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. 2013 May 30;20(1):33.
doi: 10.1186/1423-0127-20-33.

Similar dose-dependence of motor neuron cell death caused by wild type human TDP-43 and mutants with ALS-associated amino acid substitutions

Lien-Szu Wu  1 , Wei-Cheng Cheng, Che-Kun James Shen
Affiliations

Similar dose-dependence of motor neuron cell death caused by wild type human TDP-43 and mutants with ALS-associated amino acid substitutions

Lien-Szu Wu et al. J Biomed Sci. .

Abstract

Background: TDP-43, a multi-functional DNA/ RNA-binding protein encoded by the TARDBP gene, has emerged as a major patho-signature factor of the ubiquitinated intracellular inclusions (UBIs) in the diseased cells of a range of neurodegenerative diseases. Mutations in at least 9 different genes including TARDBP have been identified in ALS with TDP-43 (+)-UBIs. Thus far, the pathogenic role(s) of the more than 30 ALS-associated mutations in the TARDBP gene has not been well defined.

Results: By transient DNA transfection studies, we show that exogenously expressed human TDP-43 (hTDP-43), either wild type (WT) or 2 different ALS mutant (MT) forms, could cause significantly higher apoptotic death rate of a mouse spinal motor neuron-like cell line (NSC34) than other types of cells, e.g. mouse neuronal Neuro2a and human fibroblast HEK293T cells. Furthermore, at the same plasmid DNA dose(s) used for transfection, the percentages of NSC34 cell death caused by the 2 exogenously expressed hTDP-43 mutants are all higher than that caused by the WT hTDP-43. Significantly, the above observations are correlated with higher steady-state levels of the mutant hTDP-43 proteins as well as their stabilities than the WT.

Conclusions: Based on these data and previous transgenic TDP-43 studies in animals or cell cultures, we suggest that one major common consequence of the different ALS-associated TDP-43 mutations is the stabilization of the hTDP-43 polypeptide. The resulting elevation of the steady state level of hTDP-43 in combination with the relatively low tolerance of the spinal motor neurons to the increased amount of hTDP-43 lead to the neurodegeneration and pathogenesis of ALS, and of diseases with TDP-43 proteinopathies in general.

