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Cited by 101Multiple Sclerosis Research StudiesNeuroinflammation and Neurodegeneration MechanismsPeripheral Neuropathies and Disorders

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Myelin insulation as a risk factor for axonal degeneration in autoimmune demyelinating disease

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Schäffner, Erik, Bosch-Queralt, Mar, Edgar, Julia M., Lehning, Maria, Strauß, Judith, Fleischer, Niko, Kungl, Theresa, Wieghofer, Peter, Berghoff, Stefan A., Reinert, Tilo, Krueger, Martin, Morawski, Markus, Möbius, Wiebke, Barrantes-Freer, Alonso, Stieler, Jens, Sun, Ting, Saher, Gesine, Schwab, Markus H., Wrede, Christoph, Frosch, Maximilian, Prinz, Marco, Reich, Daniel S., Flügel, Alexander, Stadelmann, Christine, Fledrich, Robert, Nave, Klaus-Armin, Stassart, Ruth M.·2023·Nature Neuroscience
DOI 10.1038/s41593-023-01366-9PMC PMC10322724

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Abstract

Axonal degeneration determines the clinical outcome of multiple sclerosis and is thought to result from exposure of denuded axons to immune-mediated damage. Therefore, myelin is widely considered to be a protective structure for axons in multiple sclerosis. Myelinated axons also depend on oligodendrocytes, which provide metabolic and structural support to the axonal compartment. Given that axonal pathology in multiple sclerosis is already visible at early disease stages, before overt demyelination, we reasoned that autoimmune inflammation may disrupt oligodendroglial support mechanisms and hence primarily affect axons insulated by myelin. Here, we studied axonal pathology as a function of myelination in human multiple sclerosis and mouse models of autoimmune encephalomyelitis with genetically altered myelination. We demonstrate that myelin ensheathment itself becomes detrimental for axonal survival and increases the risk of axons degenerating in an autoimmune environment. This challenges the view of myelin as a solely protective structure and suggests that axonal dependence on oligodendroglial support can become fatal when myelin is under inflammatory attack.

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Figures

Fig. 1 In human MS lesions, irreversible axonal damage prevails in myelinated fibers. a, Representative electron micrograph of a human multiple sclerosis (MS) lesion with moderate demyelination activity. Common types of axonal damage are exemplified, including demyelinated axons with organelle accumulation (box 1, yellow), myelinated axons with organelle accumulation (box 2, light orange) and axons with condensed axoplasm (box 3, dark orange). Scale bars, 5 µm; 0.5 µm in boxes 1–3. b, Demyelinated and unmyelinated axons (as a percentage of all axons) in four lesions from individual human MS lesions (lesion with near-complete demyelination, active lesion with extensive demyelination, active lesion with moderate demyelination and lesion border with limited demyelination). Each point represents one lesion. c, The damage types (listed above) show an inverse correlation between the extent of demyelination in the four MS lesions and the percentage of irreversibly damaged axons (dense axons). d, The myelination status of condensed axons reveals that irreversibly damaged axons are always myelinated, independent of the lesion characteristics. Each point represents one lesion. e, Electron microscopic images of axonal pathology in human MS samples demonstrating the heterogeneity of axonal organelle accumulations and axons with highly condensed axoplasm, as well as potential transition types between both forms of axonal damage from 1 to 8. Scale bars, 1 µm.
Fig. 1 In human MS lesions, irreversible axonal damage prevails in myelinated fibers. a, Representative electron micrograph of a human multiple sclerosis (MS) lesion with moderate demyelination activity. Common types of axonal damage are exemplified, including demyelinated axons with organelle accumulation (box 1, yellow), myelinated axons with organelle accumulation (box 2, light orange) and axons with condensed axoplasm (box 3, dark orange). Scale bars, 5 µm; 0.5 µm in boxes 1–3. b, Demyelinated and unmyelinated axons (as a percentage of all axons) in four lesions from individual human MS lesions (lesion with near-complete demyelination, active lesion with extensive demyelination, active lesion with moderate demyelination and lesion border with limited demyelination). Each point represents one lesion. c, The damage types (listed above) show an inverse correlation between the extent of demyelination in the four MS lesions and the percentage of irreversibly damaged axons (dense axons). d, The myelination status of condensed axons reveals that irreversibly damaged axons are always myelinated, independent of the lesion characteristics. Each point represents one lesion. e, Electron microscopic images of axonal pathology in human MS samples demonstrating the heterogeneity of axonal organelle accumulations and axons with highly condensed axoplasm, as well as potential transition types between both forms of axonal damage from 1 to 8. Scale bars, 1 µm.CC BY · PubMed Central
