Mouse models and genotyping
Mouse experiments were approved by local ethical committee from Strasbourg University (CREMEAS) under reference number 2,016,111,716,439,395 and all experimental procedures performed in San Diego were approved by the Institutional Animal Care and Use Committee of the University of California, San Diego. Transgenic mice were generated as described in [11, 12] and [8], were bred in Charles River animal facility and housed in the Faculty of medicine from Strasbourg University with 12/12 hours of light/dark cycle (light on at 7:00 am) under constant conditions (21 ± 1 °C; 60 % humidity) and with unrestricted access to food and water.
Mice were weaned and genotyped at 21 days by PCR from tail biopsy, or at death if occurring before 21 days of age.
The following primer sequences were used to genotype mice:
hFUS-For: GAATTCGTGGACCAGGAAGGTC.
hFUS-Rev: CACGTGTGAACTCACCGGAGTCA.
FUS-For: GAT-TTG-AAG-TGG-GTA-GAT-AGT-GCA-GG.
FUS-Rev: CCT-TTC-CAC-ACT-TTA-GTT-TAG-TCA-CAG.
Heterozygous Fus∆NLS/+ knock-in mice, lacking the PY-NLS, were crossed with mice expressing human wild type FUS from a complete, autoregulatory competent, human gene to obtain following genotypes: Fus+/+, FusΔNLS/+,FusΔNLS/ΔNLS, Fus+/+/hFUS, Fus∆NLS/+/hFUS, FusΔNLS/ΔNLS/hFUS. The genetic background of all mice used in this study is C57Bl6/J. Breeding steps were performed in parallel in both laboratories. 76 mice of the F2 generation were generated in Strasbourg, and 110 mice of the F2 generation were generated in San Diego.
Mouse behavior
Survival
Survival was studied during the first hours after birth and dead new born mice were genotyped. Mice surviving the post-natal period were genotyped at 21 days and followed weekly until death or euthanized using ketamine-xylazine when they reach the following endpoints: auto-mutilation, weight loss greater than 10 % of the initial weight and when they could not turn around again within 10 s after being laid on their side.
Inverted grid
Mice were habituated for 30 min in the test room prior testing. Motor performance was assessed weekly as described previously [12] from 1 month until 22 months of age. The wire grid hanging time (or “hang time”) was defined as the amount of time that it takes the mouse to fall down from the inverted grid and was measured visually with a stopwatch. The procedure was repeated 3 times during 5 min with 5 min break between tests. All mice were returned to their homecage after completing the test. The holding impulse corresponds to hanging time normalized with mouse weight and gravitational force.
Grip test
Grip strength was measured using a Grip Strength Meter (Columbus Instruments, Columbus, OH) on cohorts (N = 12–30) made up of approximately the same number of males and females. Mice were allowed to grip a triangular bar only with hind limbs, followed by pulling the mice until they released; five force measurements were recorded in each separate trial.
Histological techniques
Mice aged of 22 months were anesthetized with intraperitoneal injection of 100 mg/kg ketamine chlorhydrate and 5 mg/kg xylazine then perfused with PFA 4 %. After dissection, spinal cord was included in agar 4 % and serial cuts of 40 μm thick were made with vibratome.
Peroxidase immunohistochemistry
For peroxidase immunohistochemistry, sections were incubated 10 min with H2O2 3 %, washed 3 times and blocked with 8 % Horse serum, 0,3 % Bovine Serum Albumin and 0,3 % Triton in PBS with 0,02 % Thimerosal. Sections were incubated with rabbit anti-FUS antibody (ProteinTech 11570-1-AP; diluted 1:100) in blocking solution overnight at room temperature. After washing sections, they were incubated for 2 h at room temperature with biotinylated donkey anti-rabbit antibody (Jackson 711-067-003; diluted 1:500) in blocking solution. Then, sections were washed, incubated for 1 h in horseradish peroxidase ABC kit (Vectastain ABC kit, PK-6100, Vector Laboratories Inc.), washed and incubated with DAB (Sigma, D5905). The enzymatic reaction was stopped by adding PBS 1X and washed with water. Finally, sections were mounted with DPX mounting medium (Sigma, O6522).
