Fig 2: ALS-Linked Mutations in FUS Cause Stress-Mediated Reduction in Axonal Protein Synthesis(A) Immunoblot demonstrating increased stress mediated by mutant FUS at early symptom onset using the phosphorylated form of eIF2α antibody (P-eIF2α) and HSF1 antibody in 12-month-old hgFUSR521C and hgFUSR521H spinal cords compared to age-matched Non-Tg and hgFUSWT. eIF2α and tubulin antibodies were used as loading controls. See also quantification in Figures S6A and S6B.(B) Expression of Atf4 in primary hippocampal neuronal cultures from hgFUSR521H compared to Non-Tg mice. Data are represented as mean ± SEM (n ≥ 3 biological replicates per group). ∗p < 0.05, two-sided unpaired Student’s t test.(C) Quantification of the amount of 35S incorporated into newly translated proteins in hippocampal neurons cultured for 15 days. Cycloheximide (25 μg/mL) addition was used as a positive control for complete protein translation inhibition. Data are represented as mean ± SEM (n = 4 biological repeats). ∗∗p < 0.01, two-sided unpaired Student’s t test.(D) Schematic of the experimental design using microfluidic compartmented chambers to distinguish neuronal cell bodies from axons.(E–G) Hippocampal neurons isolated from hgFUSR521H or Non-Tg mice (from four independent experiments with 3 or 4 mouse embryos per genotype per experiment) cultured in compartmented chambers and treated with a 10-min pulse of puromycin to label protein translation sites using puromycin antibody (green) with a neurofilament NF-H (white) antibody to stain axons (E) or with an antibody recognizing the phosphorylated form of eIF2α (P-eIF2α) (red) in (F) cell bodies and (G) neuronal processes. Scale bars, 10 μm and 50 μm, respectively.(H and I) Hippocampal neurons isolated from hgFUSR521H and hgFUSWT mice cultured in compartmented chambers immunostained for FUS (using FUS antibody in red) and protein translation (using puromycin antibody in green) in (H) cell bodies and (I) axonal processes (using NF-H antibody, white). Scale bar, 50 μm.(J–M) Quantification of the puromycin relative fluorescence intensity (protein translation) in (J) cell bodies (green signal in Figure 6F) and (K) axons (green signal in Figure 6G); quantification of P-eiF2α fluorescence intensity (stress) in (L) cell bodies (red signal in Figure 6F) and (M) axons (red signal in Figure 6G) of Non-Tg and hgFUSR521H hippocampal neurons cultured in microfluidic chambers. The bar graph represents mean ± SEM (n ≥ 30 axons or cell bodies per group from four independent experiments). ∗∗∗p < 0.001, two-sided unpaired Student’s t test.(N and O) Quantification of FUS relative fluorescence intensity in (N) cell bodies (red signal in Figure 6H) and (O) axons (red signal in Figure 6I). The bar graph represents mean ± SEM (n ≥ 50 axons or cell bodies per group). ∗∗∗p < 0.001, two-sided unpaired Student’s t test.

Fig 3: ALS-Linked Mutations in FUS Cause Stress-Mediated Reduction in Local Axonal Protein Synthesis within Sciatic Nerve Axons of Mutant FUS Mice(A) Experimental design to assess stress and local axonal translation in the sciatic nerve of 12-month-old Non-Tg, hgFUSWT, hgFUSR521C, and hgFUSR521H mice.(B) Transversal sections of mouse sciatic nerves immunostained using P-eIF2α (green) and NF-H (red) antibodies and (C) puromycin (green) antibody.(D) Longitudinal sections of mouse sciatic nerves immunostained using puromycin (green), NF-H (red), and FUS (blue) antibodies. Scale bars, 10 μm.(E–G) Quantification of the relative fluorescence intensity of the (E) P-eIF2α, (F) puromycin, and (G) FUS immunostainings within sciatic nerve axons of 12-month-old Non-Tg, hgFUSWT, hgFUSR521C, and hgFUSR521H mice. The bar graph represents mean ± SEM (n = 3 animals per group). ∗∗∗p < 0.001, two-sided unpaired Student’s t test.

