Aberrant cleavage of TDP-43 enhances aggregation and cellular toxicity

Abstract

Inclusions of TAR DNA-binding protein-43 (TDP-43), a nuclear protein that regulates transcription and RNA splicing, are the defining histopathological feature of frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-Us) and sporadic and familial forms of amyotrophic lateral sclerosis (ALS). In ALS and FTLD-U, aggregated, ubiquitinated, and N-terminally truncated TDP-43 can be isolated from brain tissue rich in neuronal and glial cytoplasmic inclusions. The loss of TDP-43 function resulting from inappropriate cleavage, translocation from the nucleus, or its sequestration into inclusions could play important roles in neurodegeneration. However, it is not known whether TDP-43 fragments directly mediate toxicity and, more specifically, whether their abnormal aggregation is a cause or consequence of pathogenesis. We report that the ectopic expression of a Ϸ25-kDa TDP-43 fragment corresponding to the C-terminal truncation product of caspase-cleaved TDP-43 leads to the formation of toxic, insoluble, and ubiquitin-and phospho-positive cytoplasmic inclusions within cells. The 25-kDa C-terminal fragment is more prone to phosphorylation at S409/S410 than full-length TDP-43, but phosphorylation at these sites is not required for inclusion formation or toxicity. Although this fragment shows no biological activity, its exogenous expression neither inhibits the function nor causes the sequestration of full-length nuclear TDP-43, suggesting that the 25-kDa fragment can induce cell death through a toxic gain-of-function. Finally, by generating a conformation-dependent antibody that detects C-terminal fragments, we show that this toxic cleavage product is specific for pathologic inclusions in human TDP-43 proteinopathies.caspase ͉ inclusion ͉ amyotrophic lateral sclerosis ͉ cell death ͉ frontotemporal lobar degeneration with ubiquitin-positive inclusions

Cytoplasmic translocation, aggregation, and cleavage of TDP-43 by enteroviral proteases modulate viral pathogenesis

“…The endogenous TDP-43 exon skipping activity in our study appeared to be already high (higher than that in their study 22 ). Thus, modest increase of exogenous TDP43 due to a shorter term of transfection (24 h in our study versus 48 h in their report 22 ) may not be sufficient to promote further increase in exon 9 exclusion. Our result is consistent with an early report with the same period of TDP-43 transfection (24 h) showing that exon skipping activity in cells expressing exogenous TDP43 is comparable to cells expressing empty-vector, but significantly higher compared with cells depleted of TDP-43.…”

“…[21][22][23] To eliminate the possibility that the above-detected fragment of TDP-43 is due to caspaseinduced cleavage, we pharmacologically inhibited caspase activity using Z-VAD-FMK. Figure 2c showed that treatment with Z-VAD-FMK did not block the generation of the~35 kDa cleavage products at 7 h post-infection.…”

“…In addition to reduced solubility, a number of studies have shown that mutated TDP-43 and caspasederived C-terminal TDP-43 fragments that are often detected in the brain of ALS and FTLD patients are highly prone to aggregation. 17,22,31,32 Furthermore, it was reported that wildtype TDP-43 and cytoplasmically restricted TDP-43 (mutated at its NLS) are also able to form protein aggregates in the cytoplasm, which are associated with increased protein phosphorylation and ubiquitination, and co-localized with SG markers. 29,40 The exact mechanism leading to decreased solubility and increased aggregation of TDP-43 following CVB3 infection is still not known, but could be related to cytoplasmic translocation of TDP-43, which allows for direct interaction of the two RNA-recognition motifs (RRM 1 and 2) on full-length TDP-43 and TDP-43-N, with cytosolic SGs containing various RNA transcripts and proteins that are involved in protein ubiquitination and phosphorylation.…”

“…22 The discrepancy is likely due to the differences in the basal levels of endogenous TDP43 activity and the amount of exogenous TDP43 transfected between two studies. The endogenous TDP-43 exon skipping activity in our study appeared to be already high (higher than that in their study 22 ).…”

“…22 The discrepancy is likely due to the differences in the basal levels of endogenous TDP43 activity and the amount of exogenous TDP43 transfected between two studies. The endogenous TDP-43 exon skipping activity in our study appeared to be already high (higher than that in their study 22 ). Thus, modest increase of exogenous TDP43 due to a shorter term of transfection (24 h in our study versus 48 h in their report 22 ) may not be sufficient to promote further increase in exon 9 exclusion.…”