Animals, Genotyping, and Breeding

Transgenic mice used in this study were as follows: (1) ALS mouse model: B6SJL-Tg (SOD1G93A) 1Gur/J (SOD1G93A) mice (referred to as SOD1G93A) obtained from Jackson Laboratory (Bar Harbor, ME, USA) with overexpression of human SOD1-G93A (named “fast” [SOD1G93A-f]), resulting in a mean lifespan around 160 days (LIT) [32, 33]; and (2) NRG1 transgenic mice: C57Bl6-Tg (Thy1-Nrg1*III)1Kan+/− that overexpress an N-terminally HA epitope-tagged NRG1 type III isoform (hereafter referred to as NRG1) under control of the Thy1.2 promoter, which is active in postnatal MNs [29]. Transgenic lines were maintained as hemizygotes by crossing transgenic males with non-transgenic females on a B6 background. For the generation of double transgenic NRG1-SOD1G93A mice, NRG1 type III females and SOD1G93A males were crossbred. Animals were housed in the Animal Facility of Universitat de Lleida and kept in a strictly controlled environment (12-h light/dark cycle and 20 ± 2 °C of room temperature). Chow and water were provided ad libitum.

Genomic DNA was extracted from a tail biopsy using the Phire Kit (ThermoFisher Scientific, Waltham, MA). The genotype of the offspring was identified using PCR with the following primers: 5′-GGCTTTCTCTGAGTGGCAAAGGACC-3′ for the forward HANI-Nrg1 transgene and 5′-GTCCACAAATACCCACTTTAGGCCAGC-3′ for reverse HANI-Nrg1 transgene. For SOD1 genotyping, the following primers were used: IMRO 113, 5′-CATCAGCCCTAATCCATCT-3′ and IMRO 114, 5′-CGCGACTAACAATCAAAGTGA-3′.

All animal experimentation procedures were performed according to the European Committee Council Directive, the Animal Care and Use and Biosecurity Committees of the Universitat de Lleida, and the norms established by the Generalitat de Catalunya (Diari Oficial de la Generalitat de Catalunya (DOGC) 2073, 1995).

Tissue Sample Preparation, Immunohistochemistry, and Imaging

Animals were deeply anesthetized with inhaled isoflurane and transcardially perfused with a physiological saline solution followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB) at pH 7.4. The lumbar spinal cord and L4 ventral roots were dissected. Spinal cord samples were then postfixed for 24 h at 4 °C in the same fixative solution and then cryoprotected at 4 °C with 30% sucrose in 0.1 M PB containing 0.02% sodium azide. Transverse cryostat sections (16 µm thick) were collected on gelatine-coated glass slides.

Cryostat sections were permeabilized with phosphate-buffered saline (PBS) containing 0.1% Triton X-100 for 30 min, blocked with either 10% normal goat serum or normal horse serum in PBS for 1 h at room temperature (RT), and then incubated overnight at 4 °C with an appropriate primary antibody mixture. The primary antibodies used are as follows: ionized calcium-binding adapter molecule 1 (Iba1, 1:500, goat polyclonal, Abcam (ab5076)), CD68 (rat anti-mouse CD68,1:500 Bio-Rad MCA1957T), SOD1 (C4F6) Anti-misfolded human SOD1 (1:100, mouse monoclonal, MediMabs 2B Scientific (MM-00070-2-P)), glial fibrillary acidic protein (GFAP, 1:1000, chicken polyclonal, Abcam (ab4674)), NRG1 (1:300, rabbit polyclonal, Santa Cruz (sc-348)), and vesicular acetylcholine transporter (VAChT, 1:500, guinea pig polyclonal, Synaptic Systems (139 105)).