Fig 4: ALS-Linked Mutations in FUS Cause a Mutant-Dependent Signature of RNAs that Includes a Reduction in mRNAs for Ion Channels and Transporters and Ribosomal Proteins and an Increase in mRNAs Encoding Chaperones(A) Unsupervised hierarchical clustering of all expressed genes in spinal cords of 18-month-old Non-Tg, mFus−/−/hgFUSWT, mFus−/−/hgFUSR521C, and mFus−/−/hgFUSR521H mice identified by RNA-seq analysis.(B) Principal component analysis (PCA) of differentially expressed genes in 18-month-old Non-Tg, mFus−/−/hgFUSWT, mFus−/−/hgFUSR521C, and mFus−/−/hgFUSR521H spinal cords.(C) Heatmap with hierarchical clustering of 1,057 genes differentially expressed in 18-month-old hgFUSR521C and hgFUSR521H mice compared to age-matched Non-Tg and mFus−/−/hgFUSWT spinal cords.(D) Functional analysis using DAVID software of the differentially expressed genes revealing the most enriched gene group changes in mFus−/−/hgFUSR521C and mFus−/−/hgFUSR521H mice compared to age-matched Non-Tg and mFus−/−/hgFUSWT animals.(E) Functional analysis using the Ingenuity Pathway Analysis (IPA) software of the differentially expressed genes revealing the most enriched gene group changes in mFus−/−/hgFUSR521C and mFus−/−/hgFUSR521H mice compared to age-matched Non-Tg and mFus−/−/hgFUSWT animals.(F) Reduced expression of ion channels and transporters essential for synaptic function in 18-month-old mFus−/−/hgFUSR521C and mFus−/−/hgFUSR521H spinal cords compared to age-matched Non-Tg and mFus−/−/hgFUSWT, validated by qRT-PCR. Data are represented as mean ± SEM (n ≥ 3 per group). ∗p < 0.05, ∗∗p < 0.01, two-sided unpaired Student’s t test. See also Figure S5A.(G) Increased expression of chaperones in 18-month-old mFus−/−/hgFUSR521C and mFus−/−/hgFUSR521H spinal cords compared to age-matched Non-Tg and mFus−/−/hgFUSWT, validated by qRT-PCR. Data are represented as mean ± SEM (n ≥ 3 per group). ∗p < 0.05, ∗∗p < 0.01 and ∗∗p < 0.001, two-sided unpaired Student’s t test. See also Figure S5B.(H) Reduced expression of genes encoding ribosomes or other components of the translation machinery in 18-month-old mFus−/−/hgFUSR521C and mFus−/−/hgFUSR521H spinal cords compared to age-matched Non-Tg and mFus−/−/hgFUSWT, validated by qRT-PCR. Data are represented as mean ± SEM (n ≥ 3 per group). ∗p < 0.05 and ∗∗p < 0.01, two-sided unpaired Student’s t test. See also Figure S5C.

Fig 5: ALS-Linked FUS Mutants Show Age-Dependent Progressive Motor Impairments without Nuclear Mislocalization or Aggregation of FUS(A) Hindlimb grip strength measured up to 24 months. Data are represented as mean ± SEM (n ≥ 12 per group). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns: non-significant using one-way ANOVA.(B) Gastrocnemius muscle sections from 24-month-old Non-Tg, hgFUSWT, hgFUSR521C, and hgFUSR521H mice with axons immunostained using a synaptophysin antibody and FluoroMyelin™ red (red) and muscle endplates using α-bungarotoxin (green). Scale bar, 50 μm.(C) NMJ innervation of the gastrocnemius from 8- and 24-month-old Non-Tg, hgFUSWT, hgFUSR521C, and hgFUSR521H mice. Data are represented as mean ± SEM (n ≥ 3 mice per group). ∗p < 0.05, ∗∗∗p < 0.001, two-sided unpaired Student’s t test.(D) Lumbar motor axons from 24-month-old Non-Tg, hgFUSWT, hgFUSR521C, and hgFUSR521H mice. Scale bar, 50 μm.(E) Size distribution per motor axon in the L5 motor root of 24-month-old Non-Tg, hgFUSWT, hgFUSR521C, and hgFUSR521H mice. Data are represented as mean ± SEM (n ≥ 3 per group). ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001, two-sided unpaired Student’s t test.(F) Total number of α-motor axons (caliber size > 4.5 μm) in the L5 motor root of 8- and 24-month-old Non-Tg, hgFUSWT, hgFUSR521C, and hgFUSR521H mice. Data are represented as mean ± SEM (n ≥ 3 per group).(G) Lumbar spinal cord sections from 24-month-old Non-Tg, hgFUSWT, hgFUSR521C, and hgFUSR521H mice immunostained using a ChAT antibody to reveal cholinergic motor neurons. Scale bar, 100 μm.(H) Total number of ChAT-positive motor neurons in lumbar spinal cords of 8- and 24-month-old Non-Tg, hgFUSWT, hgFUSR521C, and hgFUSR521H mice. Data are represented as mean ± SEM (n ≥ 3 per group). ∗p < 0.05, ∗∗p < 0.01, two-sided unpaired Student’s t test.(I) Total human and/or mouse FUS protein levels in spinal cords of 2-, 12-, and 24-month-old hgFUSWT, hgFUSR521C, hgFUSR521H, and Non-Tg mice. Tubulin was used as a loading control. Asterisk illustrates a non-specific band recognized by the antibody against mouse FUS.(J) Quantification of immunoblots shown in (I). Data are represented as mean ± SEM from two independent experiments.(K and L) Lumbar spinal cord sections from 24-month-old Non-Tg, hgFUSWT, hgFUSR521C, and hgFUSR521H mice immunostained using an antibody recognizing specifically (K) human or (L) mouse FUS. DNA is stained with DAPI. Scale bars, 100 μm and 50 μm (inset). See also Figure S2.