Fig. 2 The dynamics of axonal pathology in EAE. a, Experimental outline. EAE was induced in wild-type mice, and spinal cord samples were collected during early, peak and chronic EAE. b, Electron micrograph of an EAE lesion within the ventrolateral lumbar spinal cord at 4 dpo shows a myelinated axon with organelle accumulation (pink) and intact unmyelinated axons (yellow). Scale bar, 1 µm. Quantification of pathological axons shows that most damaged axons are myelinated. Circles in the graph represent biologically independent animals (n = 7). c, Cross-section of a spinal cord lesion at 4 dpo immunostained against SMI32 (pink) and MBP (green). Arrowheads show SMI32+ axons with surrounding MBP+ myelin. Scale bar, 2.5 µm. Quantification revealed that most SMI32+ axons are myelinated (MBP+). Unpaired two-tailed Welch’s t-test was used in b and c. d, Longitudinal image and 3D reconstruction of an EAE lesion immunostained against SMI32 (pink) and MBP (green) demonstrates continuous SMI32+ labeling along a myelinated axon. Scale bars, 5 µm. e, Top, electron microscopy and 3D reconstruction of an axon with organelle accumulation that is partially covered by myelin. Scale bars, 10 µm. Bottom, electron microscopic cross-sections at indicated segments from the top panels. Scale bars, 1 µm. Organelle accumulation is predominantly found in the myelinated part of the axon. f, Left, electron microscopy 3D reconstruction of a myelinated condensed axon. Scale bar, 10 µm. Right, cross-sections at indicated segments from the left panel (dense axon without pseudocolor). Scale bars, 1 µm. g–j, Quantification of axon pathology in electron micrographs of spinal cord lesions during early (3 dpo), peak (4 dpo) and chronic (40 d post induction) EAE. Organelle accumulations become less frequent over time (g). Condensed axons accumulate over time (no pathology in healthy spinal cord, no EAE) (h). Most condensed axons are myelinated (i). Percentage of myelinated axonal swellings decreases with EAE progression (j). One-way ANOVA, Tukey’s multiple comparisons (g,h) or Kruskal–Wallis with Dunn’s multiple comparisons post-hoc tests (i,j). Exact P values are given for early versus chronic EAE. Circles in the graphs represent biologically independent animals (no EAE, n = 4; early, n = 6; peak and chronic, n = 7). Data are means ± s.d.
Fig. 2 The dynamics of axonal pathology in EAE. a, Experimental outline. EAE was induced in wild-type mice, and spinal cord samples were collected during early, peak and chronic EAE. b, Electron micrograph of an EAE lesion within the ventrolateral lumbar spinal cord at 4 dpo shows a myelinated axon with organelle accumulation (pink) and intact unmyelinated axons (yellow). Scale bar, 1 µm. Quantification of pathological axons shows that most damaged axons are myelinated. Circles in the graph represent biologically independent animals (n = 7). c, Cross-section of a spinal cord lesion at 4 dpo immunostained against SMI32 (pink) and MBP (green). Arrowheads show SMI32+ axons with surrounding MBP+ myelin. Scale bar, 2.5 µm. Quantification revealed that most SMI32+ axons are myelinated (MBP+). Unpaired two-tailed Welch’s t-test was used in b and c. d, Longitudinal image and 3D reconstruction of an EAE lesion immunostained against SMI32 (pink) and MBP (green) demonstrates continuous SMI32+ labeling along a myelinated axon. Scale bars, 5 µm. e, Top, electron microscopy and 3D reconstruction of an axon with organelle accumulation that is partially covered by myelin. Scale bars, 10 µm. Bottom, electron microscopic cross-sections at indicated segments from the top panels. Scale bars, 1 µm. Organelle accumulation is predominantly found in the myelinated part of the axon. f, Left, electron microscopy 3D reconstruction of a myelinated condensed axon. Scale bar, 10 µm. Right, cross-sections at indicated segments from the left panel (dense axon without pseudocolor). Scale bars, 1 µm. g–j, Quantification of axon pathology in electron micrographs of spinal cord lesions during early (3 dpo), peak (4 dpo) and chronic (40 d post induction) EAE. Organelle accumulations become less frequent over time (g). Condensed axons accumulate over time (no pathology in healthy spinal cord, no EAE) (h). Most condensed axons are myelinated (i). Percentage of myelinated axonal swellings decreases with EAE progression (j). One-way ANOVA, Tukey’s multiple comparisons (g,h) or Kruskal–Wallis with Dunn’s multiple comparisons post-hoc tests (i,j). Exact P values are given for early versus chronic EAE. Circles in the graphs represent biologically independent animals (no EAE, n = 4; early, n = 6; peak and chronic, n = 7). Data are means ± s.d.CC BY · PubMed Central
Fig. 3 The dynamics of axonal pathology in CPZ-induced demyelination. a, Left, schematic outline of the experiment. Wild-type mice were fed CPZ to induce demyelination for 3 weeks (early demyelination), 5 weeks (progressed demyelination) and 12 weeks (full demyelination). The corpus callosum was analyzed. Right, representative images of the corpus callosum without CPZ treatment (Control), and with CPZ feeding for 3 weeks, 5 weeks and 12 weeks. Scale bars, 1.5 µm. b, The percentage of unmyelinated or demyelinated axons in CPZ lesions after different time periods of CPZ feeding. One-way ANOVA with Tukey’s multiple comparisons test (P values shown for comparison in chronological order). c, Although the percentage of axonal swellings first strongly increases along with CPZ lesion development, few axonal organelle accumulations characterize fully demyelinated CPZ lesions as revealed by quantification of electron micrographs. One-way ANOVA with Tukey’s multiple comparisons test (P values shown for comparison with progressed demyelination). d, The myelination status of axonal organelle accumulations during CPZ treatment. Axons with organelle accumulations become demyelinated over time. One-way ANOVA with Tukey’s post-hoc test. e, Axonal numbers in CPZ lesions show only a mild, non-significant axonal loss after 12 weeks of CPZ treatment compared to non-treated controls. Unpaired two-tailed Welch’s t-test. f, The myelination status of axons with condensed axoplasm during CPZ treatment. All condensed axons are myelinated. Note that at full demyelination, condensed axons are virtually absent and were only detected in one mouse (n = 1). Circles in all graphs in b–f represent independent animals (Control, n = 5; early demyelination, n = 3; progressed demyelination, n = 3; full demyelination, n = 3). Data represent means ± s.d.
