TDP-43 dimerizes in human cells in culture

Abstract

TAR DNA-binding protein-43 (TDP-43) is a 43-kDa nuclear protein involved in regulation of gene expression. Abnormally, phosphorylated, ubiquitinated, and aggregated TDP-43 constitute a principal component of neuronal and glial cytoplasmic and nuclear inclusions in the brains of frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS), although the molecular mechanism that triggers aggregate formation remains unknown. By Western blot analysis using anti-TDP-43 antibodies, we identified a band with an apparent molecular mass of 86-kDa in HEK293, HeLa, and SK-N-SH cells in culture. It was labeled with both N-terminal-specific and C-terminal-specific TDP-43 antibodies, enriched in the cytosolic fraction, and the expression levels were reduced by TDP-43 siRNA but unaltered by treatment with MG-132 or by expression of ubiqulin-1 or casein kinase-1. By immunoprecipitation analysis, we found the interaction between the endogenous full-length TDP-43 and the exogenous Flag-tagged TDP-43, and identified the N-terminal half of TDP-43 spanning amino acid residues 3-183 as an intermolecular interaction domain. When the tagged 86-kDa tandemly connected dimer of TDP-43 was overexpressed in HEK293, it was sequestered in the cytoplasm and promoted an accumulation of high-molecular-mass TDP-43-immunoreactive proteins. Furthermore, the 86-kDa band was identified in the immunoblot of human brain tissues, including those of ALS. These results suggest that the 86-kDa band represents dimerized TDP-43 expressed constitutively in normal cells under physiological conditions.

Fig. 1

The structural domains of the human TDP-43 protein. The domain structure of the human TDP-43 protein is shown on the top with the position of amino acid residues. The genes encoding the full-length (FL) TDP-43 protein and a panel of truncated forms, such as the C-terminal domain-cleaved protein (delC), the C-terminal domain fragment (C), the N-terminal domain fragment (N), RRM1, RRM2, the N-terminal domain plus RRM1 (NRRM1), and the C-terminal domain plus RRM2 (RRM2C), were cloned in the expression vectors as listed in Table 1

Fig. 2

The constitutive expression of an 86-kDa TDP-43-immunoreactive protein in human cell lines in culture. The detergent-soluble protein extract of HEK293, HeLa, and SK-N-SH cells exposed for 24 h to the vehicle (dimethylsulfoxide) (lane 1) or 1-μM MG-132 (lane 2) was processed for Western blot with anti-TDP43 antibody (upper panels) or with anti-ubiquitin antibody (lower panels). A small but discernible amount of the 86-kDa TDP-43-immunoreactive band is expressed in all the cells examined

Fig. 3

The characterization of the 86-kDa protein. A The immunolabeling of the protein with N- or C-terminal-specific anti-TDP-43 antibodies. Both 86- and 43-kDa proteins were extracted from the corresponding SDS-PAGE gel bands, concentrated, and processed for Western blot with the N-terminal-specific anti-TDP-43 antibody (panel a), the C-terminal-specific anti-TDP-43 antibody (panel b), or the secondary antibody alone (panel c). The lanes (1 and 2) represent the 86- and the 43-kDa protein, respectively. B The effects of transient expression of TDP-43 siRNA. SK-N-SH cells were transfected with the siRNA expression vector targeted to TDP-43 or a scrambled sequence listed in Table 1. 96 h after transfection, the protein extract was processed for Western blot with anti-TDP-43 antibody (panels a and b) or anti-HSP60 antibody, an internal control for protein loading (panel c). The lanes (1–3) represent non-transfected cells, the cells transfected with the vector of a scrambled sequence, and the cells transfected with the vector of TDP-43 siRNA, respectively. C The effects of expression of ubiqulin-1 (UBQLN1) and casein kinase-1 alpha-1 (CSNK1A1). HEK293 cells were untransfected (lanes 1 and 4) or transfected with the expression vectors of Xpress-tagged LacZ (lane 2), Xpress-tagged UBQLN1 (lane 3), V5-tagged LacZ (lane 5), or V5-tagged CSNK1A1 (lane 6). The detergent-soluble protein extract was processed for Western blot with anti-TDP-43 antibody (upper panels), anti-Xpress antibody (the left lower panel), or anti-V5 antibody (the right lower panel)

