ALS-causing cleavages of TDP-43 abolish its RRM2 structure and unlock CTD for enhanced aggregation and toxicity

Pathological TDP-43 is cleaved into various fragments. Two major groups of ∼35 and ∼25 kDa have enhanced aggregation and cytotoxicity but the underlying mechanisms remain elusive. While the ∼35-kDa fragments contain entire RRM1, RRM2 and C-terminal domain (CTD) with a middle hydrophobic segment flanked by two prion-like regions; the ∼25-kDa one cleaved at Arg208 only consists of the truncated RRM2 and CTD. Remarkably, the 25-kDa fragment was characterized to induce cell death by gain of cytotoxicity and recapitulate pathological features of TDP-43 proteinopathies. Here by NMR spectroscopy we successfully characterized residue-specific conformations and inter-domain interactions of several fragments and the results show that: 1) ALS-causing truncation at Arg208 completely eliminates the intrinsic ability of RRM2 to fold, and consequently the truncated RRM2 becomes highly disordered and prone to aggregation. 2) By disrupting inter-domain interactions upon deleting the N-terminal ubiquitin-like fold in TDP-43 (102-414), the extreme C-terminal prion-like region of CTD is released, while in TDP-43 (208-414), almost the whole CTD is unlocked. As CTD itself is prone to aggregation and highly toxic, our study suggests that at least two mechanisms, namely to abolish RRM2 structure and to release CTD, may account for enhanced aggregation and toxicity of pathologically cleaved TDP-43.