TDP-25 sequesters and impairs the proteasome.Figure 3A: Rat primary cortical neurons were transduced with GFP-TDP-25 wild-type, GFP-TDP-43 or GFP at DIV5. Due to high toxicity, GFP-TDP-43 (n=6) and GFP control samples (n=6) were harvested at DIV5+4, while GFP-TDP-25 (n=5) and additional GFP control samples (n=6) were harvested at DIV5+8. Immunoprecipitates were analyzed by LC-MS/MS. Volcano plots indicate enrichment and statistical significance (grey dots: FDR-corrected p<0.05). Proteins associated with GO term "proteasome complex" (GO:0000502) are labeled in orange and proteins associated with GO term "RNA splicing" (GO:0008380) in purple. Full data is listed in Table S1. A comparison with previous proteomic studies is shown in Fig. EV3A. B: Overlap of GFP-TDP-25 and GFP-TDP-43 interactomes. Significant interactors in FDR-based approach (see methods). C: Combined violin and box plot (box shows lower quartile, median and upper quartile, whiskers extend to the most extreme data point within 1.5 the interquartile range of the box) of the proportion of low-complexity regions (D2P2) of GFP-TDP-25-specific, GFP-TDP-43-specific or shared/common interactors. TDP-25 specific (0.144 ± 0.021, n=432 Uniprot IDs) vs TDP-43 specific (0.266 ± 0.061, n=78) vs common interactors (0.184 ± 0.02, n=481): H(1)=22.276, df=2, p=1.455*10-5, η²[H]=0.0205, Kruskal-Wallis test. TDP-25 specific vs TDP-43 specific: ***p=0.00054, TDP-25 specific vs common: ***p=0.00035, TDP-43 specific vs common interactors: *p=0.04893, Pairwise Wilcoxon Rank Sum Tests with Benjamini-Hochberg correction. D: Gene ontology (GO) enrichment analysis (biological process) in interactors specific for either GFP-TDP-25 or GFP-TDP-43. Full list of GO terms is found in Table S2. E: Immunofluorescence of hippocampal neurons transduced with GFP, GFP-TDP-25 wild-type or mutant and treated with MG132 (10 µM, 14h, DIV5+8). Representative images of GFP-TDP-25 inclusions and diffuse cytoplasmic staining. Automated quantification of GFP intensity normalized to the number of DAPI stained nuclei (omitted here for clarity) in MG132 treated cells compared to DMSO control. Counterstain to label the neuronal cytoskeleton (MAP2). Scale bar = 10 µm. Box plot as in Fig. 3C. GFP (1.23 ± 0.393, n=5 independent experiments) vs GFP-TDP-25 wt (2.11 ± 0.804, n=5) vs GFP-TDP-25 mut (2.36 ± 1.16, n=5): H(1)=7.34, df=2, p=0.02548, η²[H]=0.445, Kruskal-Wallis test. GFP vs GFP-TDP-25 wt: *p=0.048, GFP vs GFP-TDP-25 mut: *p=0.048, GFP-TDP-25 wt vs GFP-TDP-25 mut: p=0.690, Pairwise Wilcoxon Rank Sum Tests with Benjamini-Hochberg correction. F: A HEK293 cell line expressing the proteostasis reporter UbG76V-GFP was transiently transfected with tagRFP, tagRFP-TDP-25 wild-type, tagRFP-TDP-25mutant, tagRFP-TDP-43 wild-type or tagRFP-TDP-43 mutant. UbG76V-GFP levels were analyzed by flow cytometry 72 h after transfection. The relationship of binned tagRFP fluorescence and UbG76V-GFP fluorescence is plotted. Values shown are means ± SD obtained from n=4 independent experiments. For clarity, adjusted p-values of pairwise comparisons within each tagRFP intensity bin are shown in Fig. EV3C. Note that at lower expression levels (bin < 30) both TDP-25 variants induce higher UbG76V-GFP levels than their respective full-length counterparts indicative of stronger impairment of proteostasis. The full-length variants show meaningful accumulation of the reporter levels only at the high-expression levels. The mutant versions of TDP-25 as well as TDP-43 lead to an increased reporter signal at higher expression levels compared to wild-type variants.