Enhanced in vitro aggregation, but not phase separation, of TDP-43 and its C-terminal fragments generates intrinsic deep-blue autofluorescence

Abstract

As misfolding, phase-separation and aggregation of the RNA/DNA-binding protein TDP-43 are implicated in proteinopathies like ALS, condensate-type distinction is important for mechanistic elucidation and therapeutic targeting. Here, we examined if in vitro enhancement of TDP-43 aggregation can generate intrinsic deep-blue autofluorescence (dbAF) previously reported for some protein aggregates, and also upon protein damage by UV, and whether dbAF is dependent on specific condensate types of TDP-43. Using amyloid-specific thioflavin-T fluorescence, turbidimetry, AFM and fluorescence microscopy, we first investigated any in vitro enhancement of the aggregation of full-length TDP-43 and its two C-terminal fragments, TDP-43^2C (aa: 193-414) and TDP-43-low complexity domain (LCD) (aa: 274-414). Strikingly, Zn²⁺ ions, linked to ALS-associated metal dyshomeostasis, and the kosmotropic SO₄²⁻ anions enhanced in vitro solid-like aggregation of TDP-43 and TDP-43^2C, which also concurrently caused dbAF. Notably, Alexa Fluor-633-labeled TDP-43-LCD in the presence of Zn²⁺ ions formed phase-separated globular species, which failed to emit dbAF but eventually emitted dbAF upon maturation into solid-like irregular aggregates that were although not amyloid. Strikingly, TDP-43 expressed in yeast cells grown with Zn²⁺ also emitted dbAF. As non-aggregated TDP-43^2C, upon in vitro UV exposure, also emitted dbAF, possibly due to damage to tyrosine amino acid side chains, the emission of dbAF from the TDP-43 aggregates of both amyloid and non-amyloid types, but not from the phase-separated droplets, suggests that the closer proximity of amino acid reactive side chains in the solid-like aggregates may cause dbAF. Thus, dbAF may enable condensate-type distinction and a label-free detection of the enhancement of TDP-43 aggregation.