Patients
The study was approved by the ethics committee of the Università Cattolica del Sacro Cuore (Rome, Italy) on 30 July 2012, Prot nr. P740/CE/2012. A written informed consent was signed by all of the subjects. The diagnosis of ALS was made according to revise El Escorial/Airlie House Criteria. The presence of familiarity was deeply investigated. Patients with one or more affected relatives were diagnosed as familial ALS (fALS), while patients with no family history were classified as sporadic (sALS). Genetic analysis was performed on patients using massive parallel sequencing of genes associated to ALS, as previously described [20], and Repeat-Primed PCR was used to screen all patients for the C9orf72 expansion [42]. Three patients harboring the C9orf72 hexanucleotide repeat expansion (C9orf72) (2 fALS and 1 sALS), one patient harboring the p.R51C FUS pathogenic variant (fALS), two patients carrying the p.Q303H and the p.A382T variants in TARDBP (both sALS) were included in the study as well as three sporadic patients with no variants and five healthy controls.
Fibroblast primary cultures
All experiments were carried out in accordance to the approved guidelines of the ethics committee of the Catholic University, A written informed consent was obtained from patients and from healthy donors. Skin biopsies were performed using a 4-mm punch on the distal leg of the patients at NEMO Clinical Centre (Rome). Primary human dermal fibroblasts were isolated, as previously described [49]. Skin samples were dissected, transferred to a cell culture flask and cultured in BIO-AMF-2 complete medium (Biological Industries) in a 37 °C incubator. After the fibroblasts reached confluence, they were expanded up to 4th passage. Fibroblasts were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% fetal bovine serum (FBS, Euroclone) and 1% penicillin/streptomycin (Sigma) at 37 °C, 5% CO2.
Chemicals and antibodies
Niclosamide and all reagents, unless otherwise specified, were purchased from Sigma-Aldrich. Immunofluorescences (IF) and immunoblots (WB) were performed with the following primary antibodies: anti-rabbit S100A4 (1:500-IF, 1:1000-WB, Millipore), anti-rabbit mTOR and phospho-mTOR (1:100-WB, Cell Signaling), anti-mouse SQSTM1/p62 (1:1000-WB, Abcam), anti-rabbit NF-kB and phospho-NF-kB (1:1000-WB, Cell Signaling), anti-rabbit STAT3 (1:1000-WB, Cell Signaling), anti-rabbit phospho-STAT3 (1:2000-WB, Cell Signaling), anti-rabbit α-SMA (1:1000-WB, 1:500-IF, GeneTex), anti-rabbit N-cadherin (1:1000-WB, GeneTex), anti-mouse glial fibrillary acidic protein (GFAP) (1:500-IF, 1:1000-WB, Cell Signaling), anti-rabbit β-III Tubulin (1:500-IF, Cell Signaling), anti-mouse MyoG (1:200-WB, Hybridoma Bank, USA), anti-mouse platelet-derived growth factor receptor β (PDGFR-β) (1:250-WB, 1:500-IF, Santa Cruz), anti-mouse GAPDH (1:10000-WB, Calbiochem). Secondary immunoblot antibodies for WB were: anti-rabbit (1:2500) and anti-mouse (1:5000) IgG peroxidase-conjugated from Bio-Rad Laboratories (Hercules, CA, USA). Secondary fluorescent antibodies for IF were: Alexa-Flour 488-Donkey anti-rabbit (1:200), Cy3-Donkey anti-mouse (1:200) from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). DAPI was used to stain nuclei (1:1000, Thermo Fisher Scientific, Waltham, MA, USA).
Immunofluorescence and confocal analysis
FUS mice and age-matched controls were euthanized by CO2 and decapitated. Spinal cords were immediately dissected and post-fixed in 4% PFA for 12 h, incubated in 30% sucrose in PBS solution for 24 h at 4 °C and then cut into 30 µm thick slices with a freezing cryostat. Lumbar spinal cord slices from at least three animals per group were blocked for 1 h in 10% NDS in PBS, 0.3% Triton X-100 and then incubated 3 days at 4 °C with primary antibodies diluted in 2% NDS in PBS, 0.3% Triton X-100 and then for 3 h at room temperature with appropriate secondary antibody, diluted in the same solution. After two rinses, 10 min each in PBS, nuclei were stained with 1 µg/ml DAPI (Sigma-Aldrich) for 10 minutes.
