Fig. 8
TDP-43 mAbs suppress cellular uptake of recombinant TDP-43 aggregates. A Overview of TDP-43 uptake assay. Aggregation of pHrodo Green-labeled TDP-43-MBP-His6 was induced by TEV protease cleavage and thorough shaking for 30 min, followed by a 2 h incubation step at room temperature. Preformed aggregates were then incubated with mAbs for 30 min before addition to SH-SY5Y cells. Upon cellular uptake via endocytosis, the pHrodo-tagged aggregates show increased fluorescence in acidic compartments, such as early endosomes (EE), and, even more so, in late endosomes (LE) or lysosomes. After 24 h of incubation, pHrodo fluorescence was analyzed by flow cytometry in the FITC channel. B Representative flow cytometry dot plots showing uptake of pHrodo-TDP-43agg in combination with different control or TDP-43 specific mAbs in SH-SY5Y cells. Data areshown as relative fluorescence in the FITC channel (x-axis) and side scatter (SSC-A, y-axis). Relevant comparisons are overlaid. Cells were pregated to identify live singlets (not shown). Vertical dotted lines indicate gating for pHrodo+ live singlets. C Quantification of TDP-43 aggregate uptake in SH-SY5Y cells, measured as percentage of pHrodo-positive cells of total live single cells. Bar graphs represent mean + SD from n = 3–4 independent experiments. The following comparisons revealed statistically significant differences after pairwise t-test with Benjamini–Hochberg correction: pHrodo-TDP-43agg + IgG1 ctrl vs. pHrodo-TDP-43agg + 31E9 (G1): p = 0.037; pHrodo-TDP-43agg + IgG2c ctrl vs. pHrodo-TDP-43agg + 36C10 (G2c): p = 0.023; non-treated vs. pHrodo-TDP-43agg: p = 0.023; pHrodo-TDP-43mono vs. pHrodo-TDP-43agg: p = 0.023; TDP-43agg vs. pHrodo-TDP-43agg: p = 0.023; pHrodo-TDP-43agg vs. pHrodo-TDP-43agg (22 °C): p = 0.023