Fig. 3 The dynamics of axonal pathology in CPZ-induced demyelination. a, Left, schematic outline of the experiment. Wild-type mice were fed CPZ to induce demyelination for 3 weeks (early demyelination), 5 weeks (progressed demyelination) and 12 weeks (full demyelination). The corpus callosum was analyzed. Right, representative images of the corpus callosum without CPZ treatment (Control), and with CPZ feeding for 3 weeks, 5 weeks and 12 weeks. Scale bars, 1.5 µm. b, The percentage of unmyelinated or demyelinated axons in CPZ lesions after different time periods of CPZ feeding. One-way ANOVA with Tukey’s multiple comparisons test (P values shown for comparison in chronological order). c, Although the percentage of axonal swellings first strongly increases along with CPZ lesion development, few axonal organelle accumulations characterize fully demyelinated CPZ lesions as revealed by quantification of electron micrographs. One-way ANOVA with Tukey’s multiple comparisons test (P values shown for comparison with progressed demyelination). d, The myelination status of axonal organelle accumulations during CPZ treatment. Axons with organelle accumulations become demyelinated over time. One-way ANOVA with Tukey’s post-hoc test. e, Axonal numbers in CPZ lesions show only a mild, non-significant axonal loss after 12 weeks of CPZ treatment compared to non-treated controls. Unpaired two-tailed Welch’s t-test. f, The myelination status of axons with condensed axoplasm during CPZ treatment. All condensed axons are myelinated. Note that at full demyelination, condensed axons are virtually absent and were only detected in one mouse (n = 1). Circles in all graphs in b–f represent independent animals (Control, n = 5; early demyelination, n = 3; progressed demyelination, n = 3; full demyelination, n = 3). Data represent means ± s.d.CC BY · PubMed Central
Fig. 4 Lesion center and border differ with regard to axonal pathology in LPC-induced demyelination. a, Left, schematic outline of the experiment. Wild-type mice were injected with LPC in the corpus callosum to induce focal demyelination, and samples were collected at an early time point (4 d post injection) and at a late time point after demyelination (14 d post injection). Right, representative images of 4- and 14-d-old LPC lesions within the corpus callosum. b–f, Based on the extent of demyelination, lesions have been divided into efficiently demyelinated lesion centers (light yellow) and incompletely demyelinated lesion borders (dark yellow). Scale bars, 2.5 µm. b, Accordingly, the extent of demyelination reveals a higher percentage of demyelinated axons in the lesion center compared to the border. c,e, The percentage of axonal organelle accumulations shows a higher abundance in the lesion center (c), while axons with condensed axoplasm are more abundant within the lesion border (e). d,f, The myelination status of axonal pathology reveals that axonal organelle accumulations are a feature of both myelinated and demyelinated fibers (d), while condensed axons virtually always have a myelin sheath in both the lesion center and border (f). Unpaired two-tailed Welch’s t-test (b,c,e) or two-tailed Mann–Whitney test (d,f). Circles in all graphs represent biologically independent animals (4 d, n = 4; 14 d, n = 8). Data are shown as means ± s.d; numbers over black bars represent P values.
