Animals
Mouse experiments were performed under license from the United Kingdom Home Office in accordance with the Animals (Scientific Procedures) Act (1986) and approved by the UCL Queen Square Institute of Neurology Ethics Committee. Mice were housed in individually ventilated cages in a controlled temperature/humidity environment and maintained on a 12 h light/dark cycle with ad libitum access to food and water. Transgenic mice carrying the mutant SOD1G93A transgene (TgN[SOD1-G93A]1Gur) were obtained from the Jackson Laboratory [46]. Colonies were maintained by breeding male heterozygous carriers with female (C57BL/6 × SJL) F1 hybrids. Mice were genotyped for the human SOD1 transgene using DNA extracted from ear notches and primers as previously described [5,6,7]. Female and male SOD1G93A mice display distinct patterns of disease, including differences in disease onset, progression and survival [47]. Therefore, only female hemizygous transgenic mice carrying the human SOD1G93A transgene (hereafter referred to as SOD1G93A) and WT littermates were used, allowing comparisons with our previous studies [5,6,7,8]. All experimental groups contained age-matched WT and SOD1G93A littermates to minimise the potential impact of differing oestrous cycles. For axonal transport, female WT mice had a mean age of 85.13 ± 14.97 days; we did not assess WT axonal transport separately at P73 and P94 as we have previously shown that there are no significant differences in transport between 1 and 18 months in WT mice [9, 48]. Female SOD1G93A postnatal day 73 (P73) mice had a mean age of 72.74 ± 0.67 and P94 mice had a mean age of 93.70 ± 0.46.
In vivo axonal transport
Signalling endosomes were visualised in vivo by injecting the fluorescent atoxic binding fragment of tetanus neurotoxin (HCT-555), as previously described [49, 50]. Briefly, HCT (residues 875–1315) fused to an improved cysteine-rich region was expressed in bacteria as a glutathione-S-transferase fusion protein [51], cleaved and subsequently labelled with AlexaFluor555 C2 maleimide (Thermo Fisher Scientific, A-20346). 5–7.5 µg of HCT-555 alone, or in combination with 25 ng of human recombinant BDNF (Peprotech, 450–02) or 25 ng of human recombinant GDNF (Peprotech, 450–10) (pre-mixed with phosphate buffered saline) were injected into single muscles. Briefly, after anaesthesia was initiated and maintained using isoflurane, the fur on the ventral and/or dorsal lower leg was shaved, and mice were placed on a heat-pad for the duration of the surgery. A small incision was made using iris spring scissors on the ventral surface below the patella for TA, or on the lateral aspect of the dorsal surface below the popliteal fossa for lateral head of gastrocnemius (LG). Injections were performed as a single injection targeting the motor end plate region [52] in a volume of ~ 3.5 µl using a 701 N Hamilton® syringe (Merck, 20,779) for TA and LG. For soleus injections, a vertical incision was made on the skin covering the lateral surface of lower hindlimb between the patella and tarsus to expose the underlying musculature. Subsequent vertical incisions were carefully made laterally along the connective tissue between LG and TA, and the deeper soleus muscle was exposed using forceps. 1 µl injections were performed into soleus using pulled graduated, glass micropipettes (Drummond Scientific, 5-000-1001-X10), as previously described [53]. The overlying skin was then sutured, and mice were monitored for up to 1 h. 4–8 h later, mice were re-anaesthetised with isoflurane, and the skin covering the entire lateral surface of the injected hindlimb was removed, along with the biceps femoris muscle to expose the underlying sciatic nerve. The connective tissue underneath the sciatic nerve was loosened using curved forceps to enable the placement of a small piece of parafilm aiding the subsequent imaging. The anaesthetised mouse was then transferred to an inverted LSM780 confocal microscope (Zeiss) enclosed within an environmental chamber maintained at 37 °C. Using a 40x, 1.3 NA DIC Plan-Apochromat oil- immersion objective (Zeiss), axons containing retrogradely mobile HCT-555-positive signalling endosomes were imaged every 0.3–0.4 s using an 80 × digital zoom (1024 × 1024, < 1% laser power) (Fig. 1A, Additional file 1: Video S1); movies of three to five axons per animal were acquired. All imaging was concluded within 1 h of initiating anaesthesia.
