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. 2023 Feb 19:645:164-172.
doi: 10.1016/j.bbrc.2023.01.032. Epub 2023 Jan 13.

MATR3 P154S knock-in mice do not exhibit motor, muscle or neuropathologic features of ALS

Marissa Dominick  1 , Nicole Houchins  1 , Vinisha Venugopal  1 , Aamir R Zuberi  2 , Cathleen M Lutz  2 , Bessie Meechooveet  3 , Kendall Van Keuren-Jensen  3 , Robert Bowser  1 , David X Medina  4
Affiliations

MATR3 P154S knock-in mice do not exhibit motor, muscle or neuropathologic features of ALS

Marissa Dominick et al. Biochem Biophys Res Commun. .

Abstract

Matrin 3 is a nuclear matrix protein that has many roles in RNA processing including splicing and transport of mRNA. Many missense mutations in the Matrin 3 gene (MATR3) have been linked to familial forms of amyotrophic lateral sclerosis (ALS) and distal myopathy. However, the exact role of MATR3 mutations in ALS and myopathy pathogenesis is not understood. To demonstrate a role of MATR3 mutations in vivo, we generated a novel CRISPR/Cas9 mediated knock-in mouse model harboring the MATR3 P154S mutation expressed under the control of the endogenous promoter. The P154S variant of the MATR3 gene has been linked to familial forms of ALS. Heterozygous and homozygous MATR3 P154S knock-in mice did not develop progressive motor deficits compared to wild-type mice. In addition, ALS-like pathology did not develop in nervous or muscle tissue in either heterozygous or homozygous mice. Our results suggest that the MATR3 P154S variant is not sufficient to produce ALS-like pathology in vivo.

Keywords: ALS; MATR3 P154S mutation; Matrin 3; Mouse model; Neuropathology.

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Conflict of interest statement

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Figure 1:
Figure 1:. Generation of MATR3 P154S knock-in mice:
A) Heterozygous mice were generated by Jackson Lab and validated via Sanger sequencing (top). The presence of a silent mutation removed a restriction site that could be used to validate genotypes via PCR (bottom). Homozygous mice completely lacked this restriction site. B) Mutant MATR3 mice were produced at the expected Mendelian ratios in both lines 1 and 2, with no significant differences observed (chi squared test for goodness of fit). C) Longitudinal tracking of average mouse weights demonstrate the P154S mutation did not cause changes in either males or females (p<0.05, Two-Way ANOVA) (n=18–20 per group).
Figure 2:
Figure 2:. Behavioral Analysis of MATR3 P154S knock-in mice demonstrates P154S mutation does not cause motor impairments.
A) Longitudinal rotarod analysis demonstrated that MATR3P154S/+ and MATR3P154S/P154S mice do not demonstrate declines with age or significant changes from WT performance. B) Footprint analysis demonstrated the average hindlimb stride length was not changed in 18-month-old MATR3P154S/+ or MATR3P154S/P154S mice. C) Open-Field Task (left) was used to measure locomotor activity, and found that distance moved during a 10minute trial was not significantly different in MATR3P154S/+ or MATR3P154S/P154S compared to WT mice. Thigmotaxis (right) was measured with the open field, and likewise demonstrated no differences between genotypes. (p<0.05, Two-Way repeated measures ANOVA) (n=10–15 per group).
Figure 3:
Figure 3:. MATR3 P154S knock-in does not produce myopathology at 24 months-of-age.
A) Matrin 3 IF of gastrocnemius muscle demonstrates no changes of nuclear localization of Matrin 3 in MATR3P154S/+ or MATR3P154S/P154S. Scale bar=30 μm. B) Laminin IF of gastrocnemius muscle; (Graph) Quantification of fiber diameter demonstrates no changes between genotypes. Scale bar=100 μm. C) Neuromuscular junction analysis; α-bungarotoxin (red) labeling and neurofilament (green) co-localization in gastrocnemius muscle; (Graph) quantification of innervated NMJs. Scale bar=20μm. D) Western blot analysis of Matrin 3 from RIPA soluble fraction (left) and insoluble fraction (right) from gastrocnemius (n=3/genotype). One-way ANOVA was performed on quantifications.
Figure 4:
Figure 4:. MATR3 P154S knock-in does not produce neuropathology at 18 months-of-age.
A) Representative images of Matrin 3 immunofluorescence (green) from lumbar spinal cord show nuclear localization. Scale bar= 25 μm. B) Western blot analysis of Matrin 3 from RIPA soluble fraction (left) and insoluble fraction (right) from spinal cord (n=3–4/genotype). C) Representative images of ChAT IF in lumbar spinal cord (Graph) quantification of motor neuron number in ventral horn. Scale bar=100 μm D) Iba-1 IHC in lumbar spinal cord. Scale bar=100μm.
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