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Figures

Figure 1
Figure 1
Immunofluorescence staining analysis of NSC34 and Neuro2a cells overexpressing WT and MT hTDP-43. (A) Physical maps showing the domains of human TDP-43 (hTDP-43) and mouse TDP-43 (mTDP-43). NLS, nuclear localization signal; NES, nuclear export signal; GR, glycine-rich sequence; GQN, glycine (G), glutamine (Q) and/ or asparagine (N)-rich sequence. For hTDP-43, the cDNA inserts in the 3 expression plasmids encoding the WT and the two mutant forms of hTDP-43 are shown. The positions of the nucleotide substitutions are indicated by the stars. (B) Representative photographs showing the immunofluorescence staining patterns of transfected cells. NSC34 (upper two rows) and Neuro2a (lower two rows) cells were transfected with plasmids expressing the WT hTDP-43 and hTDP-43A315T, respectively, and analyzed by immunofluorescence staining at 48 hr post-transfection with use of the 2E2D3 antibody recognizing hTDP-43 and DAPI staining the nucleus. Note the predominant nuclear localization of the exogenous WT and MT hTDP-43 in both types of cells. Scale bar, 10 μm.
Figure 2
Figure 2
Apoptotic assays of transfected NSC34 and Neuro2a cells. (A) Comparison of apoptotic death induced by exogenous WT and MT hTDP-43. Cell death was assayed by measurement of the activities of caspase 3/ 7 at 24 hr and 72 hr post-transfection with the expression plasmids (5 ug/ 106 cells). Mock, cells without transfection; C, cells transfected with the pEF vector; Stau. 5 uM, cells treated with 5 uM of staurosporine for 6 hr to induce apoptosis. The folds of the caspase activities relative to that of the Mock sample were calculated and shown. Note the significant increase of the caspase 3/ 7 activities in NSC34 cells (left panel), but not Neuro2a cells (right panel), induced by the two MT hTDP-43 forms at 72 hr post-transfection. (B) Comparison of plasmid dose-dependent apoptotic death induced by exogenous WT and MT hTDP-43. Apoptotic cell death was determined by immunofluorescence staining of cleaved caspase 3. The extents of apoptotic cell death of NSC34 cells and Neuro2a cells at 72 hr post-transfection with different amounts of the expression plasmids were assayed by immunofluorescence staining with the antibodies 2E2D3 and Ac-cap 3. Means of three independent experiments (S.D.) are plotted in the upper 2 panels, with the % of hTDP-43-positive cells that are also Ac-cap 3-positive as a function of the doses of transfection. Approximately 1% of cells transfected with the pEF vector were Ac-cap3 positive (◆ in the two plots). Representative photographs are shown below the plots, with the apoptotic nuclei/ cleaved caspase 3-positive cells indicated by the arrowheads. For both the NSC34 and Neuro2a sets, two Ac-cap 3-positive cells (the boxed areas) are magnified for better visualization. Scale bar, 10 μm. *, p<0.05; **, p<0.01; ***, p<0.001.
Figure 3
Figure 3
hTDP-43 protein dose-dependent apoptotic death of NSC34 cells. (A) Higher levels of MT hTDP-43 than WT hTDP-43 at the same dose(s) of DNA transfection. NSC34 and Neuro2a cells were transfected with different doses (μg/ 106 cells) of the expression plasmids. At 72 hr post-transfection, the levels of the exogenous hTDP-43 expressed were compared by Western blotting using the anti-hTDP-43 antibody 2E2D3, with the mouse Hsp70 level as the internal control. The means of the relative levels of hTDP-43 obtained from three independent experiments (S.D.) are then plotted in the 2 panels, with the level of the WT hTDP-43 in cells with the transfection dose of 20 μg DNA/ 106 cells as 100. Note the higher levels of the MT hTDP-43 than WT hTDP-43 in both NSC34 and Neuro2a cells at all doses of transfection. *, p<0.05; **, p<0.01; ***, p<0.001. (B) The % of transfected cells that were cleaved caspase 3-positive as a function of the relative amounts of hTDP-43. The plots were derived from the combined data of Figures 2 and 3A. The amounts of hTDP-43 in NSC34 cells or Neuro2a cells transfected with 20 μg of pEF-hTDP-43N390D are assigned the value of 1.
Figure 4
Figure 4
Increased stabilities of hTDP-43 polypeptides with ALS-associated mutations. The stabilities of the exogenous hTDP-43 in transfected NSC34 (A), Neuro2a (B), and SHSY5Y (C) cells were measured by cycloheximide chase experiments as described in Methods. The Western blots of the exogenous WT hTDP-43 and MT hTDP-43A315T in transfected cells are shown as the examples of analysis. The relative amounts of hTDP-43 at different hr of cycloheximide treatment are plotted in the upper two panels. The mean values of 3 different sets of the chase experiments are used for the plots. p<0. in NSC34, Neuro2a, and SHSY5Y cells.
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References

    1. Wang HY, Wang IF, Bose J, Shen CK. Structural diversity and functional implications of the eukaryotic TDP gene family. Genomics. 2004;83:130–139. doi: 10.1016/S0888-7543(03)00214-3. - DOI - PubMed
    1. Ayala YM, Pantano S, D'Ambrogio A, Buratti E, Brindisi A, Marchetti C, Romano M, Baralle FE. Human, Drosophila, and C.elegans TDP43: nucleic acid binding properties and splicing regulatory function. J Mol Biol. 2005;348:575–588. doi: 10.1016/j.jmb.2005.02.038. - DOI - PubMed
    1. Ou SH, Wu F, Harrich D, Garcia-Martinez LF, Gaynor RB. Cloning and characterization of a novel cellular protein, TDP-43, that binds to human immunodeficiency virus type 1 TAR DNA sequence motifs. J Virol. 1995;69:3584–3596. - PMC - PubMed
    1. Wang IF, Wu LS, Chang HY, Shen CK. TDP-43, the signature protein of FTLD-U, is a neuronal activity-responsive factor. J Neurochem. 2008;105:797–806. doi: 10.1111/j.1471-4159.2007.05190.x. - DOI - PubMed
    1. Wang IF, Reddy NM, Shen CK. Higher order arrangement of the eukaryotic nuclear bodies. Proc Natl Acad Sci U S A. 2002;99:13583–13588. doi: 10.1073/pnas.212483099. - DOI - PMC - PubMed

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