Whole mount sciatic nerves were post-fixed in 4% PFA for 24 h, incubated with PBS at 4ºC for 48h and blocked with blocking buffer of 10% NDS in PBS, 0.3% Triton X-100 for 6 h at RT. Nerves were then incubated 3 days at 4 °C with primary antibodies diluted in 2% NDS in PBS, 0.3% Triton X-100 and then for 3 h at room temperature with appropriate secondary antibody, diluted in the same solution. After two rinses, 10 min each in PBS, nuclei were stained with 1 µg/ml DAPI for 10 minutes. Images were visualized by Nikon Eclipse TE200 epifluorescence microscope (Nikon, Florence, Italy) connected to a CCD camera. Images were captured under constant exposure time, gain and offset. After creating a region of interest, background was subtracted, and the average pixel intensity was determined. All images quantifications were done using ImageJ software (NIH, Bethesda, USA).
Western blot
Cell were lysed on plates in 2xLaemmli buffer and the lysates were boiled at 100°C for 5 min. Spinal cords, sciatic nerves and gastrocnemius muscles of at least 3 animals per group were dissected [1] and lysed in homogenization buffer (50 mM Tris HCl pH 7.4, 250 mM NaCl, 1 mM EDTA, 5 mM MgCl2, 1% Triton X-100, 0.25% Na-deoxycholate, 0.1% SDS, protease inhibitor cocktail). After 2 × 10″ sonication cycles, samples were incubated on ice and then centrifuged at 15000 × g for 20′ at 4 °C. Supernatants were then quantified with Bradford protein assay (Bio-Rad) and resuspended in Laemmli Buffer before SDS-PAGE (Sigma-Aldrich). Proteins were separated on 10% SDS-PAGE and transferred to nitrocellulose membranes, followed by incubation with 5% skimmed milk for 1 h and with primary antibodies at 4°C overnight. HRP-conjugated secondary antibodies (1:2,500, Jackson ImmunoResearch) were applied at RT for 1 h. ECL solution (Roche) was used for chemiluminescent detection. GAPDH was used as a control for equal loading. Following densitometry-based quantification and analysis using ImageJ software, the relative density of each identified protein was calculated.
S100A4 Silencing
Primary fibroblasts were seeded in 12-well plate at a density of 50,000 cells per well approximately 24 h before transfection and at the confluence of about 50%, the cells were transfected with two types of siRNAs for S100A4 (50 nM) (Thermo Fischer). A scrambled siRNA (100 nM) (Thermo Fischer) was used as a negative control. Transfection was performed using Metafectene (Biontex, Germany) following the manufacturer’s instructions. After transfection for 48 or 72 h cells were harvested for further experiments.
FUS transgenic mice
Adult Tg (Prnp-FUS) WT3Cshw/J mice expressing hemagglutinin-tagged human wild-type FUS (hFUS) were obtained from Jackson Laboratories. Animals were housed in our indoor animal facility at constant temperature (22 ± 1 °C) and relative humidity (50%) with 12-h light cycle (light 7 am–7 pm). Mice were maintained in hemizygousity on the same C57BL/6 genetic background. Hemizygous FUS mice were backcrossed to obtain homozygous mice, used as experimental subjects. Food and water were freely available. When animals showed symptoms of paralysis, wet food was given daily into the cages for easy access to nutrition and hydration. Mice were genotyped by PCR analysis of tissue extracts from tail tips. Hemizygous FUS mice were identified using PCR primers: Fwr5′-AGGGCTATTCCCAGCAGAG-3′, Rev5′-TGCTGCTGTTGTACTGGTTCT-3′. Homozygous FUS mice were genotyped by qPCR using the following primers: Fwr5′-GCCAGAACACAGGCTATGGAA-3′ and Rev5′-GTAAGACGATTGGGAGCTCTG-5′.
All animal experiments complied with the ARRIVE guidelines and were carried out in accordance with the European Guidelines for the use of animals in research (2010/63/EU) and the requirements of Italian laws (D.L. 26/2014). The ethical procedure was approved by the Italian Ministry of Health (protocol number 406/2019 PR). All efforts were made to minimize animal suffering and the number of animals necessary to produce reliable results.
Niclosamide in vitro and in vivo treatment
The inhibitor of S100A4 niclosamide (2′,5-dichloro-4′-nitrosalicylanilide) was solubilized in dimethyl sulfoxide (DMSO) for in vitro experiments. Control cells were treated with the equal amount of solvent. For the in vivo experiments niclosamide (20 mg/kg/d, dissolved in Cremophor ®) was administered daily from post-natal day 25 via intraperitoneal (i.p.) injections, when hFUS mice showed first signs of destabilized gait [30]. Control mice were treated with the appropriate volume of solvent solution. Survival was determined by the loss of righting reflex within 20 s after laying the mouse on its side [1].
Statistics
Data are reported as mean ± SEM. Two-tailed Student’s t test (for paired or unpaired samples as appropriate) or one-way ANOVA was used for statistical analysis. A p value less than 0.05 was accepted as a significant difference.