In vivo axonal transport analysis
Confocal “.czi” images were opened in FIJI/ImageJ (http://rsb.info.nih.gov/ij/), converted to “.tiff” and transport dynamics were then assessed semi-automatically (i.e., automated spot detection, followed by manual linking (see Additional file 1: Video S1)) using the TrackMate plugin [54]. Indeed, as determined by several parameters such as fluorescence intensity and diameter, the TrackMate automated spot detection method encloses all cargoes that fit the criteria within purple circles (Additional file 1: Video S1B). Endosomes selected for transport analysis were then manually connected across multiple adjacent frames (Additional file 1: Video S1C). This method provides single frame-to-frame velocities, which were then averaged across the entire run to give an average speed for each tracked endosome (as represented by an individual data point in Fig. 1C). Kymographs (Additional file 2: Fig. S2) were generated using FIJI/ImageJ to highlight axonal transport phenotypes, but were not used to assess axonal transport dynamics. Only thicker axons were selected for tracking [9]. Signalling endosomes with the following criteria were analysed: (1) organelles were tracked for a minimum of 10 and a maximum of 100 consecutive frames (i.e., ~ 3–40 s), including pauses. Terminal pausing carriers, which we defined by the absence of movement in ≥ 10 consecutive frames, were excluded; (2) for every individual axon, 15–40 signalling endosomes were tracked across at least 1000 frames (i.e., ~ 5–10 min); and (3) signalling endosome data representing an individual animal were comprised from at least three separate motor axons. The individual datapoints obtained from each experimental group can be found in Additional file 2: Table S1. Relative frequency curves were generated to display the relative frame-to-frame movements of all signalling endosomes per animal (e.g., Fig. 1B). For all mice included in the analysis, the speeds of all individual endosomes were plotted (e.g., Fig. 1C), the mean speeds of all endosomes per individual axon were averaged (e.g., Fig. 1D), and finally, the mean speed of all endosomes per animal was also averaged (e.g., triangles in Fig. 1E). Importantly, the mean values across all analyses (e.g., Fig. 1C–E) were similar. For example, for WT soleus transport, the mean speed of all individual endosomes was 2.55 µm/s (Fig. 1C), the mean endosome speed per axon was 2.51 µm/s (Fig. 1D) and the mean endosome speed per animal was 2.54 µm/s (Fig. 1E). Owing to statistical overpowering of individual endosome speed data, statistical tests were only performed on the mean endosome speeds per axon (Fig. 1D) and per animal (Fig. 1E). The fastest individual endosome speed per animal was considered as the maximum speed (e.g., represented by circles in Fig. 1E). A pause was defined by an endosome that moved less than 0.1 µm between consecutive frames, and the time paused (%) is determined by the number of pauses divided by the total number of frame-to-frame movements assessed per animal (e.g., Fig. 1F).
In vitro axonal transport
Mixed ventral horn cultures were prepared as previously described [6,7,8]. Briefly, ventral horns from E11.5–13.5 WT and SOD1G93A mice were dissociated, centrifuged at 380 × g for 5 min, seeded into two-chambered microfluidic devices (Fig. 3A) [7], and maintained in motor neuron media (Neurobasal (Gibco) with 2% B27 (Gibco), 2% heat-inactivated horse serum, 1% Glutamax (Invitrogen), 24.8 µM β-mercaptoethanol, 10 ng/ml ciliary neurotrophic factor (Peprotech, 450–13), 0.1 ng/ml GDNF (Peprotech, 450–10), 1 ng/ml BDNF (Peprotech, 450–02) and 1 × penicillin streptomycin (Thermo Fisher; 15140122)) at 37 °C and 5% CO2. After 6 days in vitro (DIV6), 30 nM HCT-555 and ± 50 ng/ml of BDNF was added to existing media for 45 min, then all media was replaced with fresh MN media containing 20 mM HEPES–NaOH (pH 7.4) ± 50 ng/ml of BDNF for time-lapse microscopy. Live imaging was performed on an inverted LSM780 confocal microscope at 37 °C using a 40x, 1.3 NA DIC Plan-Apochromat oil- immersion objective (Zeiss). Videos were taken at 2 frames/s for > 2.5 min. Videos were manually tracked using TrackMate [54] to determine endosome track dynamics (Fig. 3). The breakdown of each experimental group can be found in Additional file 2: Table S2.
In vitro TrkB and p75NTR western blot analysis
Mixed ventral horn cultures from E11.5–13.5 WT and SOD1G93A mouse spinal cords were prepared as above, and plated in MN media in a 12-well plate coated with poly-ornithine (1.5 mg/ml) and laminin (3 µg/ml). On DIV 6–7, each well was washed once in ice-cold PBS and lysates were prepared in RIPA buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM EGTA) with freshly added Halt™ protease and phosphatase inhibitor cocktail (1:100, Thermo Fisher), and incubated on ice for 30 min. Lysates were spun at 14800 rpm at 4 °C for 15 min, the supernatant was then resuspended in 4 × Laemmli sample buffer (15% SDS, 312.5 mM Tris–HCl pH 6.8, 50% glycerol, 10% β-mercaptoethanol, 0.1% bromophenol blue) and loaded into 4–15% Mini-PROTEAN® TGX Stain-Free™ protein gels (Bio-Rad). Western blotting was then performed using standard protocols. The primary antibodies used were TrkB (R&D Systems, AF1494, 1:500) and p75NTR (Biolegend, 239701, 1:500) (see Additional file 2: Table S3). Densitometry was performed using the bands at ~ 140 kDa for TrkB.FL (Additional file 2: Fig. S2Ai), ~ 75–100 kDa for truncated TrkB isoforms (Additional file 2: Fig. S2Ai) and ~ 70–85 kDa for p75NTR (Additional file 2: Fig. S2Aii). Post-immunoblotting Coomassie staining [55] (Additional file 2: Fig. S2Aiii) between 60–150 kDa and 60–100 kDa was used to estimate total protein for TrkB (full-length and truncated isoforms) and p75NTR, respectively. This total protein load was used as an internal reference to accurately quantitate protein levels, and data were then normalised using the sum of all data points per replicate [56].
