TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disorder characterized pathologically by ubiquitinated TAR DNA binding protein (TDP-43) inclusions. The function of TDP-43 in the nervous system is uncertain, and a mechanistic role in neurodegeneration remains speculative. We identified neighboring mutations in a highly conserved region of TARDBP in sporadic and familial ALS cases. TARDBPM337V segregated with disease within one kindred and a genome-wide scan confirmed that linkage was restricted to chromosome 1p36, which contains the TARDBP locus. Mutant forms of TDP-43 fragmented in vitro more readily than wild type and, in vivo, caused neural apoptosis and developmental delay in the chick embryo. Our evidence suggests a pathophysiological link between TDP-43 and ALS.

Fig. 1

Identification of three missense mutations in exon 6 of TARDBP. (A) DNA sequence demonstrating a single base substitution (asterisk) changing the wild-type adenine at 1009 to guanine, substituting valine for methionine (M337V). (B) Two additional mutations were found in sporadic ALS cases; 991 C>A substituting lysine for glutamine (Q33 1K), and 881 G>C substituting alanine for glycine (G294A). (C) A sequence alignment of amino acids 287-347 of TDP43 from diverse vertebrate species is shown. Identical amino acids have a black background, similar amino acids are grey and mutation sites are red.

Fig. 2

Mutation in TARDBP cosegregates with disease in kindred ALS85 (A) Linkage analysis of Affymetrix 10K SNP array data demonstrated linkage to chromosome 1p36. (B) Linkage to the region containing TARDBP was confirmed using microsatellite markers. (C) The ALS85 pedigree is shown. Affected individuals are indicated by black symbols. The proband is indicated by the arrow. Unaffected individuals have open symbols. II. 1 (vertical bar) was an obligate carrier with an anecdotal history of ALS. Slashed symbols indicate deceased subjects. Gender has been omitted for confidentiality. Microsatellite markers are shown in chromosomal order with their genetic and physical locations. Genotypes for each individual are given with inferred genotypes in parentheses. The haplotype segregating with disease is indicated by the black bar. The 1009 A>G TARDBP mutation is indicated by a red “G”.

Fig. 3

Immunoblots of cytoplasmic fractions from CHO cells transiently transfected with wild-type and mutant TDP-43. (A) Probing for the N-terminal myc tag demonstrating numerous fragments which were absent in controls (NT, non-transfected, and CAT, chloramphenicol acetyl transferase transfected). The dominant bands were at 40, 18 and 14 kD. (B) Quantitative analysis of myc-immunoreactive bands. Densitometry of bands was conducted using Odyssey. Results are presented as mean and standard error of the mean of eight experiments.

Fig. 4

Mutant TDP-43 causes chick embryonic developmental delay. Chick embryo spinal cords were transfected with plasmids encoding wild-type TDP-43 (A, D, G), mutant Q331K (B, E, H) or M337V (C, F, I). Embryo images and sections shown were taken 24 hours post electroporation. Embryos expressing TDP43_WT developed normal limb (black arrows) and tail buds (A) while those expressing mutant TDP-43 did not (B, C). Magnified images of upper limb buds are shown in insets. (J) The percentage of embryos that matured normally (reached HH stage 15 or 17 after 24 hours and 48 hours respectively) is shown. Data were generated from analysis of 49 embryos in each treatment group. (D-F) Transverse sections of chick spinal cord double-stained with anti-HA (green) and anti-myc (red) antibodies to the tags on transfected TDP-43 constructs. Unilateral TDP-43 construct expression in spinal cord neural cells occurs following embryo electroporation. The majority of the cells in the transfected region have the characteristic phenotype of neuroepithelial cells. (G-I) TUNEL staining of sections shown in D-F demonstrated apoptotic nuclei (blue arrows). (K) Quantification of apoptosis. TUNEL positive cells in five sections per embryo (two embryos per transfection) were counted.