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, 4 (9), e1000193

Novel Mutations in TARDBP (TDP-43) in Patients With Familial Amyotrophic Lateral Sclerosis


Novel Mutations in TARDBP (TDP-43) in Patients With Familial Amyotrophic Lateral Sclerosis

Nicola J Rutherford et al. PLoS Genet.


The TAR DNA-binding protein 43 (TDP-43) has been identified as the major disease protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U), defining a novel class of neurodegenerative conditions: the TDP-43 proteinopathies. The first pathogenic mutations in the gene encoding TDP-43 (TARDBP) were recently reported in familial and sporadic ALS patients, supporting a direct role for TDP-43 in neurodegeneration. In this study, we report the identification and functional analyses of two novel and one known mutation in TARDBP that we identified as a result of extensive mutation analyses in a cohort of 296 patients with variable neurodegenerative diseases associated with TDP-43 histopathology. Three different heterozygous missense mutations in exon 6 of TARDBP (p.M337V, p.N345K, and p.I383V) were identified in the analysis of 92 familial ALS patients (3.3%), while no mutations were detected in 24 patients with sporadic ALS or 180 patients with other TDP-43-positive neurodegenerative diseases. The presence of p.M337V, p.N345K, and p.I383V was excluded in 825 controls and 652 additional sporadic ALS patients. All three mutations affect highly conserved amino acid residues in the C-terminal part of TDP-43 known to be involved in protein-protein interactions. Biochemical analysis of TDP-43 in ALS patient cell lines revealed a substantial increase in caspase cleaved fragments, including the approximately 25 kDa fragment, compared to control cell lines. Our findings support TARDBP mutations as a cause of ALS. Based on the specific C-terminal location of the mutations and the accumulation of a smaller C-terminal fragment, we speculate that TARDBP mutations may cause a toxic gain of function through novel protein interactions or intracellular accumulation of TDP-43 fragments leading to apoptosis.

Conflict of interest statement

The authors have declared that no competing interests exist.


Figure 1
Figure 1. Missense mutations identified in TARDBP in familial ALS patients.
(A) Pedigrees showing family history of ALS for three probands carrying TARDBP mutations. Black symbols represent patients affected with ALS; white symbols represent unaffected individuals. Pedigrees are constructed based on family history data provided by the NINDS Human Genetics Resource Center DNA and Cell Line Repository ( The alive/dead status of individuals is unknown. Arrowheads indicate the probands. The onset age of ALS symptoms and the TARDBP mutation identified are included below each proband. (B) DNA sequence traces observed in a sample from the proband of each family. The observed single base substitution and predicted amino acid change are indicated below each chromatogram. cDNA numbering is according to the largest TARDBP transcript (NM_007375.3) and starting at the translation initiation codon. Protein numbering is relative to the largest TDP-43 isoform (NP_031401.1).
Figure 2
Figure 2. Overview of mutations identified to date in TARDBP.
Schematic overview of the 7 TARDBP exons showing coding regions in dark blue and non-coding regions in light blue (top). The TDP-43 protein structure with location of the conserved domains is shown with protein numbering according to the largest isoforms NP_031401.1 (middle). Protein sequence alignment shows strong conservation in the C-terminal region of TDP-43 (bottom). Colored boxes indicate the position of known and novel TDP-43 mutations identified in sporadic (orange) and familial (red) ALS patients. TDP-43 mutations identified in this study are underlined. Orange and red lines in TARDBP gene and TDP-43 protein indicate approximate positions of the mutations. RRM = RNA recognition motif.
Figure 3
Figure 3. Biochemical analysis of TDP-43 in lymphoblastoid cell lines of TARDBP mutation carriers.
Western blot analyses of protein lysates derived from lymphoblastoid cell lines from 3 familial ALS patients carrying different TARDBP mutations (p.M337V, p.N345K and p.I383V), 2 ALS patients (1 and 2) without TARDBP mutations and 5 healthy control individuals (Control 1–5). In lymphoblastoid cell lines derived from TARDBP mutation carriers and sporadic ALS patients an accumulation of 2 smaller C-terminal fragments of TDP-43 protein of approximately 35 and 25 kDa was observed in detergent-insoluble fractions treated with the proteasome inhibitor, MG-132. In lymphoblastoid cell lines derived from control individuals the levels of the 35 kDa fragment were substantially lower, and the 25 kDa fragment was mostly undetectable. Membranes from the soluble fraction were reprobed for beta-actin to monitor protein loading.
Figure 4
Figure 4. Proteasome inhibition increases the proteolytic cleavage of TDP-43.
Western blot analyses of H4 neuroglioma cells treated with the proteasome inhibitor, PSI (10 µM, 24 hours) and a pan-caspase inhibitor, Z-VAD-FMK (100 µM, 24 hours) separately or in combination. Treatment with PSI revealed an increase in proteolytic cleavage of TDP-43 fragments (35 and 25 kDa) and an increase in caspase-3 activity. Treatment with a pan-caspase inhibitor suppressed PSI-induced TDP-43 cleavage and caspase-3 activity. HSP70 levels were increased after PSI treatment and the levels persisted in the presence of a pan-caspase inhibitor. Similar results were obtained in 3 independent experiments.

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