Fig. 4 Lesion center and border differ with regard to axonal pathology in LPC-induced demyelination. a, Left, schematic outline of the experiment. Wild-type mice were injected with LPC in the corpus callosum to induce focal demyelination, and samples were collected at an early time point (4 d post injection) and at a late time point after demyelination (14 d post injection). Right, representative images of 4- and 14-d-old LPC lesions within the corpus callosum. b–f, Based on the extent of demyelination, lesions have been divided into efficiently demyelinated lesion centers (light yellow) and incompletely demyelinated lesion borders (dark yellow). Scale bars, 2.5 µm. b, Accordingly, the extent of demyelination reveals a higher percentage of demyelinated axons in the lesion center compared to the border. c,e, The percentage of axonal organelle accumulations shows a higher abundance in the lesion center (c), while axons with condensed axoplasm are more abundant within the lesion border (e). d,f, The myelination status of axonal pathology reveals that axonal organelle accumulations are a feature of both myelinated and demyelinated fibers (d), while condensed axons virtually always have a myelin sheath in both the lesion center and border (f). Unpaired two-tailed Welch’s t-test (b,c,e) or two-tailed Mann–Whitney test (d,f). Circles in all graphs represent biologically independent animals (4 d, n = 4; 14 d, n = 8). Data are shown as means ± s.d; numbers over black bars represent P values.CC BY · PubMed Central
Fig. 5 hMbp mice demonstrate an amelioration of the clinical disease course in EAE. a, Experimental outline. EAE was induced in wild-type and hypomyelinated hMbp mice and samples were collected during early, peak and chronic EAE. b, Left, representative electron micrographs of the ventrolateral spinal cord of control (Ctrl) and hMbp mice. Scale bars, 2.5 µm. Right, the percentage of myelinated and unmyelinated axons reveals around 10% unmyelinated fibers in the lumbar spinal cord in hMbp mice. Unpaired two-tailed Welch’s t-test; n = 3 biologically independent animals were analyzed. c, Clinical EAE scoring (1–10) shows an ameliorated disease course in hMbp mice compared to wild-type controls. Ctrl EAE, n = 13; hMbp EAE, n = 10. Two-way repeated measures ANOVA. d, Left, representative images of APP immunohistochemistry for axonal damage in EAE lesions. Arrowheads, APP+ axons. Scale bars, 10 µm. Right, the number of APP+ axons in EAE lesions reveals a reduced number of APP+ axons in hMbp mice at peak (4 dpo) and chronic EAE (40 d post induction) compared to wild-type EAE controls. Unpaired two-tailed Mann–Whitney test for each time point. e, Left, immunohistochemical images of SMI32+ MBP+ axons in EAE lesions. SMI32+ axons (arrowheads) are surrounded by MBP+ myelin sheaths. Scale bars, 5 µm. Right, quantification of SMI32+ axons reveals a reduced number of SMI32+ axons in hMbp mice at peak and chronic EAE compared to wild-type EAE controls. In d and e, peak Ctrl, n = 12; hMbp, n = 10; chronic Ctrl, n = 8; hMbp, n = 6. f, Quantification of unmyelinated or demyelinated axons in electron micrographs of EAE lesions shows that the extent of demyelination between both genotypes is similar. Note that demyelination is most prominent in the chronic EAE phase in both genotypes. Early Ctrl, n = 6; hMbp, n = 5; peak Ctrl, n = 7; hMbp, n = 4; chronic Ctrl, n = 5; hMbp, n = 5. Unpaired two-tailed Welch’s t-test per time point. Data are shown as means ± s.d. (b,d–f), apart from clinical scores, which are shown as means ± s.e.m. (c). For all graphs, numbers represent P values and circles represent biologically independent animals.
Fig. 5 hMbp mice demonstrate an amelioration of the clinical disease course in EAE. a, Experimental outline. EAE was induced in wild-type and hypomyelinated hMbp mice and samples were collected during early, peak and chronic EAE. b, Left, representative electron micrographs of the ventrolateral spinal cord of control (Ctrl) and hMbp mice. Scale bars, 2.5 µm. Right, the percentage of myelinated and unmyelinated axons reveals around 10% unmyelinated fibers in the lumbar spinal cord in hMbp mice. Unpaired two-tailed Welch’s t-test; n = 3 biologically independent animals were analyzed. c, Clinical EAE scoring (1–10) shows an ameliorated disease course in hMbp mice compared to wild-type controls. Ctrl EAE, n = 13; hMbp EAE, n = 10. Two-way repeated measures ANOVA. d, Left, representative images of APP immunohistochemistry for axonal damage in EAE lesions. Arrowheads, APP+ axons. Scale bars, 10 µm. Right, the number of APP+ axons in EAE lesions reveals a reduced number of APP+ axons in hMbp mice at peak (4 dpo) and chronic EAE (40 d post induction) compared to wild-type EAE controls. Unpaired two-tailed Mann–Whitney test for each time point. e, Left, immunohistochemical images of SMI32+ MBP+ axons in EAE lesions. SMI32+ axons (arrowheads) are surrounded by MBP+ myelin sheaths. Scale bars, 5 µm. Right, quantification of SMI32+ axons reveals a reduced