The cleavage pattern of TDP-43 determines its rate of clearance and cytotoxicity.

TAR DNA-binding protein of 43 kDa (TDP-43) and its C-terminal fragment of 25 kDa (CTF25) play critical roles in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Although the overexpression of TDP-43 in cultured cells and animals results in the production of CTF25, the cleavage site that generates CTF25 and biological significance of the cleavage remain undetermined. Here we identify Asp174 as a cleavage site for CTF25. TDP-43 is cleaved initially after Asp174, which activates caspase-3/7 to accelerate TDP-43 fragmentation. Consequently, blockage of this cleavage results in a severe delay in TDP-43 clearance and prolonged necrotic cell death. We further show that the endoplasmic reticulum membrane-bound caspase-4 is the enzyme responsible for the cleavage after Asp174 and inhibition of caspase-4 activity slows TDP-43 fragmentation and reduces cell viability. These findings suggest that caspase-4-mediated cleavage after Asp174 is an initiator of TDP-43 clearance, which is required to avoid cell death induced by overexpressed TDP-43.