Human spinal cord tissue processing
Frozen sections of lumbar spinal cord were obtained from the Victorian Brain Bank (Australia) and the Multiple Sclerosis (MS) Society Tissue Bank (UK). Tissue samples were stored at -80°C until processed for analysis. Procedures involving handling of post-mortem human tissue were approved by a University of Melbourne Human Ethics Committee (Project ID 1238124). Briefly, human spinal cord tissue samples were homogenised in a tris(hydroxymethyl)-aminomethane-buffered saline (TBS)-based homogenisation buffer and the TBS-insoluble material was collected by centrifugation (21,000 g, 4°C, 15 min). This TBS-insoluble material was then resuspended in TBS-based homogenisation buffer supplemented with 1% (v/v) triton X-100 detergent and the samples centrifuged (18,000 g, 4°C, 5 min) to produce Triton X-100 soluble protein extracts which were assessed for protein content using the Pierce BCA Protein Assay kit, and the protein concentrations across the samples were normalised to a consistent protein concentration by diluting with the Triton X-100 supplemented homogenisation buffer. 10 µg protein samples was separated by gel electrophoresis was performed as previously described (42).
All mouse experiments in this study complied with the National Health and Medical Research Council code for the care and use of animals for scientific purposes and were approved by the University of Melbourne Animal Ethics Committee (Project Number: 1413304). Mice were housed on a reverse 12/12-hour light/dark cycle with access to feed and water ad libitum. The experimenter was blinded to age and genotype until completion of the behavioural testing. The SOD1-G37R, line 42, stock # 008342 (43), were sourced from The Jackson Laboratory (Bar Harbor, USA), are on a C57BL/6J background and are hemizygous for the SOD1-G37R transgene. The SOD1-G37R mouse colony was maintained by breeding the hemizygous SOD1-G37R mouse with a non-transgenic C57BL/6J (wild-type, WT) and identifying the hemizygous SOD1-G37R mouse by PCR genotyping. For the SOD1-G37R:APLP2-/- mouse line, the F1 breeding strategy involved mating the SOD1-G37R mouse with a APLP2-/- mouse to generate the WT:APLP2+/- and SOD1-G37R:APLP2+/- progeny at a ratio of 1:1. The F2 breeding strategy involved mating WT:APLP2+/- and SOD1-G37R:APLP2+/- mouse to generate six possible genotypes of equal weighting- 1) SOD1-G37R:APLP2+/+, 2) SOD1-G37R:APLP2+/-, 3) SOD1-G37R:APLP2-/-, 4) WT:APLP2+/+, 5) WT:APLP2+/-, 6) WT:APLP2-/- at 1:6 ratios.
Mice monitoring and end-stage disease
Mouse body weights were recorded weekly and from three weeks of age (at weaning age), the health monitoring frequency was increased to three times per week and then to daily after 25 weeks of age as the MND symptoms became more progressive and severe. Mice were killed when they reached pre-symptomatic (12 weeks), symptomatic (22 weeks) and End-stage (~28 weeks) and similar numbers of control WT mice at similar ages were killed at the same timepoints for direct comparison. End-stage is defined when the mouse met displayed one or more of the following criteria points- no longer able to perform the rotarod task, unable to right itself within 15 sec of being placed on either side, when paralysis was observed in at least one hindlimb or if they lost 15 % of their maximum recorded weight. The same number of WT:APLP2+/+ and WT:APLP2-/- were killed at the same time for age matching. Mice were killed by an intraperitoneal injection of a cocktail of xylazine (16 mg/kg body weight) and ketamine (120 mg/kg body weight) and followed by opening of the pneumothorax and transcardial perfusion with PBS solution.
Neurological scoring in mouse
For each mouse, a neurological score was calculated for both hindlimbs. The neurological scores were assigned using an amended scaling system of the neurological scoring system developed at the ALS Therapy Development Institue (44). Briefly, a score of 0.5 to 1.0 was assigned when the onset motor symptoms, as defined by the mouse displaying tremoring of hind legs and partial collapse of the leg extensions from the lateral midline when suspended by its tail, a score of 1.5-2.0 was assigned when the mouse displayed a complete or partial collapse of hindlimbs, signs of hindlimb muscle atrophy or forelimb tremoring, a sign of toes curling during the tail suspension test or if they curled under at least twice during a 30 centimetre walk or if any part of a foot was dragging along the cage bottom/table), a score of 2.5-3.0 assigned when the mouse developed a very wobbly gait, prominent signs of muscle atrophy in the hindlimb, rigid paralysis or minimal joint movement, foot not being used for generating forward motion or unsteady when walking, and a score 3.5-4.0 assigned when they displayed rigid paralysis and no forward motion, paralysis in one or more limbs, or the mouse cannot right itself within 15 seconds after being placed on either side.
