Loss of ALS-associated TDP-43 in zebrafish causes muscle degeneration, vascular dysfunction, and reduced motor neuron axon outgrowth

Abstract

Mutations in the Tar DNA binding protein of 43 kDa (TDP-43; TARDBP) are associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43(+) inclusions (FTLD-TDP). To determine the physiological function of TDP-43, we knocked out zebrafish Tardbp and its paralogue Tardbp (TAR DNA binding protein-like), which lacks the glycine-rich domain where ALS- and FTLD-TDP-associated mutations cluster. tardbp mutants show no phenotype, a result of compensation by a unique splice variant of tardbpl that additionally contains a C-terminal elongation highly homologous to the glycine-rich domain of tardbp. Double-homozygous mutants of tardbp and tardbpl show muscle degeneration, strongly reduced blood circulation, mispatterning of vessels, impaired spinal motor neuron axon outgrowth, and early death. In double mutants the muscle-specific actin binding protein Filamin Ca is up-regulated. Strikingly, Filamin C is similarly increased in the frontal cortex of FTLD-TDP patients, suggesting aberrant expression in smooth muscle cells and TDP-43 loss-of-function as one underlying disease mechanism.

Fig. 1.

Overview of human TDP-43 and zebrafish orthologs and Western blot of their loss-of-function alleles. (A) Schematic representation of human TDP-43 (turquois), zebrafish Tardbp (red), and zebrafish Tardbpl protein (orange); blue bars represent nuclear localization sequence; green bars represent nuclear export sequence; RRM, RNA recognition motif. (B, Top) Tardbp specific monoclonal antibody 4A12-111 detects the 43-kDa Tardbp protein in adult brain from wild-type and tardbpl^{mde222−/−} but not from tardbp^{mde198−/−} and tardbp^{mde897−/−} fish. Asterisk marks an unspecific band. (Middle) Tardbpl-specific monoclonal antibody 5F5-11 detects the approximately 34-kDa Tardbpl protein in adult brain from wild-type, tardbp^{mde198−/−} and tardbp^{mde897−/−} but not from tardbpl^{mde222−/−} fish. (Bottom) α-Tubulin serves as a loading control.

Fig. 2.

Loss of Tardbp does not affect spinal motor neuron axon outgrowth because of alternative splicing of Tardbpl. (A) Lateral view of wild-type, maternal zygotic tardbp^−/−, and maternal zygotic tardbpl^−/− embryos at 30 h postfertilization (hpf) showing the five spinal motor neuron axonal projections (labeled 1–5) anterior to the end of the yolk extension stained with znp-1 used for quantitation. Anterior to the left. (Scale bar, 25 μm.) (B) Quantitation of the length of spinal motor neuron axons measured from the exit point of the spinal cord to the growth cone in wild-type (green), maternal zygotic tardbp^−/− (red), and maternal zygotic tardbpl^−/− embryos (orange). Error bars indicate ±SD, n ≥ 12 embryos per experiment. n.s., not significant, Student t test. (C) Western blot analysis comparing two independent transient Tardbp knockdown experiments with respective control injected wild-type siblings. (Upper) The Western blot with the Tardbp-specific antibody anti–TDP-43 (Novus) reveals a significant reduction of Tardbp (arrow), indicating successful knockdown in lanes 2 and 4. (Lower) Probing with a pool of N-terminal Tardbpl-specific monoclonal antibodies (anti-Tardbpl 8G1) reveals a robust up-regulation of Tardbpl_tv1 upon Tardbp knockdown (lanes 2 and 4). (D) Schematic representation of genomic exon-intron organization of tardbpl (light green boxes represent 5′ and 3′ UTR; orange boxes represents coding exons) and tardbpl_tv1 (only coding exons are shown in dark orange). Enlargement of the exon 5 splice donor site of tardbpl and the corresponding sequence in tardbpl_tv1. (E) Western blot analysis with the Tardbpl_tv1 specific monoclonal antibody Tardbpl_tv1 16C8-11 detects up-regulated Tardbpl_tv1 expression at 48 hpf and in adult brain upon loss of Tardbp compared with wild-type. The anti-Tardbpl_tv1 antibody is specific because no protein is detected in tardbpl^−/−. α-Tubulin serves as a loading control. Genotypes are indicated above the respective lanes. (F) Western blot analysis with an anti-human TDP-43 antibody (Sigma) that detects zebrafish Tardbp and Tardbpl_tv1. Genotypes are indicated above the respective lanes. Note that Tardbpl_tv1 is prominently detected in tardbp^−/− and runs at a slightly lower molecular weight compared with Tardbp (compare lane 3 with lane 5, labeled with an asterisk). Specificity of the antibody is demonstrated in double homozygous embryos (lane 6). α-Tubulin serves as a loading control.