Axon diameters
The axon diameters were measured following protocols established in ChAT.eGFP mice [9], using the same videos in the axonal transport analyses. Briefly, axon diameters were assessed by measuring the upper and lower positions of moving HCT-555 signalling endosomes from consecutive frames in unprocessed (i.e., not dissected, fixed, or sectioned), anatomically connected individual axons. A minimum of 10 positions were averaged for a single axon, and the mean axon diameters per animal were determined by averaging all axons from that animal (n ≥ 3 axons per animal). Similar to the in vivo transport experiments, this quantification is reliant upon intact NMJs, which can internalise HCT-555; hence, we cannot extrapolate diameters from denervated axons.
Muscle BDNF, TrkB and p75NTR western blot analysis
P73 (n = 5) and P94 (n = 5) WT and SOD1G93A mice were culled, and fresh TA and soleus muscles were immediately dissected, snap frozen in liquid nitrogen and stored at -80 °C. Protein extraction from frozen muscles was achieved by mechanically disrupting the tissue using a scalpel, followed by immersion in RIPA buffer containing freshly added HaltTM protease and phosphatase inhibitor cocktail for 15 min on ice, and then homogenised on ice using an electrical homogeniser. Lysates were incubated at 4 °C with mild agitation for 2 h, after which they were centrifuged at 21000 g for 30 min at 4 °C. 20 μl of supernatant (~ 25–40 μg of protein) was treated with 6.5% trichloroacetic acid and the resulting pellet was washed with acetone. Proteins were resuspended in 1 × Laemmli buffer and loaded on 4–12% Bis–Tris polyacrylamide gels prior to western blotting. The primary antibodies used were against BDNF (Alomone ANT-010), TrkB (Millipore, 07–225) and p75NTR (Biolegend, 839701) (all 1:1000; see Additional file 2: Table S3). Densitometry was performed on the bands at ~ 20 kDa for BDNF (Fig. 5A), ~ 140 kDa for TrkB.FL (Fig. 5B), ~ 75–100 kDa for truncated TrkB (Fig. 5B) and ~ 70–85 kDa for p75NTR (Fig. 5C). As the steady-state levels of standard housekeeping proteins, such as GAPDH and β-actin, differ between muscle types, and can be affected by age, sex, and pathology [57, 58], post-immunoblotting Coomassie staining [55] between 10–25 kDa, 70-150 kDa and 60-100 kDa was used to assess relative levels of BDNF, TrkB (full-length and truncated isoforms), and p75NTR, respectively. The total protein load was used as an internal reference to accurately quantify relative protein levels, and data were then normalised using the sum of all data points in a replicate [56]. P73 and P94 WT and SOD1G93A data points were combined as there were no timepoint-specific differences (data not shown).