number of SMI32+ axons in hMbp mice at peak and chronic EAE compared to wild-type EAE controls. In d and e, peak Ctrl, n = 12; hMbp, n = 10; chronic Ctrl, n = 8; hMbp, n = 6. f, Quantification of unmyelinated or demyelinated axons in electron micrographs of EAE lesions shows that the extent of demyelination between both genotypes is similar. Note that demyelination is most prominent in the chronic EAE phase in both genotypes. Early Ctrl, n = 6; hMbp, n = 5; peak Ctrl, n = 7; hMbp, n = 4; chronic Ctrl, n = 5; hMbp, n = 5. Unpaired two-tailed Welch’s t-test per time point. Data are shown as means ± s.d. (b,d–f), apart from clinical scores, which are shown as means ± s.e.m. (c). For all graphs, numbers represent P values and circles represent biologically independent animals.CC BY · PubMed Central
Fig. 6 Characterization of the immune response in hMbp mice in response to EAE. a, Schematic outline of the experiment. EAE was induced in wild-type and hMbp mice. The ventrolateral white matter of the spinal cord was dissected at disease peak and processed to obtain a single-cell suspension, which was then labeled with magnetically labeled CD45 antibodies to specifically sort immune cells via magnetic cell sorting. Immune cells were finally processed for single-cell sequencing analyses. b, UMAP plot for 8,954 cells from two hMbp and two wild-type mice at 4 dpo. Cells are colored and annotated by cell type. Inset, UMAP plot split and colored by genotype. DC, dendritic cell. c–f, Gene expression of several marker genes for homeostatic and activated microglia (c), activated macrophages (d), activated T cells (e) and major cytokines (f) in the microglia population. No differences were observed between the genotypes. Each point represents one cell. Data are displayed as violin plots highlighting probability density.
Fig. 6 Characterization of the immune response in hMbp mice in response to EAE. a, Schematic outline of the experiment. EAE was induced in wild-type and hMbp mice. The ventrolateral white matter of the spinal cord was dissected at disease peak and processed to obtain a single-cell suspension, which was then labeled with magnetically labeled CD45 antibodies to specifically sort immune cells via magnetic cell sorting. Immune cells were finally processed for single-cell sequencing analyses. b, UMAP plot for 8,954 cells from two hMbp and two wild-type mice at 4 dpo. Cells are colored and annotated by cell type. Inset, UMAP plot split and colored by genotype. DC, dendritic cell. c–f, Gene expression of several marker genes for homeostatic and activated microglia (c), activated macrophages (d), activated T cells (e) and major cytokines (f) in the microglia population. No differences were observed between the genotypes. Each point represents one cell. Data are displayed as violin plots highlighting probability density.CC BY · PubMed Central
Fig. 7 Axonal pathology is reduced in hMbp mice in EAE. a, Outline of the adoptive transfer EAE experiment. EAE was induced in Nur77-GFP/RFP mice, and after 11–12 d, the lymph nodes were dissected and the purified lymphocytes cultured with MOG and cytokines. Cultured T lymphocytes were then transferred to either wild-type (Ctrl) or hMbp mice to induce adoptive transfer EAE. Ab, antibody. b, Quantification of the mean fluorescence intensity of GFP of transferred CD4+ T cells as a measure for T cell activation indicated by Nur77 expression by FACS reveals no difference in T cell activation between hMbp mice and controls. Two-way ANOVA with Sidak’s multiple comparisons test. AT, adoptive transfer. c, Clinical EAE scoring (1–10) after adoptive transfer shows an ameliorated disease course in hMbp mice compared to controls, reminiscent of the findings using classical EAE (Fig. 5c, light colors). Ctrl AT, n = 14; hMbp AT, n = 18. Pairwise two-way ANOVA. d, Quantification of marker mRNA expression by rt–qPCR for microglia and T cell activation and for relevant cytokines demonstrates no differences between Ctrl and hMbp mice. n = 4 per group, unpaired two-tailed Welch’s t-test. e, The percentage of myelinated SMI32+ axons is comparable between hMbp and Ctrl mice. For peak EAE, Ctrl, n = 14; hMbp, n = 8; for chronic EAE, Ctrl, n = 12; for hMbp, n = 11. Unpaired two-tailed Mann–Whitney test for each time point. f, The percentage of axonal organelle accumulations in electron micrographs of EAE lesions shows a reduction in hMbp mice compared to controls. g, hMbp mice demonstrate a lower percentage of axons with condensed axoplasm compared to controls at peak and chronic EAE. h, The myelination status of axonal organelle accumulations in hMbp and control mice reveals no difference between genotypes. i, In both hMbp and wild-type controls, condensed axons are an almost exclusive feature of myelinated axons. In (f–i), unpaired two-tailed Welch’s t-test was used for each time point. The key for f–i is given in i. For all graphs, circles represent biologically independent animals. Data are means ± s.d. except in c, in which data are means ± s.e.m.
