Environmental and Life Sciences, Trent University, Peterborough, Ontario K9L 0G2, Canada.
Environmental and Life Sciences, Trent University, Peterborough, Ontario K9L 0G2, Canada; Department of Forensic Science, Trent University, Peterborough, Ontario K9L 0G2, Canada.
Biochim Biophys Acta Mol Basis Dis. 2021 Dec 1;1867(12):166234. doi: 10.1016/j.bbadis.2021.166234. Epub 2021 Jul 31.
TAR DNA-binding protein-43 (TDP-43) pathology, including fibrillar aggregates and mutations, develops in amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD) and limbic-predominant age-related TDP-43 encephalopathy (LATE). Hyperphosphorylation and aggregation of TDP-43 contribute to pathology and are viable therapeutic targets for ALS. In vivo inhibition of TDP-43 aggregation was evaluated using anti-TDP-43 antibodies with promising outcomes. However, the exact mechanism of antibody-based inhibition targeting TDP-43 is not well understood but may lead to the identification of viable immunotherapies. Herein, the mechanism of in vitro aggregation of phosphorylated TDP-43 was explored, and the anti-TDP-43 antibodies tested for their inhibitor efficacies. Specifically, the aggregation of phosphorylated full-length TDP-43 protein (pS410) was monitored by transmission electron microscopy (TEM), turbidity absorbance, and thioflavin (ThT) spectroscopy. The protein aggregates were insoluble, ThT-positive and characterized with heterogeneous morphologies (fibers, amorphous structures). Antibodies specific to epitopes 178-393 and 256-269, within the RRM2-CTD domain, reduced the formation of β-sheets and insoluble aggregates, at low antibody loading (antibody: protein ratio = 1 μg/mL: 45 μg/mL). Inhibition outcomes were highly dependent on the type and loading of antibodies, indicating dual functionality of such inhibitors, as aggregation inhibitors or aggregation promoters. Anti-SOD1 and anti-tau antibodies were not effective inhibitors against TDP-43 aggregation, indicating selective inhibition.
TDP-43（TAR DNA 结合蛋白-43）病理学，包括纤维状聚集物和突变，在肌萎缩侧索硬化症（ALS）、额颞叶变性（FTLD）和边缘为主的与年龄相关的 TDP-43 脑病（LATE）中发展。TDP-43 的过度磷酸化和聚集导致病理学，并成为 ALS 的可行治疗靶点。使用具有前景的抗 TDP-43 抗体评估了 TDP-43 聚集的体内抑制作用。然而，基于抗体的针对 TDP-43 的抑制的确切机制尚不清楚，但可能导致可行的免疫疗法的鉴定。本文探索了磷酸化 TDP-43 的体外聚集的机制，并测试了抗 TDP-43 抗体的抑制剂功效。具体而言，通过透射电子显微镜（TEM）、浊度吸光度和硫黄素（ThT）光谱监测磷酸化全长 TDP-43 蛋白（pS410）的聚集。蛋白质聚集体不溶，ThT 阳性，具有异质形态（纤维、无定形结构）。针对 RRM2-CTD 结构域内表位 178-393 和 256-269 的抗体特异性减少了β-折叠和不溶性聚集体的形成，在低抗体负载（抗体：蛋白比=1μg/mL：45μg/mL）下。抑制结果高度依赖于抗体的类型和负载，表明此类抑制剂具有双重功能，既是聚集抑制剂，又是聚集促进剂。抗 SOD1 和抗 tau 抗体对 TDP-43 聚集没有有效的抑制作用，表明具有选择性抑制作用。
