FIGURE 8.

Different peptide fibrils generated different band patterns of TDP-43 C-terminal fragments and protease-resistant fragments. Peptide fibrils of 274–313 and 314–353 were introduced into cells expressing WT-TDP-43 and ΔNLS-TDP-43, and aggregated TDP-43 contained in the Sar-insoluble fraction was detected by immunoblot analysis using anti-pS409/410 antibodies. A, 22–28-kDa band patterns were compared between 274–313 and 314–353 peptide fibril-induced cells. Schematic diagrams of band patterns are indicated below the blot. B, Sar-insoluble fractions were treated with 10 μg/ml trypsin for 30 min, and then the reaction was stopped by heating at 100 °C for 5 min. Immunoblot analysis using anti-pS409/410 detected remaining trypsin-resistant bands, and their pattern is indicated as a schematic diagram below the blot. C, comparison of TDP-43 in brains of patients with that in cells treated with peptide-fibril seeds. Sar-insoluble fractions prepared from brains of patients (one case of FTD and one case of ALS) and from cells treated with peptide fibrils of 274–313 and 314–353 were digested with trypsin (10 μg/ml at 37 °C for 30 min). Untreated and trypsin-treated samples were immunoblotted with pS409/410. D, extents of cell death in transfected and fibril-introduced cells were quantified by lactate dehydrogenase assay. Cell death assay was conducted 2 days after treatment with peptide fibrils. Data are means ± S.E. (n = 3).