Locomotor function was assessed using the rotarod instrument (IITC Life Science, Woodland Hills, CA, USA) set to 4 rpm and increasing to 40 rpm over 180 seconds. Prior to testing day, mice were habituated to the instrument for two consecutive days and allowed to explore the apparatus set at a constant speed of 10 rpm for 300 seconds. Following habituation, mice were trained on the rotarod for three consecutive days with the rotation speed initially set to 4 rpm and increasing to 40 rpm over 180 seconds. Mice that were still on the rotarod after 180 seconds were recorded as having no detectable locomotor deficit. Mice that could not continue on the rotarod for the 180 seconds would fall safely on to a padded base. The fall latency was recorded by the experimenter and is defined as the physical fall off the rotarod or if their hindlimbs slip off and they use their front paws remain grasping to the rotarod. Rotarod performances were performed and recorded two days per week, with three trials on each day of testing and intervals of 10 min of rest provided between each test trial. The mice were tested from 8 weeks of age up until the disease End-stage and matching time points in control mice.
Analysis of the mice’s gait was conducted using the DigiGait instrument and analysis performed using the supplied software analysis (Mouse Specifics, Inc, USA). Prior to testing the mice on this instrument, they undergo a pre-training period for 3 days to acclimatize to the instrument and a range of treadmill speeds were tested to determine the maximum speed the mice could tolerate safely. After the training period, DigiGait test recordings were taken for each mouse weekly from 7 weeks of age. Mice were placed on the DigiGait treadmill and setting the belt speed at either 10, 15, 20 cm/s and a 15 second recording taken.
Mouse Tissue collection
Mice were killed and tissues were collected when SOD1-G37R mice reached pre-symptomatic (12 weeks), symptomatic (22 weeks) and end-stage (~28 weeks) and a similar number of control WT mice of similar ages were killed at the same timepoints for direct comparison. For biochemical analysis, the brain, spinal cord, and hindlimb skeletal muscle organs were dissected free, immediately snap frozen in liquid nitrogen and then transferred to clean tubes for storage at -80C until processing. For histological analysis, the spinal cord tissue was carefully removed from the spinal cord canal and the lumbar region identified and dissected free and postfixed in freshly prepared 4% paraformaldehyde in PBS solution overnight then cryo-protected by incubation in a 30% sucrose solution (v/v in PBS) for at least 24 hours. The harvested lumber spinal cord or hindlimb skeletal muscle (which were laid flat on the end of a syringe) were embedded in Optimal Cutting Temperature compound (Tissue-Tek; Sakura FineTek) and snap frozen in a bath of isopentane pre-chilled in liquid nitrogen and then wrapped in plastic and stored at -80C until cutting for histochemistry.
Mouse tissue processing for biochemical analysis
Brain and spinal cord tissue samples were weighed and lysed at 1:5 w/v in brain lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% TritonX-100, pH 7.5) by passaging the tissue several times through a series of 18G and 22G needles followed by a homogenization step using a chilled handheld homogenizer that was kept on ice during the procedure. Harvested muscle samples were weighed, minced into very small pieces using a clean sterile scalpel blade while on a petri dish resting on ice, then transferred to a centrifuge tube and muscle lysis buffer added to 1:5 w/v (50 mM Tris-HCl, 150 mM NaCl, 0.1% TritonX-100, 0.1% SDS, 10 mM EDTA, 1 mM dithiothreitol). The tubes were placed in a chilled sonicating water bath and sonicated with three 30 second bursts. To complete homogenization, all homogenate samples were incubated on ice for 20 min then centrifuged at 15,870 g for 30 min at 4°C. The supernatant was transferred to a new tube and the protein concentration quantified using the bicinchoninic acid assay (Pierce, Rockford, IL, USA). Lysis buffer was added to the samples to normalize protein concentration across the samples to 2 mg/ml before gel electrophoresis. Proteins were resolved by SDS-PAGE electrophoresis under reducing and denaturing conditions by pre-mixing equivalent amounts of protein samples with 2X Tris-glycine SDS sample loading buffer (0.16M Tris, 4% SDS, 20% glycerol, 0.04% bromophenol blue). Samples were heated at 95°C for 5 min, allowed to cool for 5 min followed by a quick centrifugation step then loaded into 4-12% NuPAGE Bis-Tris pre-cast gel and the gels were run according to manufacturer’s instructions (Invitrogen, Australia). Resolved gels were transferred onto nitrocellulose membranes (BioRad, Australia) using the wet tank blotting system (BioRad, Australia).
