Skip to main content

ALS-linked TDP-43M337V knock-in mice exhibit splicing deregulation without neurodegeneration


Abnormal accumulation of TAR DNA-binding protein 43 (TDP-43), a DNA/RNA binding protein, is a pathological signature of amyotrophic lateral sclerosis (ALS). Missense mutations in the TARDBP gene are also found in inherited and sporadic ALS, indicating that dysfunction in TDP-43 is causative for ALS. To model TDP-43-linked ALS in rodents, we generated TDP-43 knock-in mice with inherited ALS patient-derived TDP-43M337V mutation. Homozygous TDP-43M337V mice developed normally without exhibiting detectable motor dysfunction and neurodegeneration. However, splicing of mRNAs regulated by TDP-43 was deregulated in the spinal cords of TDP-43M337V mice. Together with the recently reported TDP-43 knock-in mice with ALS-linked mutations, our finding indicates that ALS patient-derived mutations in the TARDBP gene at a carboxyl-terminal domain of TDP-43 may cause a gain of splicing function by TDP-43, however, were insufficient to induce robust neurodegeneration in mice.

Main text

Abnormal accumulation of TDP-43 has been identified as a pathological signature of amyotrophic lateral sclerosis (ALS), an adult neurodegenerative disease characterized by a selective loss of motor neurons, and a part of frontotemporal dementia (FTD) [1]. Cytoplasmic accumulation of TDP-43 with a loss of TDP-43 from nuclei, known as a TDP-43 pathology, is observed in almost all forms of ALS, including sporadic and familial ALS. To date, more than 50 mutations in the TARDBP gene, encoding TDP-43, have been identified in inherited and sporadic ALS, implicating TDP-43 dysfunction as a central component for ALS pathogenesis [2]. TDP-43 is a ubiquitously expressed DNA/RNA binding nuclear protein and plays multifunctional roles in RNA metabolism, including pre-mRNA splicing, translational control, and mRNA stability [3]. Of note, TDP-43 is known to control its own mRNA stability through binding to the 3′ UTR, indicating that the level of TDP-43 protein is tightly regulated [3]. Indeed, overexpression of wild-type TDP-43 in mice induces neurodegeneration, whereas elimination of TDP-43 leads to embryonic lethality [4, 5]. However, it is still unclear whether dysfunction in TDP-43 leads to neurodegeneration through a gain or loss of TDP-43 function. To model TDP-43-mediated neurodegeneration in mice, several lines of transgenic mice have been developed and reproduced some features of neurodegeneration observed in human ALS/FTD. However, the overexpression approach has a limitation in differentiating the role between wild-type and mutant TDP-43 in motor neuron health and disease in mice [4, 5].

Based on these backgrounds, we set out to create a knock-in mouse model carrying an ALS patient-derived mutation in the murine Tardbp gene. Of more than 50 known mutations, we chose TDP-43M337V mutation for the following reasons: TDP-43M337V protein has a long half-life in cells, the ALS patients with this mutation show earlier disease onset [6, 7], and an amino acid sequence of 241–414 including a methionine residue at position 337 is highly conserved among mouse and human. We engineered mice with n.1009 A > G (M337V) mutation in the murine Tardbp gene by utilizing CRISPR/Cas9 genome-editing technology (Additional file 1). Both homozygous and heterozygous mice carrying the allele of TDP-43M337V developed normally as recently reported (Fig. 1a, Additional file 1: Figure S1, S2) [8].

