Cell biology of TDP-43 expression. A, immunoblot analysis of TDP-43 expression from RIPA extracts of undifferentiated (NT2-) and retinoic acid-treated differentiated neuronal (NT2N) human teratocarcinoma cells; undifferentiated (PC12) and neural growth factor-treated differentiated neural (dPC12) rat pheochromocytoma cells; mouse neuroblastoma (Neuro2a) cells; human embryonic kidney (QBI-293) cells; and Sarkosyl-extracted lysate from human brain (top) and from primary mouse neurons isolated from cortex (Ctx) and hippocampus (Hipp) (bottom). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control (bottom). No glyceraldehyde-3-phosphate dehydrogenase was detected in the Sarkosyl-soluble human brain lysate due to prior extraction with less stringent buffers. B, immunofluorescence detection of endogenous TDP-43 in QBI-293 cells (red), DAPI (blue), and merged images (top) and in primary mouse hippocampal neurons (bottom). In addition, MAP2 (green in the merged image) was used to mark neuronal perikarya and dendrites. Scale bar, 20 μm. C, detection of cytoplasmic (permeabilized with 0.02% Triton X-100; top) or total (permeabilized with 0.2% Triton X-100; bottom) TDP-43 (green), β-tubulin (red), and merged images in QBI-293 cells.

TDP-43 expression and accumulation in cytoplasm of temperature-sensitive BN2 cells. A and B, endogenous TDP-43 (red) was detected in the nucleus of tsBN2 cells at 33 °C (permissive temperature). The merge image (B) of TDP-43 (red) and nuclei stain DAPI (blue) confirms that TDP-43 is in the cell nucleus. C and D, at nonpermissive temperature (39.5 °C), TDP-43 (red) was detected as punctate cytoplasmic aggregates (arrowheads) with clearing of nuclear TDP-43 (^*) and did not colocalize with DAPI (blue) in tsBN2 cells. D, merged image. Scale bars, 20 μm.

Expression of TDP-43-ΔNLS mutants led to sequestration of endogenous nuclear TDP-43. A, TDP-43 immunohistochemistry in a hippocampal section from a FTLD-U brain. Note clearing of nuclear TDP-43 (arrowheads) in neurons containing cytoplasmic TDP-43 inclusions, as compared with normal neurons (asterics). B, schematic diagram of N-terminal Myc-tagged TDP-43 protein highlighting the location of a bipartite NLS and NES. Both sets of basic aa (red) in NLS were mutated to generate three defective mutants (ΔNLS1, ΔNLS2, and ΔNLS1/2), and both sets of hydrophobic aa (blue) in NES were mutated to generate ΔNES1 and ΔNES2. C-K, double labeling of TDP-43 (red), Myc (green), and counterstaining with DAPI (blue) for nuclei. C-E, QBI-293 cells 72 h after transfection with Myc-TDP-43-WT (WT) and merged image (E) show colocalization of TDP-43 and Myc in nuclei of transfected cells. F-H, QBI-293 cells 24 h after transfection of Myc-TDP-43-ΔNLS1 (ΔNLS1). The merged image (H) shows colocalization of TDP-43 and Myc in the cytoplasm of transfected cells. I-K, QBI-293 cells 72 h after transfection of ΔNLS1. The merged image (K) shows colocalization of TDP-43 and Myc in the cytoplasm of transfected cells. Note the clearing of endogenous nuclear TDP-43 in cells expressing NLS1, as compared with nontransfected cells. Scale bars, 25 μm. ^*, nontransfected cells.

Cytoplasmic expression of human and mouse TDP-43-ΔNLS results in nuclear clearance of TDP-43. A-C, QBI-293 cells 72 h post-transfection with GFP-TDP-43-WT and Myc-TDP-43-WT. Both GFP and TDP-43 (red) were only detected in the nucleus of transfected cells. D-L, QBI-293 cells 24 h (D-F) or 72 h (G-I) post-transfection with GFP-TDP-43-WT and ΔNLS1. GFP-TDP-43-WT was localized solely to the nucleus (green), and the merged image (F) shows no colocalization of Myc-TDP-43-WT (red) and GFP (green) in cytoplasm 24 h post-transfection. Nuclei were labeled with DAPI (blue). G-L, 72 h post-transfection, GFP-TDP-43-WT was detected in cytoplasm and colocalized with ΔNLS1 (I-L). Note punctate colocalization (yellow) of ΔNLS1 and GFP-TDP-43-WT in the cytoplasm of transfected cells. M-O, QBI-293 cells 72 h after transfection with mouse FLAG-TDP-43-mWT (mWT) and double-labeled with a human-specific mouse anti-TDP-43 (green) and rabbit anti-FLAG antibody (red) with merged image (O) showing expression of endogenous human TDP-43 and FLAG-TDP-43-mWT in nuclei of transfected cells. Note the decreased levels of endogenous human nuclear TDP-43 in cells expressing FLAG-TDP-43-mWT when compared with nontransfected cells (^*). P-R, QBI-293 cells 72 h after transfection with mouse FLAG-TDP-43-ΔNLS1/2 (ΔmNLS1/2). The merged image (R) shows the presence of endogenous human TDP-43 (green) and FLAG-TDP-43-ΔNLS1/2 (red) in the cytoplasm of transfected cells. Note that there is clearing of endogenous human nuclear TDP-43 in cells expressing FLAG-TDP-43-ΔNLS1/2 (arrowhead) as compared with nontransfected cells (^*). Scale bars, 20 μm.