Muscle immunohistochemistry (IHC)
P73 (n = 3) and P94 (n = 3) WT and SOD1G93A mice were culled, and TA and soleus muscles were immediately dissected and post-fixed in 4% paraformaldehyde (PFA) for 15–60 min. Muscle fibres were teased apart in bundles of 1–10 fibres and stained with α-bungarotoxin (BTX; Thermo Fisher Scientific, B13423, 1:500) for 1 h. Fibres were then permeabilized with 2% Triton X-100 in PBS for 90 min, then immersed in a blocking solution containing 4% bovine serum albumin and 1% Triton X-100 in PBS for 30 min at room temperature. Primary antibodies (see Additional file 2: Table S3) against TUJ1 (Synaptic Systems, 302306, 1:50), synaptophysin (Syn; Synaptic Systems, 101006, 1:50), TrkB (Millipore, 07–225, 1:50), p75NTR (Promega, G3231, 1:50) and S100 (Atlas Antibodies, AMAb91038, 1:250) immersed in blocking solution were added to the teased muscle fibres for ~ 3 d at 4 °C with mild agitation, and then washed in PBS at room temperature. Secondary antibodies (see Additional file 2: Table S4) in PBS were then applied to fibres for ~ 1 h at room temperature, followed by multiple washes in PBS and then finally mounted on SuperFrost Plus slides (VWR, 631–0108) using Mowiol. Slides were dried and imaged with a LSM780 confocal microscope using a 63 × Plan-Apochromat oil immersion objective (Zeiss). A minimum of 25 NMJs were imaged per condition, comprised of fully or partially innervated, but not denervated, NMJs. Mean fluorescence was measured using FIJI/ImageJ by applying an overlapped Syn/TUJ1-BTX mask to the TrkB or p75NTR immunolabelled regions, and the mean fluorescence per animal was assessed by averaging all the individual data points. For the TrkB analysis, we assessed the WT TA (n = 6, NMJs = 182), WT soleus (n = 6, NMJs = 223), SOD1G93A TA (n = 6, NMJs = 180) and SOD1G93A soleus (n = 6, NMJs = 191) muscles. For the p75NTR analysis, we assessed the WT TA (n = 6, NMJs = 176), WT soleus (n = 6, NMJs = 224), SOD1G93A TA (n = 6, NMJs = 183) and SOD1G93A soleus (n = 6, NMJs = 198) muscles. P73 and P94 WT and SOD1G93A data points were combined as there were no timepoint specific differences (data not shown).
Sciatic nerve western blot analysis
P73 (n = 5) and P94 (n = 5) WT and SOD1G93A mice were culled, and sciatic nerves were immediately dissected, snap frozen in liquid nitrogen and stored at − 80 °C. Thawed sciatic nerves were then immersed in NP-40 lysis buffer (150 mM NaCl, 1% NP-40, 50 mM Tris–HCl, pH 8.0) with freshly added HaltTM protease and phosphatase inhibitor cocktail (10% weight/volume) (100x, Fisher, 78442). Lysates underwent mechanical disruption using a plastic pestle before being left on ice for 0.5 h and then centrifuged for 20 min at 10,000 g. Proteins were re-suspended in 4 × Laemmli buffer, and 40 µg/sample were loaded on 4–12% Bis–Tris polyacrylamide gels prior to western blotting. Primary antibodies (see Additional file 2: Table S3) against TrkB (Millipore, 07–225, 1:1000), p75NTR (Biolegend, 839701, 1:2000), ERK1/2 (CST, 9102, 1:1000), p-ERK1/2 (CST, 9101, 1:1000), AKT (CST, 9272, 1:1000), p-AKT (CST, 9275, 1:1000) and Cofilin (Cytoskeleton, ACFL02, 1:500) were used to quantify protein levels. N.B. The expression of TrkB.FL, AKT and p-AKT were below detection levels. All bands were first standardised to cofilin, and then normalised by the sum of all data points in a replicate [56]. P73 and P94 WT and SOD1G93A data points were combined as there were no timepoint specific differences (data not shown).
Sciatic nerve IHC
P73 (n = 4) WT and SOD1G93A mice were culled, and sciatic nerves were immediately dissected, post-fixed in a 4% PFA solution in PBS overnight at 4 °C, cryopreserved in a 30% sucrose solution in PBS for 2 d at 4 °C, and finally frozen in OCT (Agar Scientific, AGR1180). 30 µm longitudinal cryosections of sciatic nerves were directly mounted on SuperFrost Plus slides (VWR, 631–0108), and a hydrophobic barrier pen (Vector Laboratories, H-4001) was then applied to the slides surrounding the sectioned tissue. PBS rehydrated tissue was then blocked using 10% normal horse serum in 0.2% Triton X-100 in PBS for ~ 1 h and then primary antibodies (see Additional file 2: Table S3) specific for S100 (Merck, S2532, 1:200), TUJ1 (Synaptic systems, 302306, 1:500), TrkB (Millipore, 07–225, 1:250) and p75NTR (Promega, G3231, 1:500) were applied overnight at room temperature. After multiple PBS washes, the secondary antibodies (see Additional file 2: Table S4) in PBS were applied for 2–3 h, followed by multiple washes and then mounted with Mowiol. Slides were dried and imaged as described above. A minimum of six sciatic nerve sections were imaged per condition. Mean fluorescence was measured by applying a TUJ1 and S100 mask to the TrkB or p75NTR regions, and the mean fluorescence per animal was assessed by averaging all the individual data points.
Statistical analyses
GraphPad Prism 9 (GraphPad Software) was used for statistical analyses. Normal distribution was first ascertained by the D’Agostino and Pearson omnibus normality test, and parametric data were statistically assessed using unpaired, two-tail t-tests, one-way or two-way analyses of variance (ANOVA) with Holm-Sidaks multiple comparison tests. Non-normally distributed data were analysed by a two-tailed Mann–Whitney U test or Kruskal–Wallis test with Dunn’s multiple comparisons test.