Fig. 1
figure 1

Characterization of TDP-43M337V knock-in mice. a Schematic illustration of introducing TDP-43M337V mutation into an endogenous murine Tardbp exon 6 (left panel). The representative genotyping result is also shown (right panel). Nhe I restriction site is introduced in the mutant allele, resulting in no change of the amino acid at Nhe I site. b The expression level of Tardbp mRNA was not altered in the brains of 700-days-old TDP-43M337V/M337V (M337 V/M337 V) mice and wild-type (WT) littermates. c Alternation in splicing of genes regulated by TDP-43. The level of mRNA containing exons included by TDP-43 (Kcnip2 exon 2 and 3) was increased, while the levels of mRNA containing exons excluded by TDP-43 (Sort1 exon 17b and Sema3f exon 5) were reduced, suggesting a gain-of-function mechanism in TDP-43M337V/M337V mice. Relative expression levels of mRNA normalized to the WT control are plotted with SD (n = 3 each (b, c)) and were analyzed by unpaired t-tests. d Representative immunoblots of TDP-43 and β-actin in the brains and spinal cords of 700-days-old TDP-43M337V/M337V mice and WT littermates. Asterisk denotes a non-specific band. e and f Representative immunofluorescence images of the anterior horn in lumbar spinal cords of 700-days-old TDP-43M337V/M337V mice and WT littermates stained with anti-TDP-43 (3H8, green) and anti-ChAT (red) antibodies along with the merged images. TDP-43 was not mislocalized in motor neurons of TDP-43M337V/M337V mice (e). Low magnification images stained with anti-ChAT antibody (f). Scale bars: 20 μm (e), 100 μm (f). g Quantification of the numbers of ChAT-positive motor neurons per each anterior horn (AH) in the lumbar spinal cords of 700-days-old TDP-43M337V/M337V mice and WT littermates. For quantification, 20 AHs in three animals per each genotype were counted, and data are plotted as mean ± SD, and were analyzed by an unpaired t-test. h and i TDP-43M337V/M337V mice did not show any motor dysfunction phenotypes in the measurement of clasping score (h) and rotarod test (i). Data are plotted as mean ± SD, and were analyzed with two-way ANOVA. n = 15 for WT and 14 for M337 V/M337 V

TDP-43 plays a pivotal role in regulating alternative splicing as well as controlling the level of TDP-43 mRNA itself by a negative feedback mechanism. Therefore, we first examined whether ALS-linked TDP-43M337V mutation affects the expression level of its own mRNA in mice. Analysis of TDP-43 mRNA levels in the brains of 700-days-old homozygous TDP-43M337V mice (TDP-43M337V/M337V) revealed that there was no difference in expression level between wild-type and TDP-43M337V/M337V mice (Fig. 1b). In addition, the mRNA levels of Notch1 and Nek1, known as TDP-43 target genes, were unaffected by homozygous M337 V mutation (Additional file 1: Figure S3). We next examined whether TDP-43M337V deregulates alternative splicing of mRNAs that are known as splicing targets of TDP-43. Among the several splicing targets examined, we found a 1.49-fold increase in inclusion of Kcinp2 exon 2/3, a 0.85-fold decrease in exclusion of Sort1 exon 17b, and a 0.63-fold decrease in exclusion of Sema3f exon 5 in the brain of TDP-43M337V/M337V mice (Fig. 1c). Although there were no significant changes in other splicing targets, Poldip3 and Eif4h (Additional file 1: Figure S4), changes in splicing of Kcinp2, Sort1, and Sema3f in TDP-43M337V/M337V mice are consistent with a gain of TDP-43 function [9, 10].

Since the mislocalization of TDP-43 protein in cytoplasm is a pathological signature of ALS, we examined subcellular localization of TDP-43M337V mutant protein in the affected tissue in TDP-43M337V/M337V mice. Both mutant and wild-type TDP-43 proteins expressed at the similar level, and were predominantly localized in nucleus of brain and spinal cords of 700-days-old TDP-43M337V/M337V and wild-type mice (Fig. 1d, e), suggesting that disease-causing missense mutation in TDP-43 alone did not alter the protein level itself and was insufficient to induce protein mislocalization in mice. Moreover, carboxyl-terminal (C-terminal) fragments of TDP-43, characteristic of TDP-43 pathology, were not detected in the brains and spinal cords of TDP-43M337V/M337V mice (Fig. 1d), and there was no detectable loss of motor neurons or reactive gliosis in TDP-43M337V/M337V mice (Fig. 1e-g, Additional file 1: Figure S5). Nuclear Gems, where SMN complex resides to control splicing, are known to be regulated by TDP-43 and FUS [11,12,13]. In ventral horn neurons of TDP-43M337V/M337V mice, the number of nuclear Gems was not altered (Additional file 1: Figure S6). We further examined whether TDP-43M337V/M337V mice show motor dysfunction with aging. Measurement of rotarod and clasping scores as well as body weights revealed no difference in those scores between TDP-43M337V/M337V and wild-type mice until 18 months old (Fig. 1h, i, Additional file 1: Figure S2).

