Species: Mouse
Genes: FUS
Mutations: FUSDelta14 truncation mutation
Modification: FUS: Knock-In
Disease Relevance: Amyotrophic Lateral Sclerosis
Strain Name: B6N;B6J-Fus^tm1Emcf/H
Genetic Background: C57BL6/J
Availability: European Mouse Mutant Archive (EM:11106)

Summary

The FUSDelta14 mutation (g.13845A>G) at the splice acceptor site of intron 13 results in skipping of exon 14, out-of-frame translation of exon 15, and C-terminal truncation of the FUS protein (FUS p.G466VfsX14). This mutation removes the nuclear localization signal and most of the third RGG-rich RNA-binding domain. The FUSDelta14 mutation was first described as a de novo mutation in a woman who developed symptoms at age 20 and died 22 months later (DeJesus-Hernandez et al., 2010). Clinical presentation was typical for ALS with bulbar onset, except that the patient did not develop clinical upper-motor-neuron signs.

In this knock-in model, the point mutation g.13845A>G was introduced into the splice acceptor site of mouse FUS intron 13, and the exon 15 coding sequence was humanized (Devoy et al., 2017). Gene expression is thus under the control of endogenous mouse regulatory elements. Mice heterozygous for the FUSDelta14 mutation express FUS protein at physiological levels, develop motor impairment before 15 months of age, and exhibit progressive degeneration of motor neurons from before 12 months of age. Premature death is seen in FUSDelta14 mice beginning at 19 months of age.

In order to study expression and localization of the FUSDelta14 protein, a polyclonal antibody was raised against the novel epitope generated by the humanized frameshift peptide; this antibody recognizes FUSDelta14 but not wild-type FUS. Total FUS protein level in spinal cord was found to be similar in wild-type mice and heterozygous FUSDelta14 mice. In the heterozygous FUSDelta14 mice, levels of wild-type and mutant proteins were equivalent, but the mutant protein mislocalized. While wild-type FUS protein has a normal nuclear localization, approximately 75 percent of the mutant FUSDelta14 protein is cytoplasmic. Some of the FUSDelta14 protein found outside the nucleus co-localizes with S6 riboprotein, suggesting that it might be located in the rough endoplasmic reticulum.

FUSDelta14 mice do not show evidence of FUS aggregation. Sarkosyl-insoluble FUS is not detected in the spinal cords of 12-month FUSDelta14 mice, and ubiquitin- or p62-positive inclusions are no more frequent in the spinal motor neurons of aged FUSDelta14 mice than in aged wild-type mice. However, FUSDelta14 protein, but not wild-type protein, was recruited to stress granules in stressed fibroblasts cultured from heterozygous FUSDelta14 mice—showing a gain-of-function behavior.

Heterozygous FUSDelta14 mice have normal motor function at two and six months of age, assessed using the Locotronic horizontal ladder test and analysis of gait. Motor impairment is seen at 15 months, in the form of significantly increased numbers of hindlimb errors in the ladder test. Altered rear-stride pattern, assessed by gait analysis, is seen by 18 months of age.  Forelimb function is normal at least through 18 months. Both male and female mice were used in the behavioral characterization, with no differences between sexes observed.

Consistent with the behavioral phenotype, FUSDelta14 mice exhibit a progressive loss of motor neurons in the lumbar spinal cord, beginning by 12 months of age (14 percent reduction at 12 months, 20 percent reduction at 18 months, compared with wild-type littermates). By 18 months of age FUSDelta14 mice show a 15 percent reduction in the number of motor units innervating extensor digitorum hindlimb muscles and a decrease in the number of fully innervated endplates in hindlimb lumbrical muscles (58 percent fully innervated endplates in heterozygous FUSDelta14 mice versus 85 percent in wild-type littermates).

Transcriptomic analysis showed significant alterations in gene expression at 12 months, but not at three months, in FUSDelta14 mice, particularly affecting genes encoding mitochondrial proteins, ribosomal proteins, and the catalytic core of the proteasome.

In summary, heterozygous FUSDelta14 mice express mutant FUS protein at a physiological level and display a dominant progressive ALS-like phenotype. A frameshift-specific antibody was generated in conjunction with the FUSDelta14 mouse, which further enabled the investigation of mechanisms of toxicity of mutant FUS protein.

Modification Details

The FUS gene in C57BL6/N mouse embryonic stem cells (ESC) was modified through homologous recombination using a targeting vector designed to introduce the following changes: g.13845 A>G (mutation at splice acceptor site of intron 13); g.14230 C>T, g.14232 A>T, g.14234C>G, g.14260 A>G, and g.14266 ATTA insertion, to humanize the coding sequence of exon 15 (this vector also contained an FRT-flanked Neo cassette to select for ESCs that had successfully integrated the vector). Mice were generated by injection of the genetically modified ESCs into B6-albino donor embryos. Chimeric males were bred to B6-albino females, and progeny were crossed to Flpo-expressing mice (maintained on a C57BL6/J background) to remove the Neo cassette. The resulting Neo-negative line then was backcrossed for a minimum of four generations onto the C57BL6/J background.

Phenotype Characterization

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References

Paper Citations

1. DeJesus-Hernandez M, Kocerha J, Finch N, Crook R, Baker M, Desaro P, Johnston A, Rutherford N, Wojtas A, Kennelly K, Wszolek ZK, Graff-Radford N, Boylan K, Rademakers R. De novo truncating FUS gene mutation as a cause of sporadic amyotrophic lateral sclerosis. Hum Mutat. 2010 May;31(5):E1377-89. PubMed.
2. Devoy A, Kalmar B, Stewart M, Park H, Burke B, Noy SJ, Redhead Y, Humphrey J, Lo K, Jaeger J, Mejia Maza A, Sivakumar P, Bertolin C, Soraru G, Plagnol V, Greensmith L, Acevedo Arozena A, Isaacs AM, Davies B, Fratta P, Fisher EM. Humanized mutant FUS drives progressive motor neuron degeneration without aggregation in 'FUSDelta14' knockin mice. Brain. 2017 Nov 1;140(11):2797-2805. PubMed.

External Citations

1. EM:11106

Further Reading

No Available Further Reading