Immunofluorescence
After epitope retrieval in 10 mM citrate pH6.0 30 min at 80 °C, sections were incubated in blocking solution (5 % Horse serum, 1 % Triton in PBS) at room temperature for 30 min, then incubated overnight at room temperature in primary antibody in PBS + 0.1 % triton X100: rabbit anti-FUS antibody (total FUS) (ProteinTech, 11570-1-AP, 1:100), Rabbit anti-C-ter FUS (Bethyl, A300-294 A, 1/100), Rabbit anti-mouse FUS[8], goat anti-ChAT (Millipore, AB144P, 1/50), rat anti-ADMA FUS ([5, 6], kind gift of Pr C. Haass, Munich Germany, 1/20). After 3 rinses in PBS, sections were incubated for 2 h at room temperature with Hoechst (Sigma, B2261, 1/50.000) and secondary antibodies in blocking solution: Alexa-488-conjugated donkey anti-rabbit secondary antibody (Jackson, 711-547-003, 1/500) Alexa-488-conjugated donkey anti-rat secondary antibody (Jackson 712-545-153 1/1000) or Alexa-594-conjugated donkey anti-goat secondary antibody (Molecular Probes, A 11,058, 1/500). Finally, sections were subsequently washed with PBS 1 × (3 x 10 min) and mounted in Aqua/polymount (Polysciences 18,606).
Immunofluorescence staining was monitored with a laser scanning microscope (confocal LSM 800 Zeiss) equipped with 40 × oil objective (NA1.4). Excitation rays are sequential argon laser 488nm, diode 561nm, diode 405nm. Emission bandwidths are 500-570nm for Alexa488, 570-617nm for Alexa594, and 400-500nm for Hoechst. Single-layer images were analyzed using ImageJ freeware (http://rsbweb.nih.gov/ij/).
Tissue homogenization, fractionation and western blotting
Total protein extracts were obtained from brain homogenization using zirconium oxide beads (Bertin Technologies) in combination with Precellys Tissue homogenizer (Bertin Technologies) for 3 × 15 s, 5000 rpm in RIPA buffer (Tris-HCl pH 8 50mM, sodium chloride 150mM, sodium deoxycholate 0.5 %, SDS 0.1 %, Triton-X100 1 %). The supernatants were collected after centrifugation for 15 min, 14,000 rpm at 4 °C and the protein extracts were measured with Pierce™ BCA Protein Assay Kit (Thermo Scientific). SDS-PAGE was performed with 10 µg of total protein extracts using Mini-PROTEAN TGX gel 4–15 % (Biorad). Proteins were blotted on PVDF membrane using Mini Trans-Blot® Cell (Biorad) and blocked with 10 % non-fat milk during 1 h. Primary antibodies (Rabbit anti-hFUS (1/2000), Rabbit anti-mFUS (1/4000), Rabbit anti-FUS (total FUS) (Bethyl, A-300-293 A, 1/2000), Rabbit anti-C-ter FUS (Bethyl, A300-294 A, 1/2000), Mouse-anti-vinculin (Merk Millipore, V9131, 1/2000)) were incubated overnight at 4 °C in 3 % non-fat milk. Washing was proceeded with washing buffer (Tris pH 7.4 1 M, NaCl 5 M, Tween 20 0.1 %) and secondary antibodies (anti-rabbit HRP (Agilent, P0448, 1/5000), anti-mouse HRP (Jackson Immunoresearch, 715-035-150, 1/5000) were incubated 1h30 at room temperature. After successive washes, proteins were visualized with chemiluminescence using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Scientific, 34,577) and chemiluminescence detector.