Once previously washed with PBS, sections were incubated for 1 h with a combination of appropriate secondary fluorescent antibodies labelled with one of the following fluorochromes (1/500): Alexa Fluor 488, DyLight 549, or DyLight 649 (Jackson Immuno Research Laboratories, West Grove, PA, USA). Finally, the spinal cord sections were labelled with blue fluorescent NeuroTrace Nissl staining (1:150; Molecular Probes) and mounted using an anti-fading medium containing 0.1 M Tris-HCl buffer (pH 8.5), 20% glycerol, 10% Mowiol, and 0.1% 1,4-diazabicyclo[2,2,2]octane. The slides were then examined under a FluoView FV-500 or FluoView FV-1000 Olympus Laser-Scanning Confocal Microscope (Olympus, Hamburg, Germany). The MNs were imaged after obtaining optical sections (0.5 or 1 μm) of cell bodies. For comparisons, slides from different animals and experimental conditions were processed in parallel for immunocytochemistry and subsequent imaging. The same scanning parameters were used for the acquisition of images corresponding to different experimental groups. Digital images were analysed using the FV10-ASW 3.1 Viewer (Olympus) and the ImageJ software (US National Institutes of Health, Bethesda, MD, USA). Immunolabelled profiles of the different protein markers were examined and then manually counted on the screen for each MN soma. The area and perimeter of MN somata and both microglial and astroglial profiles physically close to MNs were also manually measured. The digital images were edited using the FV10-ASW 3.1 Viewer (Olympus) and Adobe Photoshop CS4 (Adobe Systems Inc., San Jose, CA).

Electron Microscopy and Ventral Root Axon Counting

Animals were perfused with 2% PFA and 2% glutaraldehyde in 0.1 M PB (pH 7.4) for conventional electron microscopy. Dissected tissues and the ventral roots were postfixed for 24 h at 4 °C in the above fixative solution, and, if needed, they were sectioned at 200 µm using a vibratome. The tissues were postfixed in 1% OsO4 for 2 h and then contrasted with 0.5% uranyl acetate for 30 min. Samples were processed for Embed 812 (Electron Microscopy Sciences, Hatfield, PA, USA) epoxy resin according to standard procedures. Semithin transversal sections (1 µm thick) were stained with Richardson stain and imaged using an Olympus ×60/1.4NA PlanApo oil immersion objective (Olympus) and a DMX 1200 Nikon (Tokyo, Japan) digital camera. Ultrathin sections were counterstained with Reynold’s lead citrate. All observations were performed on a transmission electron microscope JEOL JEM 1010 (Akishima, Tokyo, Japan).

SOD1 Dot and Western Blotting

Frozen lumbar spinal cords were fragmented and homogenized using an electric homogenizer (Tissue Grinder) in ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer (150 mM NaCl; 1% NP-40; 0.5% Na-deoxycholate; 0.1% SDS; 50 mM Tris-HCl [pH 7.4]), supplemented with protease inhibitor (Sigma-Aldrich, cat # P8340) and PhosSTOP (Roche). Homogenized samples were centrifuged at 800×g for 10 min at 4 °C. The protein concentrations of the supernatants were determined by BIO-RAD Micro DC protein assay (BIO-RAD, Laboratories, Inc.). For SOD1 dot blotting, 25 µg of protein were loaded in the wells of the Bio-dot protein blotting (BIO-RAD) and transferred by gravity filtering to a nitrocellulose membrane. Dot blot membranes were blocked in 3% BSA in Tris-buffered saline pH 8 (TBS), filtering it through the wells by gravity. For SOD1 western blotting, samples containing 25 µg of protein were heated at 100 °C for 5 min with an equivalent volume of sample buffer (containing 8% SDS and 2% mercaptoethanol) and loaded onto a denaturing 10% sodium dodecyl sulphate-polyacrylamide gel. The proteins were electrotransferred to a nitrocellulose membrane in Tris-glycine-methanol buffer. The membrane was blocked for 1 h at RT in a blocking solution mixture of 5% nonfat dry milk, 0.1% Tween 20, and TBS. Immunodetection was done by incubating the membranes overnight at 4 °C with pan-SOD1 (1:1000, sheep, Calbiochem (574597)), mouse monoclonal anti-actin (1:5000, Sigma-Aldrich, cat. # A5441), and rabbit polyclonal anti-GAPDH (1:10,000, Abcam, (ab181603)), the latter used for loading controls. Membranes were washed in TBS and incubated with the appropriate peroxidase-conjugated secondary antibodies (1:20,000, Cell Signaling, (cat. # 7076)) for 60 min at RT, washed in TBS tween (TBST), and visualized using the ECL Prime Western blotting Detection Reagent detection kit (GE Healthcare), as described by the manufacturer. Quantification of spot densities was performed using a Chemi-Doc MP Imaging System (BIO-RAD Laboratories, Inc.).