Fig. 4

Immunoprecipitation analysis of Flag-tagged TDP-43-binding proteins. A Coimmunoprecipitation with Flag-tagged proteins. Flag-tagged FL TDP-43 (lanes 1 and 3) or GFP (lanes 2 and 4) was expressed in HEK293 (panel a), HeLa (panel b), and SK-N-SH (panel c) cells. The detergent-soluble protein extract was processed for immunoprecipitation (IP) with anti-Flag M2 affinity gel, followed by Western blot (WB) with anti-TDP-43 antibody (upper panels) or anti-Flag M2 antibody (lower panels). The lanes (1–4) represent (1 and 2) the immunoprecipitates and (3 and 4) the corresponding input controls. B Flag-tagged TDP-43 constitutes multimeric forms. Flag-tagged FL TDP-43 was expressed in HEK293 cells, and then processed for IP with anti-Flag M2 affinity gel, followed by WB with anti-TDP-43 antibody or anti-Flag M2 antibody. Immunoprecipitates were overloaded on a 10% SDS–PAGE gel. The lanes (1, 2) represent anti-TDP-43 antibody and anti-Flag M2 antibody, respectively. Multimeric forms are indicated by stars. C Reciprocal coimmunoprecipitation analysis. Flag-tagged FL TDP-43 and Myc-tagged FL TDP-43 were coexpressed in HEK293. IP followed by WB was performed using the antibody against Myc (upper panel) or Flag (lower panel), reciprocally. The lanes (1–3) represent input control, IP with anti-Flag M2 or anti-Myc antibody, and IP with normal mouse or rabbit IgG, respectively

Fig. 5

Fractionation analysis of cellular proteins of HEK293. The cellular proteins of HEK293 separated into cytosol (lane 1), membrane (lane 2), nuclear (lane 3), and cytoskeletal (lane 4) fractions were processed for Western blot with anti-TDP-43 antibody (panel a), anti-HSC70 antibody (panel b), anti-pan-cadherin antibody (panel c), anti-vimentin antibody (panel d), and anti-histone H1 antibody (panel e). The lane (5) represents the unfractionated input control

Fig. 6

The domains involved in the intermolecular interaction of TDP-43 proteins. Flag-tagged FL TDP-43 (lanes 1 and 2) and a panel of truncated proteins (see the details in Fig. 1), including delC (lanes 3 and 4), N (lanes 5 and 6), RRM1 (lanes 7 and 8), NRRM1 (lanes 9 and 10), RRM2 (lanes 11 and 12), C (lanes 13 and 14), and RRM2C (lanes 15 and 16) were individually expressed in HEK293 cells. The protein extract was processed for IP with anti-Flag affinity gel, followed by Western blot with anti-TDP-43 antibody (lanes 1, 3, 5, 7, 9, 11, 13, and 15) or anti-Flag M2 antibody (lanes 2, 4, 6, 8, 10, 12, 14, and 16). The position (43-kDa) of the immunoprecipitated endogenous FL TDP-43 is indicated by arrow heads

Fig. 7

TDP-43 dimer serves as a seed for promoting aggregation of TDP-43 proteins. A The expression of TDP-43 tandem dimer promotes an accumulation of high-molecular-mass TDP-43-immunoreactive proteins. The vectors of Halo-tagged GFP (lane 3), TDP-43 FL monomer (lane 4), or FL tandem dimer (lane 5) were transfected or untransfected (lanes 1 and 2) in HEK293 cells with (lane 2) or without (lanes 1, 3–5) a 24-h treatment of 1-μM MG-132. The protein extract was processed for Western blot with anti-TDP43 antibody (panel a), anti-Halo tag antibody (panel b), anti-PARP antibody (panel c), anti-ubiquitin antibody (panel d), or anti-HSP60 antibody, an internal control for protein loading (panel e). B Cell imaging analysis. HEK293 cells expressing Halo-tagged TDP-43 monomer (panels a and b) or tandem dimer (panels c and d) were processed for labeling with Oregon Green (panels a and c) merged with nuclear labeling with DAPI (panels b and d). C The counting of the number of the cells with nuclear accumulation of Halo-tagged proteins. The average of cell counts of six random fields under the magnification of ×400 is shown with standard deviations

Fig. 8

The constitutive expression of TDP-43 dimer in human brain tissues. The detergent-soluble protein extract of human brain tissues of the cerebrum (CBR) (lanes 1, 3, 4, 6, 8, 11–14) and the cerebellum (CBL) (lanes 2, 5, 7, 9, 10, 15), derived from PD#1 (lanes 1 and 2), ALS#1 (lane 3), ALS#2 (lanes 4 and 5), ALS#3 (lanes 6 and 7), ALS#4 (lanes 8 and 9), MS#1 (lane 10), MS#2 (lane 11), MS#3 (lane 12), SCH#1 (lane 13), and DEP#1 (lanes 14 and 15) was processed for Western blot with anti-TDP43 antibody (upper panels) or anti-HSP60 antibody, an internal control for protein loading (lower panels)