Tissues were washed in PBS1x and lysed in NE-PER Nuclear and Cytoplasmic Extraction (Thermo Scientific, 78,835) according to the manufacturer’s instructions. Protein extracts were dosed by BCA Assay (Interchim, UP95424A, UP95425A). Thereafter proteins were denatured and SDS page was performed with 30 µg of cytoplasmic proteins and 10 µg of nuclear proteins on criterion TGX stain free gel 4–20 % (Biorad, 5,678,094). Proteins were blotted on nitrocellulose membrane using semi-dry Transblot Turbo system (BioRad, France) and blocked with 10 % non-fat milk during 1 h. Primary antibodies (Rabbit anti-hFUS ([8, 9], #14,080, 1/2000), Rabbit anti-mFUS ([8, 9], #14,082, 1/4000), Rat anti-FUS ADMA ([5, 6], kind gift of Pr C. Haass, Munich Germany, 1/500), Rabbit anti-FUS (total FUS) (Bethyl, A-300-293 A, 1/2000), Rabbit anti-C-ter FUS (Bethyl, A300-294 A, 1/2000), Sheep anti-SOD1 (Calbiochem, 574,597, 1/1000), Rabbit anti-HDAC1 (Bethyl, A300-713 A, 1/1000) ) were incubated overnight at 4 °C in 3 % non-fat milk. Washing was proceeded with washing buffer (Tris pH 7.4 1 M, NaCl 5 M, Tween 20 100 %) and secondary antibodies (anti-rabbit HRP (PARIS, BI2407,1/5000), anti-sheep HRP (Jackson, 713-035-147, 1/5000)) were incubated 1h30 at room temperature. After successive washes, proteins were visualized with chemiluminescence using ECL Lumina Forte (Millipore, France) and chemiluminescence detector (Bio-Rad, France). Total proteins were detected with stain free gel capacity (Biorad, 5,678,094) and used to normalize for protein loading. All values were normalized against nuclear levels of FUS in Fus+/+ extracts set to 1.
RNA extraction and RT-qPCR
Total RNA was extracted from spinal cord and frontal cortex using TRIzol® reagent (Life Technologies). 1 µg of RNA was reverse transcribed with iScript™ reverse transcription (Biorad, 1,708,841). Quantitative polymerase chain reaction was performed using Sso Advanced Universal SYBR Green Supermix (Bio-Rad 1,725,274) and quantified with Bio-Rad software. Gene expression was normalized by calculating a normalization factor using actin, TBP and pol2 genes according to GeNorm software [55].
Primer sequences are provided in Table S1.
RT-PCR
1 µg of RNA was reverse transcribed with iScript™ reverse transcription (Biorad, 1,708,841). Polymerase chain reaction was performed using in 25 µL microtubes with MasterMix Taq DNApolymerase (VWR International, Ref. 733–1320) using the following programs: Intron 6 retention and exon 7 skipping (5 min 95 °C, (30 s 95 °C, 30 s 56 °C, 30 s 68 °C)x 30; 5 min 68 °C), Intron 7 retention (5 min 95 °C, (30 s 95 °C, 30 s 61 °C, 30 s 68 °C)x 30; 5 min 68 °C), 10 µL of the PCR products were loaded on a 2 % agarose (Euromedex, Ref.D5-E) gel electrophoresis with Low Molecular Weight DNA Ladder (NEB, Ref. N3233L) and stained with ethidium bromide using standard procedures. For quantification, we quantified individually the signal intensities of the two bands, and computed a % of intron retention as such: (intensity of Intron + band )/ (intensity of Intron + band + intensity of Intron- band)*100. We did not quantify a percentage of exon 7 skipping as the exon 7 skipped product was below the detection threshold of the assay.
Statistics
All results from analysis are presented as mean ± standard error of the mean (SEM) and differences were considered significant when p < 0.05. Significance is presented as follows: * p < 0.05, ** p < 0.01, and *** p < 0.001. For comparison of two groups, two-tailed unpaired Student’s t –test was used in combination with F-test to confirm that the variances between groups were not significantly different. For longitudinal analysis of behavioral data, results were analyzed using a mixed effect analysis with three factors (∆NLS genotype, hFUS genotype and age) as indicated in the figure legends. Data were analyzed by using the GraphPad Prism version 8.0.