NRG1 Signalling Western Blotting

Sucrose homogenization buffer (320 mM Sucrose, 10 mM Tris (pH 7.4), 1 mM NaHCO3, and 1 mM MgCl2) with protease and phosphatase inhibitors (Roche) was used for protein extraction. Proteins were separated on 8% SDS-polyacrylamide gels and blotted onto PVDF (Hybond-P, Invitrogen). Membranes were incubated with primary antibodies: actin mouse 1:1000 (Millipore), GAPDH mouse 1:1000 (Invitrogen), ErbB2 rabbit 1:500 (Cell Signaling), p-HER2/ErbB2 (Tyr877) rabbit 1:500 (Cell Signaling), ErbB4 rabbit 1:500 (Abcam), p44/42 MAPK (Erk1/2) rabbit 1:1000 (Cell Signaling), p-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) rabbit 1:1000 (Cell Signaling), and Neuregulin-1a/b1/2 rabbit 1:250 (Santa Cruz), overnight at 4 °C. Membranes were washed 3× in 1× TBS-Tween and incubated in secondary antibodies (Alexa Fluor goat anti-mouse 680 1:10,000 (Invitrogen), Alexa Fluor goat anti-mouse 790 1:10,000 (Invitrogen), Alexa Fluor goat anti-rabbit 680 1:10,000 (Invitrogen), Alexa Fluor goat anti-rabbit 790 1:10,000 (Invitrogen)) for 1 h at RT, developed in an ODYSSEY DLx (LI-COR). Densitometric analysis of band intensities was carried out using ImageJ software.

Phenotypical Score and Behaviour Analysis

Behavioural tests were performed every 10 days starting at post-natal (P) day 90 until P160. When they reached their end stage (ES), animals were euthanised. ES is determined by the Righting Reflex (RR) test. In the ES, mice that are placed on their sides cannot right themselves to the sternum in 30 s. The following behavioural tests were performed: (1) clinical score, (2) catwalk, and (3) rotarod. A clinical score (CS) test is a visual observation of the phenotypical condition of the animal. When the animal is not visibly affected, it receives a CS of 4. When paralysis of the first hindlimb appears, the CS is 3. The time the animals achieve this value is considered the disease onset. A CS of 2 is assigned when second-limb paralysis occurs. Finally, when the animal shows signs of symmetrical paralysis, the CS is 0 [34]. CatWalk XT (Noldus, Wageningen, Netherlands) was used to assess the motor profile of mice. This test consists of an enclosed walkway on a glass plate that is traversed by a mouse from one side of the walkway to the other. Data showing the gait pattern are acquired using an electronic device and analysed with the appropriate software. As the software showed some limitations for the study of later stages of a neurodegenerative disease like ALS, the interpodal distance was measured until the animal was able to complete the test. The rotarod (Ugo-Basile, Gemonio, Italy) test was performed to evaluate mouse motor performance. Animals were placed onto the rod, which turned at 4 rpm. The test was performed 3 times for each individual, and the longest latency without falling was recorded, with an arbitrary cut-off time of 180 s.

Statistical Analysis

The data are expressed as means ± SEM. The statistical analysis was assessed by a Student’s t-test or by one- or two-way analysis of variance (ANOVA) followed by a post hoc Bonferroni’s test. The level of significance was established at p ≤ 0.05. GraphPad Prism 6 software was used for statistical analysis and graph presentations of data.