Fig. 7
Antibodies targeting RRM2 and the glycine-rich region of TDP-43 suppress aggregation and LLPS in vitro. A Schematic overview of a panel of novel TDP-43 monoclonal antibodies (mAbs) raised from immunization in Fig. 1 as indicated. The most potent mAbs are highlighted in bold. B Aggregation and condensation assays using recombinant TDP-43-MBP-His6. Upon cleavage of the solubilization tag maltose-binding protein (MBP) using TEV protease, soluble TDP-43 forms aggregates (C) or condensates (D/E), depending on the buffer conditions. Condensates were either analyzed by pelleting (D) or by confocal microscopy (E). mAbs were added to soluble TDP-43-MBP-His6 together with TEV protease. C Representative images of Alexa 488-labeled and in vitro aggregated TDP-43, treated with the indicated mAb (molar ratio TDP-43-MBP-His6: mAb = 1:2). Images were taken 48 h after TEV cleavage. Clones 30D3 and 31E9 nearly completely prevented aggregation. Overview and magnified insets on the right bottom corner are shown. Scale bar overview = 100 µm, scale bar inset = 50 µm. D Sedimentation assay to analyze condensation of cleaved TDP-43-MBP-His6 in the presence of mAbs. Supernatant (S) and condensates (C) were separated by centrifugation 2 h after TEV cleavage. Representative immunoblots and quantification of S/(C + S) ratios (bar graphs indicate mean + SD) from four replicates are shown. An asterisk indicates the residual uncleaved TDP-43-MBP-His6, the arrowhead marks the cleaved TDP-43 band. mAbs were compared to their respective isotype controls using pairwise t-test with Benjamini–Hochberg correction for multiple testing: IgG2c vs. 30D3: p = 0.017; IgG2c vs. 36C5: p = 3.24*10–4; IgG2c vs. 36C10: p = 0.016; IgG1 vs. 27A3: p = 0.021; IgG1 vs. 31E9: p = 3.87*10–4. E Confocal images of Alexa 488-coupled TDP-43-MBP-His6 and DyLight 650-labeled mAbs show liquid–liquid phase separation of TDP-43 and co-partitioning of mAbs with TDP-43 condensates after TEV protease cleavage. Scale bar = 10 µm