Expression of TDP-43-ΔNLS mutants led to aggregate formation in neuronal perikarya and neurites. Mouse hippocampal neurons were transfected with WT (A-C) or ΔNLS1 (D-F) TDP-43 constructs. Note the colocalization of TDP-43 and Myc in the nucleus (A-C) and soma (D-F) of neurons. G-I, higher magnification of a neuron expressing ΔNLS1. The merged image (J) shows colocalization of TDP-43 and Myc in the cytoplasm of a transfected neuron. Note the clearing of endogenous nuclear TDP-43 in I. J, high magnification image of axon in F marked by a dotted box. Neuritic accumulations of TDP-43 present in neurons expressing ΔNLS1 (arrows) are similar to neuritic pathology observed in FTLD-U brains (K). K, immunostaining of neuritic pathology (arrowheads) from frontal cortex of a FTLD-U brain. Scale bars, 20 μm.

Expression of Myc-TDP-43-ΔNLS mutants results in the sequestration of ubiquitinated and insoluble endogenous TDP-43. QBI-293 cells 24 h (A), or 72 h (B) post-transfection with empty vector (CTRL), Myc-TDP-43-WT (WT), Myc-TDP-43-ΔNLS1 (ΔNLS1), Myc-TDP-43-ΔNLS2 (ΔNLS2), or Myc-TDP-43-ΔNLS1/2 (ΔNLS1/2) sequentially extracted with RIPA (R) and urea buffer (U). Immunoblotting was conducted with TDP-43 antibody. Myc-TDP-43 (Myc) migrates slower than endogenous TDP-43 (Endo). Over-exposure of the immunoblot demonstrates the presence of a high M_r smear (^**) and C-terminal fragments (^*) in the urea fractions of Myc-TDP-43-NLS mutants in transfected cells. α-Tubulin was used as a loading control. C, immunoblots (IB) of immunoprecipitated (IP) QBI-293 cell lysates cotransfected with empty vector, Myc-TDP-43-WT, or Myc-TDP-43-ΔNLS1/2 and HA-tagged ubiquitin (HA-Ub) in the presence (+LAC) or absence of LAC. Note the presence of the LAC-dependent, ubiquitinated TDP-43 positive high-M_r smear and the TDP-43 C-terminal fragments.

TDP-43 is sequestered as insoluble nuclear inclusions by restricting nuclear export. A-D, immunofluorescence of endogenous TDP-43 (red) alone (A and C) or merged (B and D) with DAPI (blue) in QBI-293 cells treated with DMSO vehicle (CTRL) (A and B) or LMB (+LMB) (C and D). The arrowheads identify punctuate nuclear inclusions in C. E, QBI-293 cells were treated with DMSO vehicle or LMB and were sequentially extracted with RIPA (R) and urea buffer (U). Immunoblotting was conducted with anti-TDP-43 antibody. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. F-K, double labeling with TDP43 (red), Myc (green) antibodies, and DAPI (blue). QBI-293 cells 24 h (F-H) and 72 h (I-K) after transfection with Myc-TDP-43-ΔNES2 (ΔNES2) and merge images (H and K) show colocalization of TDP-43 (red) and Myc (green) in nuclei of transfected cells. Note the presence of punctate inclusions in the nucleus of transfected cells at both 24 and 72 h. Nontransfected cells are marked withanasterisk.LandM, QBI-293 cells 24 h (L) or 72 h (M)post-transfection with empty vector(CTRL), Myc-TDP-43-WT(WT), or Myc-TDP-43-ΔNES2 (ΔNES2) and sequentially extracted with RIPA (R) and urea buffer (U). Immunoblotting was conducted with TDP-43 antibody. Myc-TDP-43 (Myc) migrates slower than endogenous TDP-43 (Endo), and both Myc-TDP-43-ΔNES2 and endogenous TDP-43 were recovered in the RIPA-insoluble and urea-soluble fraction. Glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. Scale bars, 20μm.