2α, and activation of eIF2B by DNL343. b Confocal microscopy images showing induced expression of GFP-TDP-43^FL and GFP-TDP-43^(86-414) variant in H4 cells increased immunoreactivity of ATF4 (anti-ATF4, red) compared to GFP control. This ATF4 signal was reduced with 1 µM DNL343 treatment. Scale bar: 20 µm. c Quantification of ATF4 immunoreactivity in (b) from n = 3 (GFP Control) or 4 biological replicates (GFP-TDP-43 variants). a.u.: arbitrary units. d ECLIA-based assay result demonstrating elevated ATF4 protein levels with induced expression of GFP-TDP-43^FL and GFP-TDP-43^(86-414), compared to GFP control cells. Pre-treatment with DNL343 reduced ATF4 protein levels in each cell line (n = 6 biological replicates). e Volcano plot of differentially regulated genes in H4 cells induced with GFP-TDP-43^FL compared to GFP control. Representative ISR genes with significant expression changes are labeled as red (upregulation) or blue (downregulation) and insignificant genes as gray. Significance cutoff, adjusted p-value < 0.05. f Volcano plot of differentially regulated genes in H4 cells induced with GFP-TDP-43^(86-414) compared to GFP control. Representative ISR genes are labeled as in (e). Significance cutoff, adjusted p-value < 0.05. g Gene set enrichment analysis (GSEA) plot for ISR gene comparing truncated GFP-TDP-43^(86-414) from GFP control expressing cells. h Volcano plot of differentially regulated genes 24 hours after DNL343 treatment in H4 cells induced with GFP-TDP-43^(86-414). The same set of representative ISR genes from (e) and (f) are labeled. All data are shown as mean ± SEM (c, d). Statistical significance was determined with one-way ANOVA, with Tukey’s multiple comparison, ns P > 0.05. Source data are provided in Source Data file.