A90V TDP-43 variant results in the aberrant localization of TDP-43 in vitro

Abstract

TAR DNA-binding protein-43 (TDP-43) is a highly conserved, ubiquitously expressed nuclear protein that was recently identified as the disease protein in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). Pathogenic TDP-43 gene (TARDBP) mutations have been identified in familial ALS kindreds, and here we report a TARDBP variant (A90V) in a FTLD/ALS patient with a family history of dementia. Significantly, A90V is located between the bipartite nuclear localization signal sequence of TDP-43 and the in vitro expression of TDP-43-A90V led to its sequestration with endogenous TDP-43 as insoluble cytoplasmic aggregates. Thus, A90V may be a genetic risk factor for FTLD/ALS because it predisposes nuclear TDP-43 to redistribute to the cytoplasm and form pathological aggregates.

Figure 1. The A90V TDP-43 variant partially disrupts nuclear localization

(a–i), Two-color immunofluoresence of QBI-293 cells transfected with myc-TDP-43-WT (WT), myc-TDP-43- ΔNLS1 (ΔNLS1), or myc-TDP-43-A90V (A90V). 72-hrs following transfection, cells were stained with (a, d, g) anti-TDP-43 (red) and (b, e, h) anti-myc (green) antibodies or merged (c, f, i). Nuclei were labeled with DAPI stain (blue). Scale bar; 20 μm. (j), Quantification of the cellular localization, nuclear (nuc; black bars) vs. cytoplasmic (cyto; white bars), of TDP-43 in QBI-293 cells transfected with myc-TDP-43-WT (WT), myc-TDP-43-NLS1 (ΔNLS1), or myc-TDP-43-A90V (A90V). Asterisks (***) represents significant differences compared to myc-TDP-43-WT transfected cells (p ≤ 0.05). Error bars represent S.E.M.

Figure 2. Endogenous TDP-43 is sequestered in the insoluble fraction with the expression of myc-TDP-43-A90V

Immunoblots of RIPA (R) and urea (U) extracted fractions (a) 24-hrs or (b) 72-hrs post-transfection with empty vector (CTRL), myc-TDP-43-WT (WT), myc-TDP-43-ΔNLS1 (ΔNLS1), or myc-TDP-43-A90V (A90V) with anti-TDP-43 antibody. Alpha-tubulin was used as a loading control.