TDP-43 loss of cellular function through aggregation requires additional structural determinants beyond its C-terminal Q/N prion-like domain.
Bottom Line: To our knowledge, this is the only system that achieves full functional TDP 43 depletion with effects similar to RNAi depletion or gene deletion.As a result, this model will prove useful to investigate the loss-of-function effects mediated by TDP-43 aggregation within cells without affecting the expression of the endogenous gene.These data show for the first time that cellular TDP-43 aggregation can lead to total loss of function and to defective splicing of TDP-43-dependent splicing events in endogenous genes.
Affiliation: International Centre for Genetic Engineering and Biotechnology (ICGEB), 34012 Trieste, Italy.Show MeSH
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Mentions: In Figure 2, it is possible to observe that, in normal conditions, the endogenous TDP-43 is equally distributed in the soluble and insoluble fractions (Fig. 2, lines 1–2). Interestingly, in the presence of Flag-TDP-12xQ/N expression, the endogenous TDP-43 protein shifted almost completely into the insoluble fraction together with this mutant (Fig. 2, lines 3 and 4). Overexpression of TDP-43 alone does not cause aggregation/insolubilization per se in our stable transgenic TDP-43 in the HEK293 cells. This could have partly due to the self-regulation mechanisms that, in a situation of excess protein, shut-off the endogenous gene (6). In order to completely discard the possibility that the shift into the insoluble fraction was produced simply because of the overexpression of Flag-TDP-12xQ/N, we then performed the same experiment using a stable cell line overexpressing Flag-TDP-43 F4L. The advantage of using this particular RNA-binding mutant is that Flag-TDP-43 F4L is unable to downregulate the endogenous TDP-43 levels by the autoregulation mechanism (6). As shown in Figure 2, lanes 5 and 6, following TET induction both this mutant and the endogenous TDP-43 were distributed in the soluble (S) and insoluble (P) fractions in similar amounts as those observed with the endogenous protein alone (Fig. 2, lanes 1 and 2). There may only a slight increase of both proteins in the P fraction, but there is no sequestration of the endogenous TDP-43 in the P fraction as occurred in the presence of Flag-TDP-12xQ/N (Fig. 2, lines 5 and 6).Figure 2.
Affiliation: International Centre for Genetic Engineering and Biotechnology (ICGEB), 34012 Trieste, Italy.