Fig. 3.

tardbp^−/−;tardbpl^−/− mutants have impaired blood circulation that can be rescued. (A) tardbp^−/−;tardbpl^−/− mutants accumulate erythrocytes on the yolk (arrow) because of a lack of circulation at 2 dpf. Anterior to the left. See also Movie S1. (B) The circulation phenotype can be restored by injection of mRNA encoding human TDP-43 and zebrafish Tardbpl_tv1, but not by the shorter isoform Tardbpl. Injection of mRNA of the ALS-associated TDP-43^G348C also rescues the circulation phenotype, whereas mRNA of the ALS-associated gene FUS fails to rescue. The black bar represents uninjected double homozygous mutant siblings of each respective clutch. The expected 25% of double-homozygous mutant embryos from incrosses of tardbp^−/−;tardbpl^+/− or tardbp^+/−;tardbpl^−/− fish were set to 100%. Error bars indicate ±SD, n ≥ 182 embryos per experiment. *, P < 0.05; ***, P < 0.005, student t test.

Fig. 4.

Mispatterned vasculature of tardbp^−/−;tardbpl^−/− mutants. (A) Whole mount in vivo imaging of wild-type; Tg(kdrl:HsHRAS-mCherry)^s896 and (B) tardbp^−/−;tardbpl^−/−; Tg(kdrl:HsHRAS-mCherry)^s896 embryos at 2 dpf. Tg(kdrl:HsHRAS-mCherry)^s896 highlights the vasculature. The head is shown on the left and the ISV of the trunk on the right. (Scale bars, 20 μm.) Lateral view, anterior to the left.

Fig. 5.

Spinal motor neuron axon outgrowth phenotype of tardbp^−/−;tardbpl^−/− mutants. (A) Znp-1 antibody staining of motor neuron axons in 28-hpf-old embryos of the five somites (labeled 1–5) anterior to the end of the yolk extension. Spinal motor neuron axons of tardbp^−/−;tardbpl^−/− mutants are reduced in length compared with wild-type siblings. Anterior to the left, lateral view. (Scale bar, 25 μm.) (B) Quantitation of the length of the outgrowing spinal motor neuron axons measured from the exit point of the spinal cord to the growth cone in wild-type (green) and tardbp^−/−;tardbpl^−/− mutants (purple). Error bars indicate ±SD, n ≥ 13 embryos per experiment, ***P < 0.0015, Student t test.

Fig. 6.

Severely degenerated myocytes of tardbp^−/−;tardbpl^−/− mutants. (A) Antibody staining of 2-dpf-old wild-type and tardbp^−/−;tardbpl^−/− mutant embryos with the myosin specific antibody ZE-BO-1F4 (green) and DAPI (blue). White arrowheads indicate degenerated myocytes. (B) Antibody staining of 1.5-dpf-old wild-type and tardbp^−/−;tardbpl^−/− mutant embryos with α-actinin (green), vinculin (red), and DAPI (blue). (A and B) Anterior to the left, lateral view. (Scale bars, 20 μm.) (C) EM pictures of skeletal muscle of a tardbp^−/−;tardbpl^−/− mutant embryo shows a highly disorganized pattern of thinner myofibrils (f) with disorganized network of sarcoplasmic reticulum (open arrowheads). Large part of the sarcoplasmic reticulum is misaligned and dilated (filled arrowheads). Individual myofibrils are not well separated from another and mitochondria can now be found within the muscle fibers at (black arrow). (2 dpf) (Scale bars, 500 nm.)

Fig. 7.

Filamin C mRNA is up-regulated in FTLD-TDP patients but not in Alzheimer’s disease (AD) and healthy aged-matched control patients. Relative mRNA levels of Filamin C long and short splice variants in human FTLD-TDP and Alzheimer’s disease patients and healthy aged-matched controls, determined by quantitative RT-PCR and normalized to GAPDH and YWHAZ. *P < 0.05, **P < 0.01, Kruskal–Wallis test with Dunn’s multiple comparison test.