Enhancedin vitroaggregation, but not phase separation, of TDP-43 and its C-terminal fragments generate deep-blue autofluorescence

As misfolding and aggregation of the RNA/DNA-binding protein, TDP-43, are linked to devastating TDP-43 proteinopathies like amyotrophic lateral sclerosis (ALS), distinction of the nature of the aggregated TDP-43 species being liquid-like non-pathogenic or solid-like pathogenic is important for mechanistic elucidation and therapeutic targeting. Here, we examined if in vitro enhancement of the TDP-43 aggregation can generate or enhance intrinsic deep-blue autofluorescence (dbAF) previously reported for a few other protein aggregates and whether dbAF is emitted by all or only liquid-like or solid-like TDP-43 aggregates. Using thioflavin-T fluorescence, turbidimetry, atomic force microscopy and fluorescence microscopy of Alexa Fluor-labelled protein, we first tested the in vitro enhancement of the aggregation of the full-length TDP-43 and its two C-terminal fragments (CTFs), TDP-43 2C (aa: 193-414) and the TDP-43-low complexity domain (LCD) (aa: 274-414). We find that presence of metal ions, Zn 2+ or Mn 2+ , that are also linked to ALS-associated metal dyshomeostasis, or addition of a kosmotropic anion, SO 4 2- , enhance the in vitro solid-like aggregations of the full-length TDP-43 and TDP-43 2C that also concurrently enhance emission of dbAF. In contrast, Alexa fluor-633-labeled-TDP-43-LCD underwent a quick phase separation into globular structures in presence of Zn 2+ ions and the phase-separated species failed to emit dbAF but upon further incubation when matured into solid-like irregular, but non-amyloid nature aggregates, it emitted dbAF. Strikingly, we find that the TDP-43 aggregates of both amyloid and non-amyloid nature, but not the oligomers or the phase-separated droplets of TDP-43, manifest dbAF. Overall, the observed in vitro enhancement of aggregation leading to concurrent enhancement of dbAF can enable a label-free easy detection and may facilitate distinguishing of potentially pathogenic versus non-pathogenic TDP-43 aggregates.