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 (http://ccr.coriell.org/ninds). 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).
Table 1.
Patients included in the TARDBP sequencing analyses.
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.
Table 2.
Allele sharing in p.M337V families.
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.
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.