Zn2+ modulates in vitro phase separation of TDP-432C and mutant TDP-432C-A315T C-terminal fragments of TDP-43 protein implicated in ALS and FTLD-TDP diseases

Abstract

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 Zn2+ levels. Recently, Zn2+ 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 Zn2+ is lacking, we in silico predicted potential Zn2+ binding sites in TDP-43 and estimated their relative solvent accessibilities. Zn2+ 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 Zn2+ promotes the in vitro thioflavin-T-positive aggregations of C-terminal fragments (CTFs) termed TDP-432C and TDP-432C-A315T that encompass the RRM2 and CTD domains. Also, while the Alexa-fluor fluorescently labelled TDP-432C and TDP-432C-A315T proteins manifested liquid-like spherical droplets, Zn2+ caused a solid-like phase separation that was not ameliorated even by carboxymethylation of the free cysteines thereby implicating the other Zn2+-binding residues. The observed Zn2+-promoted TDP-43 CTF's solid-like phase separation can be relevant to the Zn2+ dyshomeostasis in ALS and FTLD-TDP.