Antibody-Mediated Oligodendrocyte Remyelination Promotes Axon Health in Progressive Demyelinating Disease

Abstract

Demyelination underlies early neurological symptoms in multiple sclerosis (MS); however, axonal damage is considered critical for permanent chronic deficits. The precise mechanisms by which axonal injury occurs in MS are unclear; one hypothesis is the absence or failure of remyelination, suggesting that promoting remyelination may protect axons from death. This report provides direct evidence that promoting oligodendrocyte remyelination protects axons and maintains transport function. Persistent Theiler's virus infection of Swiss Jim Lambert (SJL)/J mice was used as a model of MS to assess the effects of remyelination on axonal injury following demyelination in the spinal cord. Remyelination was induced using an oligodendrocyte/myelin-specific recombinant human monoclonal IgM, rHIgM22. The antibody is endowed with strong anti-apoptotic and pro-proliferative effects on oligodendrocyte progenitor cells. We used (1)H-magnetic resonance spectroscopy (MRS) at the brainstem to measure N-acetyl-aspartate (NAA) as a surrogate of neuronal health and spinal cord integrity. We found increased brainstem NAA concentrations at 5 weeks post-treatment with rHIgM22, which remained stable out to 10 weeks. Detailed spinal cord morphology studies revealed enhanced remyelination in the rHIgM22-treated group but not in the isotype control antibody- or saline-treated groups. Importantly, we found rHIgM22-mediated remyelination protected small- and medium-caliber mid-thoracic spinal cord axons from damage despite similar demyelination and inflammation across all experimental groups. The most direct confirmation of remyelination-mediated protection of descending neurons was an improvement in retrograde transport. Treatment with rHIgM22 significantly increased the number of retrograde-labeled neurons in the brainstem, indicating that preserved axons are functionally competent. This is direct validation that remyelination preserves spinal cord axons and protects functional axon integrity.

Keywords: Antibody; Axons; Brainstem; Magnetic resonance spectroscopy; Multiple sclerosis; N-acetyl-aspartate; Protection; Remyelination; Theiler’s murine encephalomyelitis virus.

Fig. 1

A single dose of rHIgM22 improves brainstem NAA concentrations. Brainstem MRS was collected from TMEV-infected SJL mice at 90 days post-infection prior to treatment, and at 5 and 10 weeks after treatment. a In the rHIgM22-treated group, NAA levels increased at 5 and 10 weeks to 9.51 mM (p = 0.025 and p = 0.04, respectively, one-way ANOVA repeated measures). In a saline-treated group, at 5 weeks, NAA concentrations dropped, and by 10 weeks were significantly lower (p = 0.077 and p = 0.012, respectively, one-way ANOVA repeated measures). In the control IgM-treated group, no significant difference in NAA concentrations (p = 0.17) was found. Initial NAA concentrations were not different across groups (p = 0.76, one-way ANOVA). The thick-dashed cayenne colored line represents the average baseline NAA concentrations from uninfected healthy mice (N = 6) and the thin dotted black colored line represents the average baseline NAA concentrations from all treatment groups. Symbols represent the treatment groups: rHIgM22 (red triangles), control IgM (blue circles), and saline (green boxes). b Changes in individual NAA concentrations were calculated at the 10-week point versus before treatment. NAA levels were considered improved if the difference (NAA_10 weeks − NAA_before) was greater than or equal to two times the baseline SEM. NAA levels improved in 1 of 10 in the control IgM-treated group, 1 of 11 in the saline-treated group, and 8 of 11 in the rHIgM22-treated group. c Brainstem pathological scores (means ± SEM) were similar across treatment groups (p = 0.43). d The individual brainstem NAA concentration plotted against the brainstem pathological score showed no correlation (p = 0.51), *p < 0.005, one-way ANOVA

Fig. 2

rHIgM22 promotes spinal cord remyelination. Data is expressed as a percentage of quadrants with the pathological abnormality as a function of all spinal cord quadrants examined. After the last MRS scan, 10 weeks post-treatment, mice were sacrificed, and spinal cords were removed and processed for morphologic analysis. a Mice from all three treatment groups showed similar levels of spinal cord inflammation and demyelination pathology. rHIgM22 treatment increased the extent of remyelination as compared to control groups (p < 0.001, one-way ANOVA). Typical images of demyelination (b) and remyelination (c) obtained from the antero-lateral spinal cord columns, areas with frequent disease, are shown. Arrows represent examples of remyelinated axons

Fig. 3

A single dose of rHIgM22 preserves axons in the spinal cord. When axons of all calibers were analyzed, a greater number were counted in the rHIgM22-treated group as compared to the saline-treated group (a). Symbols in a represent the treatment groups: rHIgM22 (red triangles), control IgM (blue circles), and saline (green boxes). Considering axons of different calibers, a greater number of b small-caliber (1–3.99 m²) and c medium-caliber (4–10 m²) axons were counted in the spinal cords of rHIgM22-treated mice. The number of d large-caliber axons (>10 m²) was not statistically different between groups of mice (p = 0.55). Bars represent the average of absolute number of myelinated axons ± SEM. e A positive correlation (p = 0.013, R ² = 0.19) exists between brainstem NAA concentrations collected at the 10-week time point from all mice and the number of mid-thoracic level axons. Individual brainstem NAA concentrations are plotted against f small-caliber (p = 0.11, R ² = 0.08), g medium-caliber (p = 0.005, R ² = 0.23), and h large-caliber (p = 0.4, R ² = 0.02) axons. Dotted lines represent 95 % confidence band of the best-fit linear regression line

Fig. 4

rHIgM22 improves number of retrograde-labeled brainstem neurons. FluoroGold-labeled neurons were counted in brainstem sections that correspond to the (2.5 × 2.5 × 2.5) mm³ voxel used for collecting MRS data. a Example of a cluster of fluorescently labeled neurons in the brainstem is shown. Extensive labeling of cell bodies as well as axons and dendrites can be easily appreciated. b The number of labeled neurons in the uninfected mice (N = 3) is shown as a reference. rHIgM22 treatment increased the number of retrograde-labeled brainstem neurons compared to the saline-treated group (p = 0.011, t test). The average number of retrograde-labeled brainstem neurons ± SEM per treatment group is shown. Symbols represent the treatment groups: rHIgM22 (red triangles) and saline (green boxes). c Number of fluorescently labeled neurons correlated positively and significantly to the number of axons (p = 0.008, R ² = 0.24)