Figure 4. Overexpression of the ΔNLS mutant TDP-43 (TDP-43NLSm) induces exon repression when expressed at higher levels than wildtype TDP-43.

To mimic mislocalization of TDP-43 into the cytoplasm, we used mutant forms of TDP-43. We wanted to explore to what extent this studied protein can passively migrate into the nucleus and repress exons. We used QBI-293 cells that have a stable cassette with TDP-43 tagged with GFP (iGFP-WT) and a TDP-43 mutated form in its nuclear localization signal (iGFP-NLSm), both lines under the control of the Doxycycline (Dox) inducible promoter. Cultures were exposed to Dox for different times (0, 24, 48, 72 hours). (A) Exon skipping events were seen when wild type or NLS TDP-43 forms were induced. (B) Quantification from RT-PCR results in iGFP-NLSm Dox induction shows that exon repression only reached ~9% exon skipping compared to ~95% in iGFP-WT. (C) Protein quantification shows TDP-43 endogenous self-regulation and total levels of TDP-43 for both WT and NLSm reached approximately ~2 times the normal levels of TDP-43. (D and E) Approximate levels of total nuclear TDP-43 based on estimates of TDP-43^NLSm passive diffusion into the nucleus. (F) To evaluate to what extent TDP-43^NLSm was passively diffusing in the nucleus, we transfected HEK-293 cells with TDP-43^WT-P2A-GFP or TDP-43^NLSm-P2A-GFP under the control of a constitutive promoter and FACS isolated seven fractions according to GFP intensity. (G) We isolated nuclei from all seven fractions and detected TDP-43 by immunoblotting, TDP-43 endogenous is reduced while TDP-43^WT or TDP-43^NLSm are increasing. Raw intensity value in arbitrary units is plotted and the bar graph shows the ratio between overexpressed TDP-43^NLSm and TDP-43^WT, which suggest that TDP-43^NLSm passive diffusion rate to the nucleus increases as the expression of this protein also progressively increases. (H) Double band RT-PCR from whole cell fractions showed the progressive increase of exon skipping when either TDP-43^WT or TDP-43^NLSm are overexpressed. (I) TDP-43^WT can almost completely repress exons evaluated (~95%), however, TDP-43^NLSm only reached up to ~40% of exon repression. Using these data, we estimate the proportion of TDP-43^NLSm in the Dox-inducible system, dotted line (D). Levels of total TDP-43^WT and total predicted TDP-43^NLSm in the nucleus are plotted together with their respective exon expression levels (E).