Zn modulates in vitro phase separation of TDP-43 and mutant TDP-43-A315T C-terminal fragments of TDP-43 protein implicated in ALS and FTLD-TDP diseases.

TDP-43 proteinopathy is implicated in the neurodegenerative diseases, ALS and FTLD-TDP. Metal ion dyshomeostasis is observed in neurodegenerative diseases including ALS. Previously, mice expressing A315T familial ALS TDP-43 mutant showed elevated spinal cord Zn levels. Recently, Zn was observed to modulate the in vitro amyloid-like aggregation of the TDP-43's RRM12 domains. As a systematic knowledge of the TDP-43's interaction with Zn is lacking, we in silico predicted potential Zn binding sites in TDP-43 and estimated their relative solvent accessibilities. Zn binding sites were predicted in the TDP-43's N-terminal domain, in the linker region between RRM1 and RRM2 domain, within RRM2 domain and at the junction of the RRM2 and C-terminal domain (CTD), but none in the 311-360 region of CTD. Furthermore, we found that Zn promotes the in vitro thioflavin-T-positive aggregations of C-terminal fragments (CTFs) termed TDP-43 and TDP-43-A315T that encompass the RRM2 and CTD domains. Also, while the Alexa-fluor fluorescently labelled TDP-43 and TDP-43-A315T proteins manifested liquid-like spherical droplets, Zn caused a solid-like phase separation that was not ameliorated even by carboxymethylation of the free cysteines thereby implicating the other Zn-binding residues. The observed Zn-promoted TDP-43 CTF's solid-like phase separation can be relevant to the Zn dyshomeostasis in ALS and FTLD-TDP.