TDP-43 PFFs preparation and in vitro TDP-43 seeding assay

TDP-43 PFFs preparation from purified TDP-43 monomer was performed as reported previously [14, 22]. Briefly, human derived TDP-43 protein (ab156345, 1 mg/ml, purity: > 90%) purchased from Abcam Company was resuspended in double-distilled water at a concentration of 0.5 mg/ml, and then the samples were incubated at 25 °C with agitation at 1400 rpm for 2 h. Sequentially, the TDP-43 PFFs were sonicated by an ultrasonic cell disruptor at 10% of its peak amplitude (Scientz-IID, Ningbo, China) for 50 s and then frozen and stored in a − 80 °C freezer. To observe TDP-43 fibrils, ThT staining was performed as followed: 25 µM ThT solution was mixed with TDP-43 PFFs solution (0.5 mg/ml) by pipetting and incubated for 5 min at room temperature. Fluorescence (440 nm excitation, 480–1000 nm emission) was measured in a plate reader (ARVO MX, PerkinElmer Life Sciences). After ThT staining, we used a TEM to observe the nature of TDP-43 PFFs. 5 µl of the sample was applied on a hydrophilic, 200 mesh, carbon-coated copper grids (Yasheng Electronics Technology Co, Ltd., Zhengzhou, China) and then stained with 2% uranyl acetate. After washing with distilled water, the grids were allowed to air-dry. Images were obtained by transmission electron microscopy (JEOL USA, Inc., Peabody, MA, USA). Then, we mixed prepared TDP-43 PFFs with TDP-43 monomer solution, and treated the mixture with ThT fluorescence assay. The fluorescence was detected at intervals of 30 min for 7 days with 440 and 480 nm of excitation and emission wavelength. During the reactions, no plate shaking was performed. Additionally, the fibril nature of the TDP-43 PFFs was further confirmed by analysis of PK (2.0 µg/ml, Solarbio, China) resistance using a dot-blot test.