Fig 2: Truncated TrkB and p75NTR levels are increased in SOD1G93A muscles. Representative immunoblots for A BDNF, B TrkB.FL and truncated TrkB, and C p75NTR, comparing wild-type (WT) and SOD1G93A tibialis anterior (TA) and soleus (Sol) muscles. Control tissue (Ctrl) from age-matched WT spinal cord (P94) was included. The difference in migration patterns suggests that TrkB.FL, but not truncated TrkB, undergoes distinct post-translational modifications in skeletal muscles. Immunoblot quantifications: D BDNF (genotype p = 0.95, muscle ***p < 0.001, interaction p = 0.08); E TrkB.FL (genotype p = 0.476, muscle p = 0.342, interaction p = 0.507); F truncated TrkB (genotype ***p < 0.001, muscle p = 0.239, interaction p = 0.384); and G p75NTR (genotype **p = 0.008, muscle p = 0.932, interaction p = 0.14) (n = 10). Data were compared by two-way ANOVA and Holm-Šídák's multiple comparisons tests. *p < 0.05, **p < 0.01, ***p < 0.001. Means ± SEM are plotted for all graphs. Black (P73) and grey (P94) circle borders indicate age-matched mice

Fig 3: Silencing of PTPN23 leads to p75NTR accumulation in vacuole-like compartments. (A) Confocal images of a-p75NTR accumulation assay in scrambled and PTPN23 shRNA treated N2A-FLAG-TrkB cells (n=3). Cells were incubated for 30 min with anti-p75NTR antibody and chased for 30 min with 100 ng/ml BDNF. Following acid wash and fixation, receptor localisation was revealed using Alexa Fluor 555-conjugated secondary antibody. Arrowheads indicate enlarged endocytic compartments. Images show maximum intensity Z-stack projections, acquired at 0.5 µm spacing. Scale bar: 10 µm; inset: 5 µm. (B) Diameters of 100 endosomes (per n/condition) were measured and grouped using 0.5 µm binning, in three independent experiments. Endosomes were classified as endo-lysosomes (<1.5 µm) or vacuoles (>1.5 µm). ****P<0.0001, ?2 (two-tailed chi-square) test for trend (n=3). (C) Quantification of cells containing endo-lysosomes or vacuole-like compartments (n=3); cells lacking a-p75NTR accumulation (~50% of cells) were counted but excluded from the analysis. ***P<0.001, unpaired Student's t-test. (D) Transmission electron microscopy images showing three representative classes of organelles containing accumulated a-p75NTR-gold (arrowheads). Early and late endocytic compartments were indistinguishable between scrambled and PTPN23-KD cells, and representative images taken from PTPN23-KD and scrambled cells, respectively; vacuoles were observed only in PTPN23-KD cells. Scale bars: 500 nm; insets: 100 nm.

Fig 4: TrkB.T1 and p75NTR are increased in SOD1G93A sciatic nerves. A Immunoblots of TrkB.T1, p75NTR, p-ERK1/2, ERK1/2, and cofilin in WT and SOD1G93A sciatic nerves. N.B. TrkB.FL levels were below detection. Quantification: B TrkB.T1 (**p = 0.007); C) p75NTR (***p < 0.001); and D p-ERK1/2-ERK1/2 ratio (p = 0.948), by unpaired, two-tailed t-tests (n = 10). Black (P73) and grey (P94) circles indicate age-matched mice. Immunostaining of: E TrkB; and F p75NTR in axons (TUJ1) and Schwann cells (S100). White and yellow arrowheads identify TrkB/p75NTR staining in TUJ1 and S100 regions, respectively. Scale bar = 25 µm. Immunostaining quantification: G TrkB (genotype p = 0.204, cell type p = 0.239, interaction p = 0.384); and H p75.NTR (genotype *p = 0.018, cell type p = 0.053, interaction p = 0.204). Levels in axons or Schwann cells, as assessed by two-way ANOVA and Holm-Šídák's multiple comparisons tests (n = 4). Means ± SEM are plotted for all graphs. *p < 0.05, **p < 0.01, ***p < 0.001

Fig 5: TrkB and p75NTR levels are comparable at the NMJ between tibialis anterior (TA) and soleus muscles, and are not altered in disease. NMJ immunostainings of A (i) TrkB and B (i) p75NTR receptors at the pre- and post-synaptic compartments as well as in terminal Schwann cells as identified by ii) Syn/TUJ1, iii) ?-BTX, and iv) S100 staining, respectively. The boxed area in A-Bi-v ‘ is enlarged in A-Bi-v“. Scale bars in v' = 10 µm, scale bars in v" = 5 µm. Immunostaining quantifications of: C TrkB (genotype p = 0.868, muscle p = 0.109, interaction p = 0.781); and D p75.NTR (genotype p = 0.091, muscle p = 0.108, interaction p = 0.603) (n = 6). Data were compared by two-way ANOVA and Holm-Šídák's multiple comparisons tests. Means ± SEM are plotted for all graphs. Black (P73) and grey (P94) circle borders indicate age-matched mice. See also Additional file 2: Fig. S6