Fig. 7 Axonal pathology is reduced in hMbp mice in EAE. a, Outline of the adoptive transfer EAE experiment. EAE was induced in Nur77-GFP/RFP mice, and after 11–12 d, the lymph nodes were dissected and the purified lymphocytes cultured with MOG and cytokines. Cultured T lymphocytes were then transferred to either wild-type (Ctrl) or hMbp mice to induce adoptive transfer EAE. Ab, antibody. b, Quantification of the mean fluorescence intensity of GFP of transferred CD4+ T cells as a measure for T cell activation indicated by Nur77 expression by FACS reveals no difference in T cell activation between hMbp mice and controls. Two-way ANOVA with Sidak’s multiple comparisons test. AT, adoptive transfer. c, Clinical EAE scoring (1–10) after adoptive transfer shows an ameliorated disease course in hMbp mice compared to controls, reminiscent of the findings using classical EAE (Fig. 5c, light colors). Ctrl AT, n = 14; hMbp AT, n = 18. Pairwise two-way ANOVA. d, Quantification of marker mRNA expression by rt–qPCR for microglia and T cell activation and for relevant cytokines demonstrates no differences between Ctrl and hMbp mice. n = 4 per group, unpaired two-tailed Welch’s t-test. e, The percentage of myelinated SMI32+ axons is comparable between hMbp and Ctrl mice. For peak EAE, Ctrl, n = 14; hMbp, n = 8; for chronic EAE, Ctrl, n = 12; for hMbp, n = 11. Unpaired two-tailed Mann–Whitney test for each time point. f, The percentage of axonal organelle accumulations in electron micrographs of EAE lesions shows a reduction in hMbp mice compared to controls. g, hMbp mice demonstrate a lower percentage of axons with condensed axoplasm compared to controls at peak and chronic EAE. h, The myelination status of axonal organelle accumulations in hMbp and control mice reveals no difference between genotypes. i, In both hMbp and wild-type controls, condensed axons are an almost exclusive feature of myelinated axons. In (f–i), unpaired two-tailed Welch’s t-test was used for each time point. The key for f–i is given in i. For all graphs, circles represent biologically independent animals. Data are means ± s.d. except in c, in which data are means ± s.e.m.CC BY · PubMed Central
Extended Data Fig. 1 Human multiple sclerosis lesions. a, Representative electron micrograph of a lesion with near-complete demyelination. Note the virtual absence of myelinated axons. A demyelinated axon with organelle accumulation is shown in higher magnification. Scale bar = 5µm / blow-up = 1µm. b, A representative image from an active lesion with extensive demyelination demonstrates a few myelinated axons, ongoing phagocytosis and many demyelinated axons. A demyelinated axon with organelle accumulation is shown at higher magnification. Scale bar = 5µm / blow-up = 1µm. c, A representative image of a lesion border with a large proportion of myelinated axons. Note the myelinated axons with a pronounced condensation of the axoplasm, representing irreversible axon damage (high magnification image). Scale bar = 5µm / blow-up = 1µm.
Extended Data Fig. 1 Human multiple sclerosis lesions. a, Representative electron micrograph of a lesion with near-complete demyelination. Note the virtual absence of myelinated axons. A demyelinated axon with organelle accumulation is shown in higher magnification. Scale bar = 5µm / blow-up = 1µm. b, A representative image from an active lesion with extensive demyelination demonstrates a few myelinated axons, ongoing phagocytosis and many demyelinated axons. A demyelinated axon with organelle accumulation is shown at higher magnification. Scale bar = 5µm / blow-up = 1µm. c, A representative image of a lesion border with a large proportion of myelinated axons. Note the myelinated axons with a pronounced condensation of the axoplasm, representing irreversible axon damage (high magnification image). Scale bar = 5µm / blow-up = 1µm.CC BY · PubMed Central
Extended Data Fig. 2 Axonal organelle accumulations predominate in myelinated segments. Images of longitudinal, partially demyelinated axons show that organelles accumulate specifically in not or not yet demyelinated parts of the axons. Similar observations were made in a, EAE; b, Multiple sclerosis; c, Cuprizone and d, Lysophosphatidylcholine (LPC) -induced demyelination. Scale bar = 3µm.