Western blot analysis
Briefly, the membranes were incubated in blocking buffer (5% skim milk in PBST (0.05% Tween-20 in PBS) for one hour at room temperature and then probed using an anti-APP antibody 22C11 (APP 66-81) produced in house(45) and an anti-APLP2 95/11 (is a rabbit polyclonal antiserum raised against recombinant APLP2(28-693) protein (46) and expressed in Pichia pastoris (as previously described) (47). GAPDH was detected using Cell Signaling Technology antibody #2118, with HRP-linked anti-rabbit IgG (Cell Signaling Technology, 7074). Blots were incubated in primary antibody diluted in PBST overnight at 4°C, The next day, the membranes were washed in PBST buffer, incubated with a secondary antibody conjugated to horseradish peroxidase for two hours at room temperature and washed in TBST. Immunoreactivity was detected by using the enhanced chemiluminescence reagent (ECL-plus, GE Healthcare, UK) and imaged on a ChemiDoc digital imaging system (BioRad, Australia). Protein expression levels were quantitated by densitometry analysis of band intensities using Image J/Fiji software (ver. 1.52e, NIH). The intensity value for each immune-reactive band was normalised to its corresponding housekeeping loading control to account for variability in protein loading across samples.
Immunohistochemistry of mouse spinal cord sections
Serological transverse tissue sections (1:10) of the lumbar spinal cord were cut at 20 µm thickness (serially) using a cryostat machine (Leica) and were collected and adhered to superfrost plus slides (Fisher Scientific). Immunohistochemistry was prepared by briefly washing sections with PBS buffer three times (5 min per wash), permeabilised (0.3% Triton X in block buffer for 20 min) and blocked (10% goat serum in PBS) for 1 hour. Tissue sections were incubated with primary antibodies to GFAP (Merck Millipore, 1:500), IBA1 (Wako, 1:500), APP (22C11, 1:50, produced in-house) diluted in blocking buffer overnight at 4°C in a humidifier chamber. The next day, antibodies were removed and slides washed with PBS buffer (three times, 10 min each) before incubation in a secondary goat anti-mouse or goat anti-rabbit antibody (conjugated to horseradish peroxidase (HRP)) for 2 hours at room temperature. Tissue sections were PBS washed (three times, 10 min each) then incubated in DAB enhancement solution (ImmPACT DAB peroxidase-HRP substrate, SK-4105, Vector labs, Australia). Tissue sections were rinsed with distilled water three times then dehydrated in a series of ethanol solutions, cleared in xylene solution two times and mounted in Safety mounting medium (Trajan, Grale). Spinal cord sections were imaged using a digital slide scanner (Panoramic SCAN II, 3Dhistech, Hungary) using a Carl Zeiss Plan Apochromat 20×/NA 0.8 objective (Zeiss, Germany) and the acquired images of tissue sections were viewed using Case Viewer software (ver 2.2, 3Dhistech, Hungary).
Nissl staining of Mouse Spinal cord tissue sections and neuron analysis
Nissl stains were performed to assess neurons in the lumbar sections of the spinal cord. 20 µm thick frozen tissue sections were air dried at RT for 1 h on glass slides, washed in PBS then soaked in 1:1 v/v alcohol/chloroform solution overnight, then rehydrated in a series of ethanol solutions (100%, 90% and 70%) and distilled water. Slides were stained with 0.1% cresyl violet solution (0.1g cresyl violet acetate, 100 ml distilled water, 0.3 ml glacial acetic acid) for 1 hour in a 37 ºC oven. The slides were rinsed in distilled water for three seconds and dehydrated in a series of ethanol solutions (70%, 90% and 100%), cleared in xylene solution two times (5 min each) and mounted in Safety mounting medium (Trajan, Grale). The spinal cord sections were imaged using a digital slide scanner Panoramic SCAN II machine using a Carl Zeiss Plan Apochromat 20×/NA 0.8 objective (Zeiss, Germany). The acquired images were viewed using Case Viewer software (ver 2.2, 3Dhistech, Hungary). For each Nissl stained spinal cord section, the left and right ventral horn regions were selected as regions of interest (ROIs). Neurons were analysed using the manual thresholding command, followed by cell segmentation and particle analysis with neurons having a soma diameter of less than 10 µm excluded from further analysis. A total of seven spinal cord sections were analysed for each animal and the distance between each serial section analysed was at least 200 µm apart.
