In vitro structural determination and analysis of full‐length aggregated TDP‐43 protein

Abstract

TDP-43 protein has been causally linked to amyotrophic lateral sclerosis (ALS), most often in the form of amyloid-like aggregates and characterized by changes in structural conformations that induce pathogenesis. Our research has shown that TDP-43 adopts β-sheet structures throughout in vitro aggregation, as it yields ThT-positive results. Further, these aggregating conditions have identified amyloid-like fibrils and other aggregates through morphological analyses using transmission electron microscopy (TEM) and the presence of insoluble particulates via turbidity. This study characterized the misfolding of TDP-43 in vitro, mimicking biologically-relevant aggregated TDP-43. Data indicate that wild-type, full-length TDP-43 aggregates under agitated, temperature-controlled environmental conditions and results in a fibrillar-forming, ThT-positive yield. After incubation at 37 °C, TDP-43 had aggregated significantly, showing fibril formation synonymous to similar literature on ALS-type TDP-43 proteins. Further research should focus the influence of post-translational modifications (PTMs) on TDP-43 aggregation. This study demonstrates the feasibility of studying TDP-43 aggregates in vitro and provides a valuable model for future aggregation and pathogenicity studies for TDP-43 and other, similar proteins.