Fig 2: Extended ex vivo injury selectively targeting glial membranes results in secondary axonal degeneration.Triangularis sterni nerve–muscle preparations from Glial (A) and WT (B) mice were treated ex vivo with anti-GM1 Ab or anti-sulfatide Ab, respectively, and a source of complement (injury, Inj) or Ab alone (control, Con) for 20 hours. (A) Anti-GM1 Ab (orange) and complement (green) deposition along the distal motor nerve was strongly enriched at the paranodes (arrowheads) in injured compared with control tissue. Loss of axonal integrity along the distal nerve was monitored by presence of neurofilament H immunostaining (NFH, magenta) and cytosolic CFP (blue). (B) The experiment was repeated in WT mice using an anti-sulfatide Ab; the results reflect those reported in A. Scale bars: 10 μm (A) and 20 μm (B). Results are represented as the mean ± SEM. n = 3/treatment: 25–54 NoRs/mouse (median = 39) were analyzed. **P < 0.01, ***P < 0.001 compared with control by 1-tailed Student’s t test.

Fig 3: Distal motor nerve axonal integrity remains intact following selective glial membrane targeting in vivo.WT, Neuronal, and Glial mice were dosed i.p. with 50 mg/kg anti-GM1 Ab followed 16 hours later with 30 μL/g normal human serum (NHS) (injury, Inj) or NHS only (control, Con). Respiratory function was monitored and diaphragm distal nerves assessed by immunoanalysis 5 hours after NHS delivery. (A) Injured Neuronal mice displayed the most severe respiratory phenotype: a pinched, wasp-like abdomen (arrowheads) and significantly reduced tidal volume (TV) measured using whole-body plethysmography (EMMS). Injured Glial mice also had significantly reduced TV compared with baseline. Representative respiratory flow charts for each treatment group show reduced TV and an increase in respiratory rate. Serum analysis indicates that circulating anti-GM1 Ab could be detected in Neuronal and Glial but not WT mice. Results are represented as the mean ± SEM, n = 4/genotype/treatment. (B) Complement deposition and axonal integrity (neurofilament H [NFH] occupancy) were compared at the diaphragm motor nerve terminals (MNTs) and along distal nerves. Representative images illustrate complement deposits (green) overlying the MNT, identified by bungarotoxin (BTx, orange), in injured Neuronal mice, and on the distal nerve in injured WT and Glial mice. Scale bar: 10 μm. Results are represented as the mean ± SEM. n = 4/genotype/treatment: 68–133 MNTs/mouse (median = 103) and 7–30 NoRs/mouse (median = 15) were analyzed. *P < 0.05, **P < 0.01, ***P < 0.001 by repeated measures 2-way ANOVA with Bonferroni post-hoc tests (A) or 2-way ANOVA with Tukey post-hoc tests (B).

Fig 4: Distal motor nerve integrity following selective targeting and acute injury of neural membranes ex vivo.Triangularis sterni nerve–muscle preparations from WT, Neuronal, and Glial mice were treated ex vivo with anti-GM1 Ab and a source of complement (injury, Inj) or anti-GM1 Ab alone (control, Con). (A) Loss of axonal integrity due to injury at the motor nerve terminal (MNT, identified by α-bungarotoxin, BTx, orange, asterisk) and node of Ranvier (NoR, orange, arrowheads) was monitored by presence of neurofilament H immunostaining (NFH, magenta). Membrane attack complex (MAC) complement pore deposition (green) was present in all injured preparations compared with control. (B) Ethidium homodimer–positive (EthD-2–positive, orange) cells overlying MNT (magenta, asterisk) were compared among treatment groups. Representative images show the presence of complement deposition (green) in all injured tissue. (C and D) The sites where ankyrin B (AnkB) or AnkG immunostaining should be located are indicated by arrowheads. The presence of normal (black bars, statistical comparisons indicated with asterisks) or abnormal (gray bars) AnkB and AnkG immunostaining was compared to associated controls for each genotype. A lengthened gap between AnkB domains is shown in a representative image from injured Neuronal tissue. Weakened, uneven AnkG staining in injured Glial tissue is shown in the representative image. Scale bars: 20 μm (A), 50 μm (B), and 5 μm (C and D). Results are represented as the mean ± SEM. n = 3/genotype/treatment: 10–46 NoRs/mouse (median = 21, NFH); 11–29 neuromuscular junctions (NMJs)/mouse (median = 18, EthD-2); 10–26 NoRs/mouse (median = 23, AnkG); and 12–31 NoRs/mouse (median = 21, AnkB) were analyzed. *P < 0.05; ***P < 0.001; ###P < 0.001 (for abnormal AnkB and AnkG immunostaining in C) compared with control by 2-way ANOVA with Tukey’s post hoc test.

Fig 5: C2 inhibition in an ex vivo mouse anti-GM1 mAb-mediated AMAN model. TS muscle from GalNAc-T−/−-Tg(neuronal) mice was incubated in 100 μg/mL anti-GM1 mAb, 100 μg/mL of C2 inhibitor or control mAb and 40% NHS for 1 hour at 32°C. The NHS-only control mice received PBS and NHS. (A) C1q deposits were detected at significantly more distal nerves in the control mAb (P < 0.001) and C2 inhibitor group (P < 0.01) compared with NHS-only. (B) C3c deposits were detected in the control mAb group but not in the NHS-only or C2 inhibitor groups (P < 0.001), n = 3/treatments. (C and D) Axonal integrity was assessed at the nerve terminal and distal NoR by staining for the axonal structural proteins NFH and Nav clusters, respectively. (C) There was a significant loss of NFH staining overlying the nerve terminal in the control mAb group compared with all other treatment groups (P < 0.001 compared with NHS-only; P < 0.01 compared with C2 inhibitor). n = 4 NHS-only and control mAb; n = 3 C2 inhibitor. (D) Nav clusters were significantly disrupted in the control mAb group (P < 0.001) but were protected to comparable levels to the NHS-only control in the C2 inhibitor treatment group. n = 3/treatments. (E) NFH staining was protected in the C2 inhibitor group. Representative images demonstrate C3c deposits overlying the nerve terminal (identified by BTx) and the consequent loss of NFH staining in the control mAb group. C3c is absent from the NHS-only and C2 inhibitor groups and NFH is present overlying the nerve terminal. Scale bar = 10 μm. (F) Illustrative images show Nav clusters (indicated by arrowheads) at the node (identified by a gap in MBP staining) in NHS-only and C2 inhibitor groups. C3c deposits are detected at the node in the control mAb group, accompanied by a loss of Nav clusters. Scale bar = 5 μm. Results represented as average ± SEM. Each datapoint represents an individual animal. Statistical significance is assessed by performing a one-way ANOVA.