Neuromuscular junction assessment in mouse tissue
Serological longitudinal sections (1:5) of TA muscle cut at 10 µm thick were collected on superfrost plus slides (Fisher Scientific). Muscle sections prepared for immunofluorescence were fixed in 4% PFA for 1 hour and briefly washed with PBS buffer three times (5 min per wash). All tissue sections were incubated with permeabilisation buffer (0.3% TritonX-100, 10% goat serum in PBS) for 60 min, followed by blocking buffer (10% goat serum in PBS) for 60 min and then in primary antibody diluted in blocking buffer (see Table 2.5 for antibody list) in a humidifier chamber overnight while at 4°C. The following day, diluted antibody was removed, sections washed with PBS buffer three times (10 min per wash) then incubated in a fluoro-tagged secondary antibody (goat anti-mouse or goat anti-rabbit) prepared in blocking buffer and containing DAPI (2 µg/mL) for 1 hour at room temperature and in the dark. Neuromuscular junctions (NMJs) were visualised by adding FITC-alpha-bungarotoxin (CFTM 488A, Biotium, Australia) at 1µg/ml diluted in blocking buffer solution. All tissue sections were washed with PBS (three times, 10 min each) and then covered with antifade mounting medium (Prolong Gold, Invitrogen) and mounted with a glass coverslip. The slides were air dried at room temperature for at least 24 hours in the dark before imaging. The stained tissue sections were visualised through a 20X objective using a Zeiss Axioplan 2 microscope and images taken with a Coolscope snap camera and Zen 2 software (Zeiss, Germany). The same exposure settings were used for all mice genotypes and exported in a TIF format, and the fluorescence intensity levels quantified using Image J/Fiji software (ver. 1.52e, NIH).
The innervation status of each NMJ was assessed by determining the level of co-localisation between the muscle fibre end plate (α-bungarotoxin, green) and axon terminal of motor neuron (synaptophysin, red). Co-localisation levels were measured using Imaris software (ver. 9.1, Bitplane, USA) and greater than 150 NMJs were examined in each animal for analysis. For each NMJ, two separate surfaces were created for each channel to allow the creation of a co-localisation surface containing the overlapped region of the two surfaces. The volume of the three surfaces was used to calculate the percentage of co-localization or NMJ innervation.
ATPase staining for mouse muscle tissue
The muscle fibre types in the gastrocnemius (GA) muscle located in the mouse hindlimb, were analysed by histochemical staining for myosin ATPase activity based on a previously published protocol (48). Briefly, serological transverse sections (1:10) of the GA muscle was cut at 20 µm thickness and adhered to Superfrost plus glass slides (Fisher Scientific) by incubation at room temperature for 1 hour. Slides were washed in PBS then fixed in 4% paraformaldehyde solution (prepared in PBS) for 1 hour and then washed with PBS three times. Two sets of muscle slides were incubated in Myosin ATPase activity buffer with 1 slide set incubated in buffer at pH 4.3 and the second slide incubated in a buffer set at pH 10.2. For Myosin ATPase pH 4.3 treatment, slides were pre-incubated in 0.1M acetate buffer at pH 4.3 for 10 min and for Myosin ATPase pH 10.2, slides were pre-incubated in Baker’s Formal Calcium solution for 1 min. Slides from both sets were then briefly washed in distilled water and placed in sodium barbital solution and incubated at 37°C for 30 min. Slides were brought to room temperature and washed in distilled water three times, incubated in fresh 1% CaCl2 for 10 min followed by incubation in 2% CoCl2 for 10 min. Slides were washed thoroughly in distilled water and the colour allowed to develop for 15 seconds in freshly prepared 1% ammonium sulfide solution in a fume cupboard and then slides were washed with distilled water. Slides were dehydrated through the series of 70%, 90% and 100% alcohol solutions, cleared with xylene and finally mounted in safety mounting medium (Trajan, Grale) with a glass coverslip.
Mouse Muscle fibre typing analysis
Stained muscle sections were imaged using a Zeiss Axioscope 2 light microscope through a 10X objective and images were acquired using Axiocam 503 colour camera with Zenpro software 2011 (Zeiss, Germany). Acquired images were exported in TIF format and imported into Image J/Fiji software (ver. 1.52e, NIH) for further analysis. Total fibre numbers and the composition of each fibre type group were counted manually using the cell counter function. For each muscle fibre type, the cross-sectional area of each muscle fibre cell was measured using ROI function. Over 150 muscle cells were measured per animal.
All data are expressed as mean ± SEM with p values of 0.05 or less considered as significant. A student’s t-test was used to compare between two experimental groups. Multiple comparisons were assessed using a one-way analysis of variance with Bonferroni’s post-hoc test to compare different genotypes within the same sex groups. A two-way analysis of variances was used when assessing between different genotypes, age and sex, followed by Tukey’s post-hoc tests. To determine the time in which mice showed a reduction in their rotarod performance, a split line regression model using GenStat (ver. 6, VSN International, UK) analysis package was used to fit the performance curve for each animal followed by Student’s t-test using GraphPad Prism software (Ver. 7, San Diego, CA, USA) to compare between 2 sex groups. All other statistical analyses were performed using GraphPad Prism software.