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. 2022 Jan 24;28(1):104–116. doi: 10.1038/s41591-021-01615-z

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as 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 images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a, Table relating the indicated mouse genotypes to WT and Δ14 FUS protein expression in MN and non-MN cells. b, Numbers of ChAT-positive MNs at lumbar levels 4 and 5 in 1-year-old WT/WT (left), MN-Δ14/WT (middle, c14/WT; ChAT-Cre and Δ14/WT) and MN-Δ14/Δ14 (right, c14/c14; ChAT-Cre, Δ14/c14; ChAT-Cre and Δ14/Δ14) animals normalized to the wild-type controls. Each of the MN-Δ14/Δ14 genotypes shows fewer lumbar MNs in comparison to either of MN-Δ14/WT genotypes or WT/WT group, using one-way ANOVA followed by Fisher’s least significant difference. Data are shown as mean ± s.d. n = 3 animals per genotype. c, Density of Iba1-positive microglial cells at lumbar levels 4 and 5 in 1-year-old WT/WT (left, black), MN-Δ14/WT (middle, c14/WT; ChAT-Cre and Δ14/WT) and MN-Δ14/Δ14 (right, c14/c14; ChAT-Cre, Δ14/c14; ChAT-Cre and Δ14/Δ14) animals. Each of the MN-Δ14/WT genotypes show an increase in microglial density in comparison to WT/WT animals. Each of the MN-Δ14/Δ14 genotypes has increased microglial density in comparison to either of MN-Δ14/WT genotypes or WT/WT group. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test. Data are shown as mean ± s.d. n = 3 animals per genotype. d, Immunostaining of lumbar spinal cord sections of adult MN-P517L/Δ14 (P517L/c14; ChAT-Cre) animals using anti-ChAT (green), anti-FUS-WT (Abcam[1-50], yellow), anti-FUS-Δ14 (cyan) and mouse monoclonal anti-FUS-P517L (red) antibodies. Note anti-FUS-P517L staining in all cells, anti-FUS-Δ14 staining only in ChAT-positive cells and anti-FUS-WT staining only in ChAT-negative cells. Scale bar, 100 µm. e, Percentage of completely innervated NMJs (that is, not partially or completely denervated) in TA (left) and soleus (right) muscles of CTRL (FUS WT-expressing control, black) and MN-P517L/Δ14 (P517L/c14; ChAT-Cre, green) animals. f, MN numbers at lumbar level 4 and 5 of CTRL (FUS WT-expressing control, black) and MN-P517L/Δ14 (P517L/c14; ChAT-Cre, green) animals normalized to the controls. g, Histogram of MN soma cross-sectional areas of CTRL (FUS WT-expressing control, black) and MN-P517L/Δ14 (P517L/c14; ChAT-Cre, green) animals. MNs with soma area ≥475 µm² were classified as alpha-MNs, and MNs with soma area <475 µm² were classified as gamma-MNs. Inset shows the numbers of ChAT-positive gamma-MNs (left) or alpha-MNs (right) at lumbar levels 4 and 5 in 2-year-old CTRL (FUS WT-expressing control, black) and MN-P517L/Δ14 (P517L/c14; ChAT-Cre, green) animals normalized to the controls. *P < 0.05, **P < 0.01 and ***P < 0.001, using Welch’s t-test. Data are shown as mean ± s.d. n = 3 animals per group. For e and f, *P < 0.05, **P < 0.01 and ***P < 0.001, using two-way ANOVA with Sidak´s post hoc test. Data are shown as mean ± s.d. n = 3 animals per group. CTRL, littermate c14/WT and/or c14/c14 animals depending on the breeding scheme.