The present study demonstrates that homozygous TDP-43M337V mice generated by CRISPR/Cas9 show splicing deregulation of some TDP-43 target mRNAs without apparent deterioration in motor function and pathology until 20 months old. Recently, homozygous TDP-43Q331K knock-in mice showed a reduced number of parvalbumin-positive interneurons and cognitive dysfunction with phenotypic heterogeneity [9]. Homozygous TDP-43G298S or TDP-43M337V knock-in mice showed very mild denervation of hindlimbs at 2.5 years of age [8]. Besides, heterozygous TDP-43M323K mice, generated by N-ethyl-N-nitrosourea (ENU) random mutagenesis, showed modest neurodegenerative phenotype [10]. These mutant mice uniformly show very mild phenotypes, likely because the 2-years-life span of rodents may be insufficient to induce neurodegeneration derived from splicing deregulation caused by mutant TDP-43.

Our TDP-43M337V/M337V mice showed splicing deregulation of TDP-43 target mRNAs, Kcinp2, Sort1, and Sema3f, suggesting that M337 V mutation causes a gain of function in TDP-43. Gain of TDP-43 function is also suggested in TDP-43Q331K and TDP-43M323K mice [9, 10]. All three missense mutations discussed here are located in the low complexity region at the C-terminal of TDP-43, suggesting that ALS-causing TDP-43 mutations in the C-terminal region may cause gain of TDP-43 function, at least, at an initial disease stage. This point makes a good contrast with the role of N-terminal TDP-43 fragment in dominant-negative function in TDP-43 [14]. In our study, the mRNA and protein levels of TDP-43 were unchanged in TDP-43M337V mice, while they were moderately upregulated in TDP-43Q331K [9]. This difference may explain the more modest phenotype of our TDP-43M337V mice.

All knock-in mice carrying ALS-linked missense mutations in TDP-43 do not show robust TDP-43 pathology even in homozygous mutant mice. Perhaps, additional conformational change of TDP-43 protein may be needed to develop TDP-43 pathology. Finally, our results from TDP-43M337V knock-in mice further strengthen the findings that mutations at the C-terminal region of TDP-43 likely cause a gain of TDP-43 splicing function at an initial stage of the disease, which may be followed by the loss of TDP-43 function due to a loss of TDP-43 proteins from nuclei.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.



Amyotrophic Lateral Sclerosis


Choline acetyltransferase


Frontotemporal dementia


Survival of motor neurons


TAR DNA-binding protein 43


  1. Lee EB, Lee VM, Trojanowski JQ. Gains or losses: molecular mechanisms of TDP43-mediated neurodegeneration. Nat Rev Neurosci. 2011;13(1):38–50.

    Article  Google Scholar 

  2. Buratti E. Functional significance of TDP-43 mutations in disease. Adv Genet. 2015;91:1–53.

    Article  CAS  Google Scholar 

  3. Ling SC, Polymenidou M, Cleveland DW. Converging mechanisms in ALS and FTD: disrupted RNA and protein homeostasis. Neuron. 2013;79(3):416–38.

    Article  CAS  Google Scholar 

  4. Da Cruz S, Cleveland DW. Understanding the role of TDP-43 and FUS/TLS in ALS and beyond. Curr Opin Neurobiol. 2011;21(6):904–19.

    Article  Google Scholar 

  5. Philips T, Rothstein JD. Rodent Models of Amyotrophic Lateral Sclerosis. Curr Protoc Pharmacol. 2015;69:5 67 1–5 21.

    Article  Google Scholar 

  6. Corcia P, Valdmanis P, Millecamps S, Lionnet C, Blasco H, Mouzat K, et al. Phenotype and genotype analysis in amyotrophic lateral sclerosis with TARDBP gene mutations. Neurology. 2012;78(19):1519–26.

    Article  CAS  Google Scholar 

  7. Watanabe S, Kaneko K, Yamanaka K. Accelerated disease onset with stabilized familial amyotrophic lateral sclerosis (ALS)-linked mutant TDP-43 proteins. J Biol Chem. 2013;288(5):3641–54.

