A partial deletion of the Tardbp 3'UTR affects TDP-43 regulation and leads to motor dysfunction in mice

Abstract

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that causes the selective loss of motor neurons. A histopathological hallmark of ALS is the cytoplasmic aggregation of TDP-43, a ubiquitously expressed RNA-binding protein involved in transcription and splicing regulation. To prevent abnormal accumulation, TDP-43 controls its expression levels through an autoregulatory feedback loop. While most ALS studies have focused on pathogenic variants that impair the protein function of TDP-43, the mechanisms underlying endogenous TDP-43 dysregulation mediated by non-coding elements, including the 3' untranslated region (3'UTR), remain incompletely understood. In this study, we generated a mouse model carrying a targeted deletion of the Tardbp 3'UTR that encompasses the TDP-binding region, polyadenylation signals, and alternative intronic sequences. Our findings demonstrate that the Tardbp 3'UTR is essential for normal mouse development. Loss of this region led to decreased Tardbp mRNA expression and embryonic lethality after gastrulation. Young heterozygous mice were phenotypically normal with no overt disruption in TDP-43 autoregulation. However, aged heterozygous mice displayed mild locomotor dysfunction accompanied by a modest increase in spinal cord TDP-43 protein levels and a reduction in motor neuron numbers. These findings indicate that regulatory elements within the Tardbp 3'UTR play a pivotal role in normal development and contribute to TDP-43 pathology relevant to ALS.

Keywords: 3’UTR regulation; TDP-43 autoregulation; amyotrophic lateral sclerosis; motor dysfunction; mouse model.

Fig. 1.

Generation and phenotypic characterization of the ∆UTR mouse. (A) Schematic diagram of the Tardbp gene locus. The Tardbp 3’UTR, including TDPBR, pA1, and pA2 sites, was partially deleted using the CRISPR/Cas9 system to generate the Tardbp^∆UTR allele. (B) Gross morphology of the 6-month-old Tardbp^∆UTR/+ and WT littermates. (C) Body weights of Tardbp^∆UTR/+ mice were comparable to WT. Sample sizes: 6 months (n=14 each), 12 months (n=13 each), and 18 months (n=11 each). Bar graphs represent mean ± SD. Statistical significance was assessed by Student’s t-test. (D) At E9.5, three homozygous embryos showed abnormal morphology compared to a WT littermate (left). (E, F) At E7.5, WT embryos reached the late-streak and cylinder stage, whereas homozygous embryos exhibited growth retardation and remained at the early-streak stage. (G, H) At E6.5, WT embryos initiated primitive streak formation, while homozygous embryos showed delayed gastrulation. Scale bars, 500 µm (D), 100 µm (E–H).

Fig. 2.

Tardbp expression in embryonic and central nervous tissues. Tardbp mRNA expression was examined in E9.5 embryos using two primer pairs: one detecting both WT and ∆UTR alleles (total expression) (A, C, E) and the other specific to WT allele expression (B, D, F). (A, B) Total Tardbp and WT allele-specific expression in WT, Tardbp^∆UTR/+, and Tardbp^{∆UTR/∆UTR} embryos at E9.5. Total and WT allele expression in the brain (C, D) and spinal cord (E, F) of Tardbp^∆UTR/+ and WT at both 6 and 18 months. (G) Western blot analysis of TDP-43 protein using TDP-43 and α-tubulin antibodies in the brain and spinal cord. The upper band corresponds to full-length TDP-43 (arrow). (H) Quantification of TDP-43 protein levels in the brain and spinal cord. Bar graphs represent mean ± SD and statistical significance was assessed using Student’s t-test (*P<0.05, **P<0.01, and ***P<0.001).

Fig. 3.

The behavioral assessment of Tardbp^∆UTR mice. (A) Wire maneuver test at 6, 12, and 18 months of age. WT and heterozygous mice were scored from 0 (grip securely) to 4 (fall immediately), and the percentage of mice at each score is shown. (B) Grip strength measurements of the forelimbs (left) and all limbs (right), and (C) Rotarod test were conducted at the same time points. Sample sizes: 6 months (n=14 each), 12 months (n=13 each), and 18 months (n=11 each). In (B) and (C), bar graphs represent mean ± SD and individual values are indicated as open circles (WT) and open triangles (Δ/+). Statistical significance was assessed by Student’s t-test (*P<0.05).

Fig. 4.

Loss of motor neurons in the Tardbp^∆UTR spinal cord. (A) ChAT (+) motor neurons in the spinal cord were observed at 10× (left) and 40× magnification (right). At higher magnification, nuclei were stained with DAPI. (B) Quantification of ChAT (+) motor neurons in the ventral horns of lumbar spinal cord of WT and Tardbp^∆UTR/+ mice (n=3 each). (C) KB staining was used to visualize myelin and nerve cells in the lumbar spinal cord of WT and Tardbp^∆UTR/+ mice. (D) Quantification of α motor neurons in (C) (n=3 each). Bar graphs represent mean ± SD. Statistical significance was assessed by Student’s t-test (*P<0.05). Scale bars, 200 µm (A, left), 50 µm (A, right), 100 µm (C).