Extended Data Fig. 2 Axonal organelle accumulations predominate in myelinated segments. Images of longitudinal, partially demyelinated axons show that organelles accumulate specifically in not or not yet demyelinated parts of the axons. Similar observations were made in a, EAE; b, Multiple sclerosis; c, Cuprizone and d, Lysophosphatidylcholine (LPC) -induced demyelination. Scale bar = 3µm.CC BY · PubMed Central
Extended Data Fig. 3 Quality control (QC) and cell type determination in single-cell RNA sequencing of immune cells in EAE. a, QC Violin plots showing UMI counts, feature counts, and the ratio of mitochondrial gene reads and chosen cut-offs. Single dots in the graphs represent single cells. N=1 biologically independent animal per group. Box bounds are according to the standard tukey format set as default values in ggplot2. Upper box bound: 75% percentile / centre mark: 50% percentile (median) / lower box bound: 25% percentile. Whiskers/Outliers/95% percentiles are not shown. b, Pairsplot of first 10 dimensions from a principle component (PC) analysis colored by genotype demonstrates no differential clustering between wildtype (black) and hMbp mice (green). c, Dot plot displaying the genes used for the identification of immune cell types. d, UMAP plots showing marker gene expression in the appropriate clusters.
Extended Data Fig. 3 Quality control (QC) and cell type determination in single-cell RNA sequencing of immune cells in EAE. a, QC Violin plots showing UMI counts, feature counts, and the ratio of mitochondrial gene reads and chosen cut-offs. Single dots in the graphs represent single cells. N=1 biologically independent animal per group. Box bounds are according to the standard tukey format set as default values in ggplot2. Upper box bound: 75% percentile / centre mark: 50% percentile (median) / lower box bound: 25% percentile. Whiskers/Outliers/95% percentiles are not shown. b, Pairsplot of first 10 dimensions from a principle component (PC) analysis colored by genotype demonstrates no differential clustering between wildtype (black) and hMbp mice (green). c, Dot plot displaying the genes used for the identification of immune cell types. d, UMAP plots showing marker gene expression in the appropriate clusters.CC BY · PubMed Central
Extended Data Fig. 4 Subclustering of microglia and macrophage populations in EAE. a-c, Subclustering of the microglial cell populations revealed five subclusters in wildtype and hMbp mice at EAE disease peak (for an outline of the experiment see Fig. 4g), namely homeostatic microglia (hMG), disease-associated microglia (daMG2 and daMG3), a functional phenotype characterized by a gradient between hMG and daMG (hMG/daMG) and replicating microglia (repl. MG). a, Dotplots indicate relevant marker gene expression of the individual subpopulations as previously identified by Jordão et al.39. The colors indicate the scaled expression of marker genes while the dot size reflects the percent of cells expressing the marker gene. b, UMAPs visualizing the integrated subset of microglial for wildtype and hMbp mice independently, demonstrating a similar profile for both genotypes. c, Stacked bar plots representing the relative contribution of the five individual microglia subclusters to the total population, shown for wildtype and hMbp mice. d-f, Subclustering of the macrophage population identified five subclusters in wildtype and hMbp mice, namely Arg1+, Nos2+, Cxcl10+, Saa3+ and Ifit1 + clusters. d, Dotplots indicate relevant marker gene expression of the individual subpopulations as previously identified by Giladi et al.40. The colors indicate the scaled expression of marker genes and the dot size reflects the percentage of cells expressing the marker gene. e, UMAPs visualizing the integrated subset of macrophages for wildtype and hMbp mice, respectively, demonstrating similar profiles for both genotypes. f, Stacked bar plots representing the relative contribution of the five individual macrophage subclusters to the total population, shown for wildtype and hMbp mice.
Extended Data Fig. 4 Subclustering of microglia and macrophage populations in EAE. a-c, Subclustering of the microglial cell populations revealed five subclusters in wildtype and hMbp mice at EAE disease peak (for an outline of the experiment see Fig. 4g), namely homeostatic microglia (hMG), disease-associated microglia (daMG2 and daMG3), a functional phenotype characterized by a gradient between hMG and daMG (hMG/daMG) and replicating microglia (repl. MG). a, Dotplots indicate relevant marker gene expression of the individual subpopulations as previously identified by Jordão et al.39. The colors indicate the scaled expression of marker genes while the dot size reflects the percent of cells expressing the marker gene. b, UMAPs visualizing the integrated subset of microglial for wildtype and hMbp mice independently, demonstrating a similar profile for both genotypes. c, Stacked bar plots representing the relative contribution of the five individual microglia subclusters to the total population, shown for wildtype and hMbp mice. d-f, Subclustering of the macrophage population identified five subclusters in wildtype and hMbp mice, namely Arg1+, Nos2+, Cxcl10+, Saa3+ and Ifit1 + clusters. d, Dotplots indicate relevant marker gene expression of the individual subpopulations as previously identified by Giladi et al.40. The colors indicate the scaled expression of marker genes and the dot size reflects the percentage of cells expressing the marker gene. e, UMAPs visualizing the integrated subset of macrophages for wildtype and hMbp mice, respectively, demonstrating similar profiles for both genotypes. f, Stacked bar plots representing the relative contribution of the five individual macrophage subclusters to the total population, shown for wildtype and hMbp mice.CC BY · PubMed Central
Extended Data Fig. 5 Working model on the role of myelin in inflammatory demyelinating disease. Myelinated axons are physically isolated from the environment by the myelin sheath (1) and therefore dependent on oligodendroglial support, for example metabolic exchange and detoxification (2). An autoimmune attack against myelinating oligodendrocytes by immune cells (3) perturbs glial support (4). Axons that are trapped in myelin insulation by dysfunctional oligodendrocytes are specifically vulnerable for irreversible degeneration (5). Rapid demyelination, in turn, acutely secures axonal survival (6).
Extended Data Fig. 5 Working model on the role of myelin in inflammatory demyelinating disease. Myelinated axons are physically isolated from the environment by the myelin sheath (1) and therefore dependent on oligodendroglial support, for example metabolic exchange and detoxification (2). An autoimmune attack against myelinating oligodendrocytes by immune cells (3) perturbs glial support (4). Axons that are trapped in myelin insulation by dysfunctional oligodendrocytes are specifically vulnerable for irreversible degeneration (5). Rapid demyelination, in turn, acutely secures axonal survival (6).CC BY · PubMed Central

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  1. Narciso, M. S., Hokoç, J. N. & Martinez, A. M. B. Watery and dark axons in Wallerian degeneration of the opossum’s optic nerve: different patterns of cytoskeletal breakdown? An. Acad. Bras. Cienc. 73, 230–243 (2001).
  2. Bittner, S., Afzali, A. M., Wiendl, H. & Meuth, S. G. Myelin oligodendrocyte glycoprotein (MOG35–55) induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. J. Vis. Exp. 86, e51275 (2014).

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Metadata

Authors
Schäffner, E., Bosch-Queralt, M., Edgar, J. M., Lehning, M., Strauß, J., Fleischer, N., Kungl, T., Wieghofer, P., Berghoff, S. A., Reinert, T., Krueger, M., Morawski, M., Möbius, W., Barrantes-Freer, A., Stieler, J., Sun, T., Saher, G., Schwab, M. H., Wrede, C., Frosch, M., Prinz, M., Reich, D. S., Flügel, A., Stadelmann, C., Fledrich, R., Nave, K.-A., & Stassart, R. M.
Affiliations
  • University Hospital Leipzig (DE)
  • Max Planck Institute for Multidisciplinary Sciences (DE)
  • Universitätsmedizin Göttingen (DE)
  • Leipzig University (DE)
  • University of Augsburg (DE)
  • German Center for Neurodegenerative Diseases (DE)
  • Medizinische Hochschule Hannover (DE)
  • University of Freiburg (DE)
  • National Institutes of Health (US)
Corresponding author
  • Fledrich, Robert
  • Nave, Klaus-Armin
  • Stassart, Ruth M.
ORCID
Venue
Nature Neuroscience
Publisher
Springer Science and Business Media LLC
Published
2023-06-29
Volume
26
Issue
7
Pages
1218-1228
ISSN
  • 1097-6256 (ISSN-L)
  • 1546-1726
DOI
10.1038/s41593-023-01366-9
PMC
PMC10322724
OpenAlex
W4382630803
Cited by
101
References
58
Citations by year
2026: 32 · 2025: 35 · 2024: 26 · 2023: 7
FWCI
19.91
Type
journal article
Language
en
Update notices
addendum (2025-03-11)
Open access
Licence
Locations
  • Nature Neuroscience (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • ENLIGHTEN (Jurnal Bimbingan dan Konseling Islam) (acceptedVersion, cc-by-sa)
  • GoeScholar The Publication Server of the Georg-August-Universität Göttingen (Georg-August-Universität Göttingen) (submittedVersion, cc-by)
  • DZNE Pub (submittedVersion, cc-by)
  • OPUS (Augsburg University) (submittedVersion, cc-by)
Funders
  • Dr. Miriam and Sheldon G. Adelson Medical Research Foundation
  • Deutsche Forschungsgemeinschaft (SFB 1052, 209933838, TRR274/1 2020, SFB/TRR 274, TRR-274, EXC 2067, STA1389/5-1, EXC 2067‐1, 390729940, EXC 2067/1390729940, 408885537)
  • Bundesministerium für Bildung und Forschung (01EJ2203C)
  • Boehringer Ingelheim Stiftung
  • Medical Research Council (MR/X013073/1)
  • National Institute of Neurological Disorders and Stroke
  • H2020 European Research Council (101021345, 948857)
Topics
  • Health Sciences › Medicine › Clinical Neurology › Multiple Sclerosis Research Studies
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Health Sciences › Medicine › Clinical Neurology › Peripheral Neuropathies and Disorders
Keywords
myelin, axonal degeneration, multiple sclerosis, autoimmune demyelinating disease, oligodendrocytes, myelination, autoimmune encephalomyelitis
MeSH terms
Animals, Axons, Encephalomyelitis, Autoimmune, Experimental, Humans, Multiple Sclerosis, Myelin Sheath, Risk Factors, Mice

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