    Article  CAS  Google Scholar 

  8. Ebstein SY, Yagudayeva I, Shneider NA. Mutant TDP-43 Causes Early-Stage Dose-Dependent Motor Neuron Degeneration in a TARDBP Knockin Mouse Model of ALS. Cell Rep. 2019;26(2):364–73.e4.

    Article  CAS  Google Scholar 

  9. White MA, Kim E, Duffy A, Adalbert R, Phillips BU, Peters OM, et al. TDP-43 gains function due to perturbed autoregulation in a Tardbp knock-in mouse model of ALS-FTD. Nat Neurosci. 2018;21(4):552–63.

    Article  CAS  Google Scholar 

  10. Fratta P, Sivakumar P, Humphrey J, Lo K, Ricketts T, Oliveira H, et al. Mice with endogenous TDP-43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis. EMBO J. 2018;37:e98684.

  11. Yamazaki T, Chen S, Yu Y, Yan B, Haertlein TC, Carrasco MA, et al. FUS-SMN protein interactions link the motor neuron diseases ALS and SMA. Cell Rep. 2012;2(4):799–806.

    Article  CAS  Google Scholar 

  12. Tsuiji H, Iguchi Y, Furuya A, Kataoka A, Hatsuta H, Atsuta N, et al. Spliceosome integrity is defective in the motor neuron diseases ALS and SMA. EMBO Mol Med. 2013;5(2):221–34.

    Article  CAS  Google Scholar 

  13. Ishihara T, Ariizumi Y, Shiga A, Kato T, Tan CF, Sato T, et al. Decreased number of Gemini of coiled bodies and U12 snRNA level in amyotrophic lateral sclerosis. Hum Mol Genet. 2013;22(20):4136–47.

    Article  CAS  Google Scholar 

  14. Nishino K, Watanabe S, Shijie J, Murata Y, Oiwa K, Komine O, et al. Mice deficient in the C-terminal domain of TAR DNA-binding protein 43 develop age-dependent motor dysfunction associated with impaired Notch1-Akt signaling pathway. Acta Neuropathol Commun. 2019;7(1):118.

    Article  Google Scholar 

Download references


We thank Mr. Takashi Arai in RIKEN Center for Brain Science RRD for support on developing TDP-43M337V mice, Center for Gene Research, Nagoya University for sequence analyses, and Center for Animal Research and Education (CARE), Nagoya University for support on animal experiments.


This work was supported by JSPS KAKENHI Grant Number 18H02740, 18H04860, 19KK0214 (to KY), and 17H04986 (to SW) as well as Uehara Memorial Foundation and Takeda Science Foundation (to KY).

Author information

Authors and Affiliations



SW and KY designed the study. SW, KO, and YM performed the experiments with support from OK, AS, and FE under the supervision of KY. ET and SW established the TDP-43M337V mice. SW, KO, and KY interpreted the data and wrote the manuscript. All authors approved the manuscript.

Corresponding author

Correspondence to Koji Yamanaka.

Ethics declarations

Ethics approval

All experiments using genetically modified mice were approved by the Animal Care and Use Committee and the recombinant DNA experiment committee of Nagoya University and RIKEN.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Additional file 1.

Material and methods. Figure S1. Direct sequencing of Tardbp gene exon 6 in heterozygous TDP-43M337V knock-in mice. Figure S2. Body weights were not affected in TDP-43M337V knock-in mice. Figure S3. Relative expression levels of Notch1 and Nek1 mRNAs were not altered in the brain of aged homozygous TDP-43M337V mice. Figure S4. Splicing was not altered in Eif4h or Poldip3, which are also regulated by TDP-43, in the brain of aged homozygous TDP-43M337V mouse brains. Figure S5. Gliosis was not observed in ventral horn of aged (700 days-old) homozygous TDP-43M337V mice. Figure S6. The number of Gems was not affected in ventral horn neurons of aged (700 days-old) homozygous TDP-43M337V mice.

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Watanabe, S., Oiwa, K., Murata, Y. et al. ALS-linked TDP-43M337V knock-in mice exhibit splicing deregulation without neurodegeneration. Mol Brain 13, 8 (2020).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: