Article Figures & Data
Figures
- Figure 1.
P75NTR expression is increased in injured nerves of rat EAN. A, B, Representative immunohistochemical images of p75NTR and neurofilament (NfM) staining on transverse sections of cauda equine from healthy adult (control, A) and EAN rats (B). Immunoreactivity for p75NTR protein is substantially greater in cauda equina sections of EAN rats than healthy controls. Scale bar, 20 μm. C, p75NTR mRNA expression in cauda equina and sciatic nerves of health and EAN rats via qPCR analysis. P75NTR mRAN is significantly upregulated in both cauda equina and sciatic nerves of EAN rats than healthy controls (data: mean ± SEM, relative to 18S then normalized against the healthy control, two-way repeated measures ANOVA *p < 0.01, n = 3–4/group).
- Figure 2.
Cyclo-dPAKKR ameliorates EAN in a dose-dependent manner. Time course of disease severity in rats subjected to EAN following daily administration of either vehicle control or Cyclo-dPAKKR from day 1 (data: mean ± SEM, n = 6–7/group, Mann–Whitney U test). A, Cyclo-dPAKKR reduced disease severity in rat EAN in a dose-dependent manner (3 mg/kg group: *p < 0.01, 10 mg/kg group: **p < 0.001). B, EAN rats treated with cyclo-dPAKKR beyond the disease peak (day 17) exhibited a significantly faster recovery compared with vehicle controls (*p < 0.001).
- Figure 3.
Cyclo-dPAKKR limits the extent of myelin and axonal pathology in EAN. Analyses of cauda equina or sciatic nerves isolated from age-matched healthy control (no EAN) or rats subjected to EAN at day 17 or day 24 after daily treatment with Cyclo-dPAKKR (10 mg/kg) or vehicle control (data: mean ± SEM, n = 3–4/group/time point, two-way repeated measures ANOVA *p < 0.05). A, B, Western blot (A) and densitometric analyses (B) of Western blot bands from cauda equina and sciatic nerve lysates of EAN rats at day 17. C, D, Immunohistochemistry against the myelin marker MBP (C) and quantitation of MBP intensity (D) in cauda equina sections from EAN rats. Scale bar, 50 μm. E, Representative toluidine blue staining images of cauda equine. Scale bar, 10 μm. F, Quantification of the percentage of myelinated axons in cauda equine. There are significantly more myelinated axons in animals treated with Cyclo-dPAKKR than vehicle controls. G, Quantification of the axonal density in cauda equina. Axonal density in EAN rats is expressed as a percentage of axonal density in health control rats. Axonal density in vehicle-treated animals was significantly reduced by almost 60–70% (30–40% of healthy control), whereas Cyclo-dPAKKR administration protected the axonal loss (∼60% of healthy control). H, Representative electron micrographs of myelinated axons in cauda equina. Scale bar, 2 μm. Panel a’ (a higher magnification image from panel a) shows macrophage-mediated demyelination, as evident by the last vestige of compact myelin being stripped from an axon by a macrophage filled with compact myelin debris. Panel b’ (a higher magnification image from panel b) shows the normal appearing myelin sheath penetrated with macrophages (arrows indicate myelin fragments). I, Representative immunohistochemical images of IBa1 (a marker for macrophages) in transvers sections of cauda equina of rats subjected to EAN at disease peak (day 17). Scale bar, 50 μm. J, Quantification of IBa1+ macrophages from (F) reveals that Cyclo-dPAKKR significantly reduced macrophage infiltration in injured nerves compared with vehicle controls.
- Figure 4.
Cyclo-dPAKKR protected against axonal degeneration in EAN. Analyses of cauda equina isolated from rats subjected to EAN at day 17 or day 24 after daily treatment with Cyclo-dPAKKR (10 mg/kg) or vehicle control, or age-matched healthy control rats (no-EAN). A, B, Representative immunohistochemical images of APP (A) and quantitation of APP+ axonal spheroids (B) in longitudinal sections of cauda equina isolated from rats subjected to EAN. Administration of Cyclo-dPAKKR resulted in significantly fewer APP+ axonal spheroids compared with vehicle controls (data: mean ± SEM, n = 3–4/group/time point, two-way repeated measures ANOVA *p < 0.01, scale bar, 20 μm). C, Representative immunohistochemical images of pan-Nav (red) and Caspr (green) expression in longitudinal sections of cauda equina of rats subjected to EAN or health controls. Scale bar, 20 μm, arrows indicate heminodes. D, E, Quantitation of the density of pan-Nav-expressing nodes (D) and Caspr-expressing paranodes (E) at nodes of Ranvier in the cauda equina of rats subjected to EAN or health controls (data: mean ± SEM, n = 3–4/group/time point, *p < 0.05). F, Quantitation of the density of heminodes at nodes of Ranvier from C showing significantly fewer heminodes in cyclo-dPAKKR-treated animals compared with controls (data: mean ± SEM, n = 3–4/group/time point, *p < 0.05).
- Figure 5.
Conformational constraint in Cyclo-dPAKKR determines its therapeutic effect on EAN. A, The sequence structure of the cyclic pentapeptide Cyclo-dPAKKR and its control peptide (cyclo-AdPKKR). Cyclo-dPAKKR is a circular peptide containing a tripeptide motif (KKR) from BDNF shown to bind to p75NTR. Cyclo-AdPKKR (control peptide) is also a cyclic pentapeptide containing the same p75NTR-binding motif as Cyclo-dPAKKR, but with altered position of Ala and DPro. The five amino acid residues for both peptides are indicated. B, Time courses of EAN disease severity following daily intraperitoneal delivery of either vehicle, cyclo-dPAKKR (10 mg/kg/d) or control peptide (cyclo-AdPKKR, 10 mg/kg/d) in mice after disease induction from day 1. Clinical scores: 0, normal; 1, less lively; 2, mild tail and hindlimb paresis; 3, mild ataxia and limb paresis; 4, severe ataxia and limb paresis; and 5, limb paralysis. Cyclo-dPAKKR, but not control peptide, significantly delayed the onset of disease and reduced EAN severity compared with control peptide (data: mean ± SEM, two-way repeated measures ANOVA ****p < 0.0001, n = 6 mice/group). C, Running time before failure in a DidgiGait treadmill running task at moderate speed (15 cm s−1). Cyclo-dPAKKR significantly prolonged running capacity in EAN compared with control peptide or vehicle control (data: mean ± SEM, two-way repeated measures ANOVA *p < 0.05, n = 6 mice/group).
- Figure 6.
Cyclo-dPAKKR ameliorates EAN in a p75NTR-dependent manner. A, C, E, Time courses of EAN disease severity following daily intraperitoneal delivery of either cyclo-dPAKKR (10 mg/kg/d) or control peptide (cyclo-AdPKKR, 10 mg/kg/d) in p75NTR HET or WT littermate control mice after disease induction from day 1 (n = 5–6 mice/genotype/group). B, D, F, Running times before failure in a DidgiGait treadmill running task (speed, 15 cm s−1). A, B, Cyclo-dPAKKR significantly reduced mean clinical scores (A) and improved running capacity (B) in WT mice compared with HET mice (data: mean ± SEM, two-way ANOVA with repeated measures, ****p < 0.0001 and *p < 0.05). C, D, No significant difference in clinical scores (C) and running times (D) between WT and HET mice treated with the control peptide (cyclo-AdPKKR). E, F, No significant difference in clinical scores (E) and running times (F) between for HET mice treated wither with cyclo-dPAKKR or control peptide.
- Figure 7.
Cyclo-dPAKKR reduces demyelination and axonal degeneration in a p75NTR-dependent manner. Histologic analyses of longitudinal sciatic nerve sections of EAN mice following daily treatment of either cyclo-dPAKKR (10 mg/kg) or control peptide (cyclo-AdPAKKR, 10 mg/kg) in p75NTR HET or WT littermate control mice from day 1. All tissues were collected at day 23 (disease peak; data: mean ±SEM, 95% confidence interval, ANOVA with Tukey’s post hoc testing n = 3 mice/genotype/group, *p < 0.05, **p < 0.01, ***p < 0.001). A, B, Representative SCoRe images (A) and quantification (B) of SCoRe signal as a percentage of total area measured. WT mice treated with cyclo-dPAKKR display a greater percentage area of SCoRe reflected signal than control peptide, indicating more myelinated axons. Cyclo-dPAKKR exerts no significant influence on HET mice compared with control peptide (scale bar, 200 μm). C, D, Representative images (C) of Caspr immunostaining and quantification (D) of node distance between adjacent Caspr+ paranodes. WT mice treated with cyclo-dPAKKR demonstrate significantly shorter mean node distance compared with mice treated with control peptide. Cyclo-dPAKKR exerts no significant influence on HET mice compared with control peptide (scale bar, 1 μm, minimum 70 nodes per mouse). E, F, Representative images (E) of APP and βIII-tubulin double immunostaining and quantitation (F) of the percentage of APP+ axonal area colocalizing with βII-tubulin. Cyclo-dPAKKR treatment significantly reduced the percentage of APP+ colocalization with βII-tubulin compared with the control peptide in WT, but not in HET mice (scale bar, 5 μm; scale in inset, 20 μm).
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