Aggregation of TDP-43: A Dual Pathway to Forming Cylindrins and Fibrils and a Contribution to the Etiology of Amyotrophic Lateral Sclerosis.

The pathological aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) is a hallmark of amyotrophic lateral sclerosis, and mutations within its low-complexity domain are known to influence its aggregation propensity and toxicity. Previous studies from our group and others have shown that TDP-43 located at the C-terminus of TDP-43 is toxic and can form higher-order oligomers and fibrils. Of particular interest are the hexamers, which adopt a cylindrin structure that has been strongly correlated to neurotoxicity. In this study, we used a combination of ion mobility spectroscopy-mass spectrometry (IMS-MS), atomic force microscopy (AFM), and molecular dynamics simulations to probe the oligomer distribution resulting from the earliest times (the first 5 to 15 min) of incubation at varying concentrations for three different TDP-43 mutations: wild-type (WT), A315T, and G314V. In this way, it was possible to trace the oligomer distributions at the initial stages of aggregation while avoiding the complication from aggregation-induced sedimentation over long periods. We found that both WT and A315T rapidly form stable hexamers and higher-order oligomers at low concentrations. As the concentration is increased, the IMS-MS oligomer distribution changes to favor small oligomers over the hexamers and higher-order oligomers for both WT and A315T. AFM shows that this shift in oligomer distribution is due to the formation of fibrils that are seeded by trimers and tetramers. This complex concentration dependence is attributed to two different kinetic paths: one at low concentration that favors the formation of hexamers/cylindrins and one at high concentration that favors fibril formation. Furthermore, the G314V mutation is nontoxic and does not show evidence of the two kinetic paths as hexamers are not formed at any concentration whereas fibril formation is observed at all concentrations.